Investigations Into the Mechanisms of Vitamin C

INVESTIGATIONS INTO THE MECHANISMS OF VITAMIN C

UPTAKE IN RODENT AND HUMAN LENS EPITHELIAL CELLS

MARK E. M. OBRENOVICH

Submitted in partial fulfillment of the requirements for the Degree of Doctor of Philosophy

Department of Pathology

CASE WESTERN RESERVE UNIVERSITY

August, 2008

CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES

We hereby approve the dissertation of

______MARK E. M. OBRENOVICH ______

candidate for the Ph.D. degree *.

(signed)______MARK A. SMITH______(chair of the committee)

______VINCENT M. MONNIER______

______ROBERT B. PETERSEN______

______CLIVE R. HAMLIN______

______QUINZHONG KONG______

______RAM H. NAGARAJ______

(date) ______April 24, 2008____

*We also certify that written approval has been obtained for any proprietary material contained therein.

Table of Contents

List of Tables …….…………………………….……….…………...………………...... 5

List of Figures …….…………………………….…………………….……………...... 6

Acknowledgements ……….…………………………….…..………….……………...... 9

List of Abbreviations …….……………….……………………….………………...... 10

Abstract ……….………….…………………….…………………………………...... 14

Chapter 1. INTRODUCTION: Background and Significance

1.1. Introduction to Chemical Mechanisms of Aging in the Human Lens ………...... 16

1.1.1. Development of age-related opacifications ……..……….…...... … 19

1.1.2. The unique lens structure ………….………………………...... 23

1.1.3. Models of cataractogenesis …….……………….…………………...... 25

1.2. Sources of damage to lens crystallins during aging …….……………...………..... 31

1.2.1. Oxidative damage ……….……………………...……………...…...... 33

1.2.2. Photo-oxidative damage …..……….………………………………...... 37

1.2.3. Carbonyl damage ……….…………………………………………...... 43

1.2.4. Crosslinking ………..……………………….………………………...... 47

1.2.5. Protein fragmentation, truncation and deamidation …...….………...... 50

1.2.6. Denaturation, solubility and conformational changes involving crystallins

……………………………………………………………………………...…… 55

1.3. Damage to crystallins by the Maillard Reaction ……….……….……………....… 57

1 1.3.1. Glucose ……………………….……………………….….…………….. 59

1.3.2. Oxoaldehydes .…………………………………..………..…………...… 61

1.3.3. Ascorbic acid degradation products …………..……..……………...... 63

1.3.4. Lipid peroxidation products ….……..…………………………….…...... 71

1.4. Protective Mechanisms; defense against lenticular damage ….…....….…...... 74

1.4.1. Endogenous protective mechanisms ………….…………………….…... 77

1.4.2. Intrinsic chaperone function of crystallins …..……………………..….... 78

1.4.3. Glutathione ………………………………………………….….....….…. 80

1.4.4. Protective enzymatic mechanisms to crystallin damage ….....……...... 87

1.5. Ascorbic Acid transport and biologic roles ……………...…..……...…...……...... 87

1.5.1. Function of vitamin C transporters and putative substrate transport ..…... 90

1.5.2. SVCT1 and SVCT2 cellular distribution ………………...…………..…. 92

1.5.3. SVCT2 structural and functional domains ………….…………..….….... 93

1.5.4. SVCT2 Inhibition and secretion studies of ascorbate …....……....….….. 97

Chapter 2. Relative Suppression of ASA Uptake Between Mouse and Human Lens

Epithelial Cells: A Comparative Study.

2.1. INTRODUCTION ………….………………………………….……………...... 105

2.2. METHODS ……………..……………………….………...... …………….….... 106

2.2.1. Materials …..……………………………………………...... 107

2.2.2. Cell culture ..…………………………………..………..………...... 107

2.2.3. Lens explant culture ………………………..……………...... 108

2.2.4. Measure of ASA transport ……………………………...... 109

2 2.2.5. Determination of 14C-ASA and 14C-DHA cellular uptake …...... 110

2.2.6. Inhibition of glutathione synthesis ..……………………...….……...... 112

2.2.7. Expression and cellular transfection methods ……….………....…….... 112

2.2.8. Transient transfection of lens explants ……..……..…………...... 114

2.2.9. Statistical Methods………….………....…….…………....…….…...... 115

2.3. RESULTS ……..……...………………………………..………………………... 115

2.3.1. Comparative effects of glycemic stress on F-ASA and F-DHA uptake and

GSH homeostasis in HLE B-3, 17EM15 and JAR cells …………..…………...115

2.3.2. Confirmation of suppression of ascorbic acid uptake in mouse lens

epithelial cells ………...…………………………..…………………………... 119

2.3.3. Effect of overexpression of the human SVCT2 transporter into mouse

17EM15 cells ….……………………...... 122

2.3.4. Effect of overexpression of the human SVCT2 transporter into cultured

mouse lenses …………………………………………………….….……...…. 124

2.4. DISCUSSION ……………………………….....…………………..……...... 125

2.5. SUMMARY ……………………………………………………….…………..… 131

Chapter 3. Exploring mechanisms of mouse and human SVCT2 function and regulation

3.1. INTRODUCTION …………………………………………...... 134

3.2. METHODS ………………………………………………………………..…...... 134

3.2.1. Materials …………………………………………...……..…...... 135

3.2.2. Cell culture ……………………………………..………..…………...... 136

3.2.3. RNA isolation, RT-PCR and qPCR ………..………..……………...... 138

3 3.2.4. Construction of human and mouse SVCT2 expression vectors ….....…..138

3.2.5. Western blotting ………………….…..…………………………...... 139

3.2.6. Cellular transfection methods ….………………….…...... 140

3.2.7. Transport assay with L-14C-ascorbic acid ….…………..……...……..... 142

3.2.8. UV-exposure on mouse lens epithelial cells ………..…………..…...... 144

3.2.9. Statistical methods …..…………………………..………………..….... 144

3.3. RESULTS …..……………………………………………………….………... 145

3.3.1. Comparative expression of SVCT2 in mouse and human LECs …….… 145

3.3.2. Comparison of sequence and structural motifs, in silico ………….….... 147

3.3.3. Comparative functional studies of mSVCT2 and hSVCT2 …...……….. 150

3.3.4. Effect of UV exposure on ASA uptake in endogenous mouse lens epithelial

cells ……..………...……...... …. 153

3.3.5. Effect of overexpression of the human vs. mouse transporters into various

cell lines ……………………………………………..………..………….....….155

4.1. DISCUSSION ………..…………………………………..…………………… 157

5.1. CONCLUSIONS AND FUTURE STUDIES ………………..…..………….... 162

BIBLIOGRAPHY ……………….....…………...……………...... … 168

4 List of Tables

Table 1.1. Wavelengths of UV light incidenting on structures of the eye, protecting the

retina from UV damage .…..………………………….……………...…….. 37

Table 1.2. Endogenous protective mechanisms for the lens and other eye structures .....75

Table 2.1. Intracellular glutathione levels under glycemic stress and BSO treatment ..117

Table 2.2. NMR Chemical shift of identified and unknown Fluorocompounds ……... 118

Table 3.1. Comparison of SVCT2 motifs between mouse and human …….…...... 147

List of Figures

Figure 1.1. Lens Architecture and fiber cell distribution ………………….…..……..... 21

Figure 1.2. 19F NMR Ascorbic acid, oxidation and degradation product tracers ..…..... 29

Figure 1.3. Select Pathways of the Maillard Reaction ……..……………………..….... 57

Figure 1.4. Ascorbate reaction to form ascorbate radical and the dismutation into

ascorbic acid and dehydroascorbic acid ..…………………….…….…..… 63

Figure 1.5. Aged lenses on left, compared to explants reacted with equimolar reducing

sugars or ascorbic acid or dehydroascorbic acid …...………...………...... 64

Figure 1.6. AGEs formed from ascorbic acid or degradation products of DHA …….... 68

Figure 1.7. Important antioxidant systems in the lens …………...... ………….....…… 76

Figure 2.1. F-ASA and F-DHA are both taken up into cells by a sodium-dependent

mechanism ……...…………………………………………………....…. 111

Figure 2.2. F-ASA and F-DHA uptake is via SVCT2 …………………...... …….…... 116

Figure 2.3. Comparative effect of lens epithelial cell type and glycemic stress on

intracellular concentrations of F-ASA F-ascorbic acid ..…….…….…… 119

Figure 2.4. A. Effect of high (25 mM) vs. low (5.5 mM) glucose concentration on 14C-

ASA uptake into mouse 17EM15, 21EM15 and human HLE-B3 cells ... 121

Figure 2.4. B. Percentage of sodium dependent 14C-ASA uptake determined in transport

buffer vs. sodium-free buffer under conditions of high or low glucose ... 121

Figure 2.5. Comparative effect of 14C-ASA uptake into 17EM15 mouse cells transfected

with pcDNA/hSVCT2 plasmid vs. mock transfection, and uptake into mouse

6 21EM15 and rabbit lens epithelial cells ……………………………...... 123

Figure 2.6. Effect of transfection of 17EM15 mouse lens epithelial cells with the denaA-

hSVCT2 construct on 14C-ASA uptake with or without sodium ………. 124

Figure 2.7. Effect of 24 hr transfection of cultured whole mouse lens explants with dena-

SVCT2 construct co-transfected with GFP reporter ……………..……... 125

Figure 2.8. Sequence alignment of mouse vs. human SVCT2 ….……………….…... 129

Figure 3.1. Vectors used in cloning mouse and human SVCT2……………...….….... 139

Figure 3.2. Transfection efficiency with GFP co transfected reporter construct…….... 141

Figure 3.3. A. Time course of 14C-ASA uptake with non-transfected mouse and human

lens epithelial cells …….………………………………...………..…….. 146

Figure 3.3. B. Comparative effect of lens epithelial cell type and passage on mRNA

levels by qPCR …..……………………………………………….…...… 146

Figure 3.3. C. Western Blot of 17Em15, HLE-B3 and CHO cells with antibodies to

human SVCT2 …..….…………………..…………………….……..….. 147

Figure 3.4. A. SVCT2 constructs and pCEP constructs CHO cells ………..……....… 151

Figure 3.4. B. M17 neuroblastoma cells transfected with humanSVCT2-pcDNA or

mouse SVCT2-pcDNA …………...…………………..………………… 151

Figure 3.4. C. Effect of transient transfection of pCEP-hSVCT2, pCEP-mSVCT2 and

pcDNA3.1-hSVCT2 plasmids in HEK-293T Kinetics over 30 minutes .. 152

Figure 3.5. Effect of transient transfection of hSVCT2-pcDNA and mSVCT2-pcDNA

with and without calcium in the transport buffer on uptake of ASA .…... 153

Figure 3.6. A. 14C-ASA uptake in stable cell lines A) SVCT2 constructs and pCEP

constructs CHO cells ……………..………..………………………...... 154

7 Figure 3.6. B. M17 neuroblastoma cells transfected with SVCT2-pcDNA constructs..155

Figure 3.7. Overexpression in HEK-293 cells using the d. Dena-hSVCT2 compared to

CMVpromoter-driven constructs, pCEP or pcDNA ……...……….….… 156

Figure 3.8. A. Effect of UV irradiation on 14C-ASA uptake 17EM15 vs. non-transfected

cells …………………………………...…………………………...... 157

Figure 3.8. B. Time course of 14C-ASA uptake in UV-exposed 17EM15 cells …...…. 157

ACKNOWLEDGEMENTS

My most sincere appreciation goes to my parents first, Stanley and Elizabeth Obrenovich, for the support only parents could give and then to my collaborators and those most important to the successful completion of the work, specifically, Dorjee Tamang, David

Kehres, Yi Li and Ayuna Dagdanova. Next, thanks to the workers in the Monnier,

Petersen and Smith labs, specifically, Sandy Richardson for helpful advice. Also, thanks to Drs. Aubrey de Grey, Derrik Abbott, Purnima Jaiswal, Gjumrakch Aliev, Don

Anthony, James Anderson, John Lowe, Feng Lin, Nina Singh, Shu Chen, Alan Tartakoff.

For financial support: The NIA, NIH, JDRF and NEI T-32 Training Grant and Susan

Brady-Kalnay, John Porter, Eric Pearlman and the Department of Ophthalmology cores for their valuable services. Finally, and importantly my sincere thanks to my many mentors, Drs. Vincent Monnier, Robert Petersen, Mark Smith, Gjumrakch Aliev, Luigi

Messineo, Nino Camardese and my dear Alla Zilichickhis. I thank you for the good mentoring I received over the years.

List of Abbreviations

10KMWCO Ten thousand molecular weight cut off 17EM15 Emory mouse lens epithelial 2,3-DKG 2,3-diketogulonic acid 21EM15 Emory mouse lens epithelial 2-AAA alpha-aminoadipic acid-delta-semialdehyde 3-DG 3-deoxyglucosone 3-HKG 3-hydroxykynurenine 3-OH 3-hydoxy αA, and αB Alpha Crystallins AAA 2-aminoadipic acid AAS alpha-aminoadipic semialdehyde ACTH Adrenocorticotrophic hormone AF Autofluorescence AG Aminoguanidine AGE(s) Advanced glycation end product(s) ANOVA analysis of variance AP-1 Activator protein-1 AQC 6-aminoquinolyl-N- hydroxysuccimidylcarbamate AQP0 Aquaporin 0 AR Aldose Reductase ARVO Association for Research in Vision ASA Ascorbic Acid Asc- Ascorbyl radical Asn Asparagine Asp Aspartic ATP Adenosine Triphosphate βB1, βB2, βB3, βA3/A1, βA2, and βA4 Beta Crystallins BBB Blood–brain barrier BDI Bleomycin-detectable iron BP base pair BSO Buthionine sulfoximine C2C12 Mouse myoblast Caco-2 Human, colon, adenocarcinoma intestinal CD Circular dichroism cDNA complementary DNA CHO Chinese Hamster Ovary CK-2 Casein kinase II CL Corpus Luteum CML Carboxymethyl-lysine COS-1 Monkey Kidney Fibroblast

10 Cu2+ Copper DAG Diacylglycerol DHA Dehydroascorbic acid DLS Dynamic light-scattering DNA Deoxyribonucleic acid DOPA Dopamine EC Endothelial cells EDTA Ethylene diamine tetraacetic acid F-ASA 6-fluoro-6-deoxy-ascorbic acid F-ASA F-ascorbic acid F-DHA F-dehydroascorbic acid Fra-1 fos-related antigen-1 γA, γB, γC, γD, γE, γF, γS Gamma Crystallins GAPDH Glyceraldehyde 3-phosphate dehydrogenase gDNA genomic DNA GGS Gamma-glutamyl semialdehyde GLUT Na+-independent glucose transporters GO Glyoxal GODIC 2-ammonio-6-(2-[(4-ammonio-5-oxido-5- oxopentyl)amino]-4,5-dihydro-1H-imidazol- 5-ylidene amino) hexanoate GOLA Glycine amide GSH Glutathione H2O2 Hydrogen Peroxide HbA1c Hemoglobin A1c HBO Hyperbaric oxygen HEK-293 Human Embryonic Kidney HEPES N-2-hydroxyethylpiperazine-N0-2 ethanesulfonic acid HgCl2 Mercury chloride HLE-B3 Human Lens Epithelial HMP Hexose monophosphate shunt pathway HMW High-molecular-weight IACUC Institutional Animal Care and Use IOVS Investigative Ophthalmology & Visual Science IP3 Inositol-3-Phosphate JAR Choriocarcinoma JNK c-Jun N- terminal kinase K2P 1-(5-amino-5-carboxypentyl)-4-(5-amino-5- carboxypentyl-amino)-3-hydroxy-2,3- dihydropyridinium kDa Kilodalton L6C5 Rat myoblast LEC Lens Epithelial Cell LH Lutenizing Hormone

11 LLC-PK1 Pig, kidney Fibroblastic-like Cell LM1 Vesperlysine A LMW Low-Molecular- Weight L-NAME L-nitroarginyl methyl ester M17 Neuroblastoma MAPK MAP Kinase MDA malondialdehyde MDCK Madin-Darby canine kidney MetSOX Methionine sulphoxide). MG-H1 and -H2 Methylglyoxal hydroimidazolones MGO Methylglyoxal MIP Membrane intrinsic protein mmol millimole MODIC 2-ammonio-6-(2-[(4-ammonio-5-oxido-5- oxopentyl) amino]-4-methyl-4, 5-dihydro- 1H- imidazol-5-ylidene amino) hexanoate mRNA messenger ribonucleic acid NADPH Nicotinic-adenine-dinucleotide monophosphate Ni+ Nickel nmol Nanomole NMR Nuclear magnetic resonance NO Nitric oxide OP-lysine 2-ammonio-6-(3-oxidopyridinium-1 -yl)hexanoate ORF Open Reading Frame ORN Ornithine PCEC Pig Coronary Endothelial Cell PDI Protein Disulfide Isomerase PDI Protein disulfide isomerase PEI Polyethylimine PG Prostaglandin PGE2 Prostaglandin E2 PGF2 Prostaglandin F2 PKC Protein kinase C PMA Phorbol 12-myristate 13-acetate pmol Picomole PO4- Phosphate PTM Post-translational modifications qPCR quantitative Polymerase Chain Reaction N/N 1003A Rabbit Lens Epithelial RNA Ribonucleic acid RNS Reactive nitrogen species ROS Reactive oxygen species RT-PCR Reverse Transcriptase Polymerase Chain Reaction

12 S.D. Standard Deviation SOD Superoxide dismutase STZ Streptozotocin SVCT Sodium-dependent vitamin C transporters Ta Annealing temperature Tg Transgenic TPA Tissue plasminogen activator TR-iBRB Retinal cells Trp Tryptophan TUNEL Terminal deoxynucleotidyl transferase mediated dUTP-X nick end labeling U-14C Uniformly labeled Carbon umol Micromole UTP Uridine Triphosphate UTR Untranslated region UV-A Ultraviolet radiation A UV-B Ultraviolet radiation B UV-C Ultraviolet radiation C UVEC Umbilical vein endothelial cell WI Water-insoluble proteins WS Water-soluble proteins WT Wild-type Zn2+ Zinc

Investigations into the Mechanisms of Vitamin C Uptake in Rodent and Human

Lens Epithelial Cells

Abstract

MARK E. M. OBRENOVICH

Vitamin C, Ascorbic Acid (ASA), was first isolated in 1928 by the Hungarian

biochemist and Nobel Prize winner Albert Szent-Gyorgyi (323). Szent-Gyorgi described

this compound ASA, as a carbohydrate derivative “Hexouric acid”. ASA is a water-

soluble antioxidant, somewhat unstable organic acid that is easily oxidized and destroyed when in aqueous solutions, and when dry, by oxygen or alkali and high temperature conditions. In the reduced state, ASA is an essential nutrient, cofactor and antioxidant.

Now, new evidence comes from carbohydrate research, which has focused largely on nonenzymatic glycation as a type of carbonyl stress in animals, first ascribed to glucose,

which implicates ascorbic acid as a contributor in the same glycation-derived post- translational modifications to protein or damage to DNA, lipids and most other biomolecules. Of particularly importance are the oxidation and degradation compounds

of ASA that are implicated in pathological processes, similar to glycation but in a process now referred to as ascorbylation, because they also accumulate during aging and in age-

14 related diseases, including diabetes, Alzheimer disease, other conformational diseases

and in cataracts. Nonenzymatic ascorbylation, like glycation, is also implicated in

normal aging and can form many if not the same advanced glycation endproducts

(AGEs). AGEs, especially the crosslinks, have been hypothesized to be a major

contributing factor in many pathological processes. Lens and collagen have been

suggested to be the major tissues affected by glycation-mediated damage. However, it is

the lens where inherently high levels of ascorbic acid are believed to be the major agent of carbohydrate damage as compared to rodents, which have low ocular levels of vitamin

C. Unlike most other animals, humans cannot synthesize vitamin C, rendering dietary sources an obligate necessity. Regardless of whether or not vitamin C is synthesized de novo, its uptake into tissues must be largely facilitated by active transport through the sodium-dependent vitamin C transporters (SVCT1 and 2) in order to be concentrated against a gradient. The possibility of anionic channels and passive transport has not been entirely ruled out as part of the explanation for the homeostasis of ASA in tissues.

Nevertheless, we have turned our attention to understanding the mechanism of ascorbate transport in ocular tissues and that body of work is described herein.

15 CHAPTER 1

INTRODUCTION: Background and Significance

1.1. Introduction to Mechanisms of Aging Human Lens

The vertebrate ocular systems is a useful model system to examine aging and the

molecular mechanisms by which age-related modifications, such as glycation and

carbonyl stress, or photo-oxidation or oxidative stress, affect long-lived tissues and

proteins. Aging, as a process, occurs uniquely in the lens because of low oxygen tension in nuclear regions, immune privilege and avascular nature. These unique structural

properties largely uncouple the lens from the systemic circulation biochemically and

physically aid in studies as a pseudo-closed system. That is not to say that small

molecules from the general circulation can’t pass into the lens, rather, the lens is unique

enough to impart a more amenable system to study in vivo changes to protein that occur

during aging. Regardless, it is certain that the features of cataract need to be distilled to

the basic functional components necessary to maintain, or facilitate, lens transparency.

How these findings could be used to prevent or delay a condition that will affect all

individuals, if they live long enough, will only become more clear with time.

Nevertheless, cataractogenesis is seen as a multifactorial process because of a few

genetic forms of the disease and clinically we find many and varied forms of the disease

affecting various lens regions. However, there is no data to support the notion that most

human cataracts are multifactorial in origin. What seems clear is that some changes are

16 inextricable from aging, namely, those changes that result from byproducts of oxidation and metabolism. Aggregation, pigmentation, increased fluorophores, gradual yellowing and sodium potassium ATPase damage are age-related changes, which, in part, are thought to be distinct from cataractogenesis, but are nonetheless associated with it. The similarities between lenticular aging and cataract are numerous. However, cataracts generally can be of diverse etiology, whereas aging is inevitable and universal, albeit multifactorial in etiology. Although aging is a risk factor for nuclear and cortical cataracts, cataractogenesis is considered distinct from aging. In the lens, aging is generally associated with the breakdown of the lens microarchitecture (334). Some have suggested cataracts arise from early developmental abnormalities (97), which may lead to later structural or functional changes and alterations in lens growth or differentiation at key stages, thereby predisposing some individuals to form cataracts.

Despite a remarkable resiliency to aging processes and environmental stressors, the lens undergoes numerous stochastic and developmental post-translational modifications to its crystallin proteins, which are the long-lived component proteins of the lens. Similar processes that occur with age can occur as well with cataract, such as the end result of stochastic processes. The age-dependent chemical modification, oxidation and the crosslinks of the lenticular component proteins are among the major pathways for the development of lens opacity. However, the lens changes associated with aging and cataract are considered distinct (338). One of the earliest observable changes in age-related nuclear cataract may involve presbyopia (316), i.e. the loss of accommodation and a massive increase in stiffness in the lens affecting nearly everyone

17 by middle age (207). Truscott, proposed the genesis of nuclear cataract lies with the

onset of a barrier within the lens, which restricts the ability of small molecules, such as

antioxidants, to penetrate into the centre of the lens, thereby predisposing the nucleus to

oxidation and post-translational modification. Oxidation is one hallmark of age-related cataract and is characterized by a loss of protein sulfhydryl groups, including the oxidation of methionine residues. However, the mechanism may lie in a breakdown of the microenvironment, flux of calcium into the lens, along with reactive small low molecular weight compounds, such as ASA and glucose. Moreover, during the course of certain diseases, in particular perturbations in metabolic states, such as during diabetes

(316) where the redox balance of the lens is impaired and reduced glutathione concentrations are depleted (191), some deleterious protein modifications occur at an accelerated rate (34). Unfortunately, with time, these same reactions occur in relatively healthy individuals as well as a direct consequence or side effect of normal metabolism.

Taken together, with exposure to UV-irradiation in the ocular system, the accumulating modifications are part of an inevitable, albeit slow, normal aging process that engenders largely irreversible damage with time.

This introductory section is aimed at discussing our current understanding of select biochemical processes known to occur in the vertebrate lens with time. These processes, along with concomitant damage, are the foundation for the work undertaken in the remainder of the thesis. Many currently recognized biochemical mechanisms of cataractogenesis, its progression and the various types of lens opacities involved have been extensively reviewed. The mechanisms of cataractogenesis are diverse and even

18 include some very unusual sources, such as diarrhea (129, 132). Herein, we focus largely

on one important contributing factor to the process, namely, glycation of lens crystallins

through the Maillard or browning reaction, a process that increases with advancing age.

In order to transition from cataract to the basis of this thesis, we need to first understand the mechanisms through which these modifications occur, particularly the processes of glycation or ascorbylation. It is important to discuss these changes in terms of the lens micorarchitecture and then the forms of protein modification that occur through metabolism. By understanding the mechanisms involved in the formation of deleterious post-translational modifications that are known to occur within the aging lens, it is hoped that effective treatment approaches or interventions can be developed. Elucidating the molecular biochemistry underlying cataractogenesis might aid in finding pharmacological or dietary means to retard, or even reverse, cataract formation. At the same time, these discoveries could have relevance to various other age-related diseases with processes in common.

Effort in this regard is of significance because, for many long-lived mammalian species, especially humans, these modifications lead to cataract formation, which is a major cause of visual disability worldwide (335). With increases in world population and the number of aging individuals, together with increasing dietary and environmental risk factors like type II diabetes, cataracts constitute an increasingly important area of interest for the World Health Organization and world governments alike. According to data obtained in 1984, even a delay of 10 years would have reduced, by roughly 45%, the total number of cataract blind by approximately 10 million individuals (165). In developed

19 countries, where phacoemulsification, extraction and replacement of cataractous lenses

are not readily available, cataracts are much less of a problem. However, in countries

where these interventions are not available, cataracts are an important cause for concern

and disability in the elderly. Age-related cataractous changes affect vision by interfering

with retinal imaging by causing scattering and absorption of light, as well as through autofluorescence (AF). If one could correct for the loss of light in the lens, then vision obviously would be improved. One goal of our lab is to identify and eliminate the cause of these light scattering modifications and risk factors through medical or dietary intervention at the appropriate time during the course of the disease.

1.1.1. Development of Age-related Opacifications

From an evolutionary standpoint, the lens is a highly specialized organ that is found ubiquitously in both vertebrates and invertebrates. The adult human lens is normally a pale-yellow, ovoid shaped highly structured organ of up to 10mm in diameter and 4-5 nm thick, weighing around 0.2-0.3 gm that becomes increasingly pigmented with age. The lens is composed of epithelial and fiber cells and is encapsulated in a collagenous capsule. Aging leads to ultrastructural changes that are evident in the lens

and capsule, which include the formation of dense elements that can delaminate from the polar-regions (294). The lens is suspended in a watery aqueous humor and receives

trophic support via the Ciliary Body, which links the lens to the systemic circulatory

system. With age, the lens itself becomes increasingly permeable to water (103). As the

lens continues to develop over one’s lifetime, it grows in concentric rings through an

20 annular-like process, akin to the growth rings of an onion. One difference is the

asymmetric distribution of the lens-forming epithelial cells that are largely on the anterior

surface of the lens. By age 70, the time at which cataracts are most evident, the growth

rate of the lens gradually diminishes, although increases in lens weight have been noted

(130). The development of the lens results in unique features, making it amenable to temporal, spatial and regional classification.

The lens can be divided grossly into three regions, a germinative region of cell division, a region of elongation and a region of differentiation into mature lens fiber cells.

Further, the lens can be divided from the core to periphery into nuclear, perinuclear and cortical regions, of which the cortical region is younger (Figure 1.1). The lens fiber cells and epithelial cells are surrounded by a collagenous capsule and extracellular matrix.

This capsule layer, which contains glycoprotein, laminin and fibronectin, is secreted by the epithelium and is devoid of living cells (245, 257). Also, the lens contains some lipids, largely sphingomyelin, cholesterol and glycolipids, which are concentrated in the plasma membranes of the cortical and nuclear layers (272) and in the lens epithelium.

Epithelial Cells Anterior

Nucleus

Cortex Cortex

Posterior Fiber Cells

Figure 1.1. Lens Architecture and fiber cell distribution. Fiber cells are composed mostly of crystallins, which, from their temporal origin to terminal differentiation, are not turned over but do change in the relative abundance of crystallin protein isoforms (267).

As the lens matures, it undergoes a process called fiber-cell differentiation, giving rise to the so-called fiber cells that are derived from the epithelial cells (45, 360). The fiber cell deposition process is an additive one, wherein old fiber cells are not turned over but are continuously added to the ones already present at birth. This process imparts unique properties to the lens, making it particularly amenable to aging studies of long- lived proteins. In the mammalian lens, fiber cell differentiation is characterized by distinct molecular and morphological changes including exit from the cell cycle, cell elongation, loss of select organelles, e.g., mitochondria and nuclei, the accumulation of fiber-cell-specific proteins, beta- and gamma-crystallins, and intermediate filaments, such as filensin and CP49 (45). As the lens ages it undergoes nuclear compression and becomes harder or sclerotic, and its cortical fiber cells begin to approximate those of the

22 older cortical regions and show irregularities such as disruptions in cell contact or adhesive properties, the formation of inclusions and vacuoles and fiber cell destruction

(160). Among the forms of age-related cataracts, nuclear cataract accounts for ~60% of the age-related cataracts, ~30% for cortical cataract and the remaining 10% are largely posterior subcapsular cataracts (159).

The Unique Structure the Lens

The lens has many interesting properties, including a remarkable structure and development. Especially notable is how the lens is able to remain translucent for many years, yet its long-lived proteins are not known to turn over (361), as are the proteins of other long-lived tissues, such as cartilage or skin, albeit at a reduced rate in these tissues as well. The unique composition and longevity of the lens is commonly assumed to be due to long-term retention of its native structure and intrinsic function of some component proteins.

The lens is extremely protein-rich, about 35-50% protein, measured as wet mass

(249). In fact, it contains the highest protein concentration of any tissue. The lens can be separated into water-soluble (WS) and water insoluble (WI) fractions. It undergoes normal age-related syneresis, which is the release of bound water into the bulk of the tissue, resulting thus in an increase in wet weight with advancing age and with cellular proliferation. Among the water-soluble proteins, the crystallins account for 80-90% of transparent lens protein (248). They are not turned over but rather are packed in a

23 particular arrangement that conveys their refractive and translucent properties.

Four major groups of crystallins (alpha, beta, gamma, delta) and some minor crystallins have been identified. In the mammalian lens, not including enzyme crystallins, they are (αA, and αB; βB1, βB2, βB3, βA3/A1, βA2, and βA4;γA, γB, γC,

γD, γE, γF, and γS). The crystallin proteins of the lens are largely structural proteins, rich

in beta sheet structure and are highly packed and organized. This high-density packing of

crystallins minimizes light scattering and results in more internal reflection of light

through the lens and the generation of an enhanced clear image on the retina (80).

One key factor to a long maintenance of transparency can be attributed to inherent

thermodynamic stability. Through a process called gene sharing, many diverse forms of

crystallins are now known, which often differ among species and are found outside the

lens where their role is a non-refractive one and include activities such as metabolic

enzyme cofactors or as stress proteins. For example, the small heat shock protein alpha

crystallins (A and B) have chaperone-like activity. Because of their homology to

chaperone proteins, new function has been attributed to these proteins (144, 147). This

activity will be discussed in the defense to damage section below as it is ascribed to be

but one protective mechanism against cataractogenesis.

Since the lens is arguably the largest avascular organ, it is conceivable that it

evolved to create a complex alternative to systemic circulatory system, dependent on the

flux of ions and solutes to serve the role of a circulatory system in the eye and lens.

24 Evidence for this rudimentary circulatory system that couples the movement of ions and

numerous transporter proteins has led others to conclude the evolution of an internal

micro-circulatory system consisting of ions coupled to fluid movement as well (201).

Such a system would involve a tight regulatory process. Indeed, the lack of a blood

supply is a strong argument for the existence of such a system, where the flux of ascorbate, glucose and free amino acids, which is tightly modulated by calcium and

coupled with sodium/potassium movement. In cases where this process fails there is an association with impaired cellular homeostasis and cataractogenesis. In that regard, the regulation of amino acid transport appears to be compromised in cataractous lenses.

Previous studies (33, 315) demonstrated that while normal lenses displayed tight

regulation of the levels of lenticular free amino acids, the majority of cataract lenses did

not, but rather, contained excess proteogenic amino acids. Conversely, these results

could be interpreted as an increase in degradation of proteins in these lenses (338).

Nevertheless, good models that take into account protein modification, loss of transport

and ion flux as well as impaired redox homeostasis are needed when considering treating

this disease.

1.1.3. Models of Cataractogenesis

There are many currently postulated biochemical causes of cataractogenesis, the

progression of cataract, and various lens opacities. However, obtaining suitable human

lenses is an increasing problem due to the rapidly expanding use of phacoemulsification

and extraction of cataracts in the United States and other developing countries.

25 Establishing models of cataractogenesis has been difficult and numerous models have been explored from those associated with the breakdown of the lens micorarchitecture or cellular membranes to chemical modifications and cross-linking of proteins. Differences between animals and humans are numerous, and lenses vary greatly in their total lenticular protein (310) and crystallin isoform content (7).

Some obstacles and benefits to murine models include the intrinsic dichotomy that exists between humans and mice. Mice and men share similarly low levels of aldose reductase enzyme (AR), however, mice are short-lived. However, rat, rabbit and mouse lenses contain no UV filters. Further, for models of cataractogenesis via ascorbylation and glycation due to breakdown products of ascorbic acid metabolism, the low lenticular levels of ASA make murine models unacceptable. There is evidence of significant biochemical changes associated with sugar cataract formation despite very low levels of aldose reductase (136).

The best rodent model to study effect of photo-oxidation on cataractogenesis, until recently was the guinea pig. This species can be made scorbutic by ASA depletion in the diet. Its lenses contain UV filters such as high levels of NADPH that can absorb

UV-A in the same range as the kynurenins. These filters approximate the human pathway but are not identical. However, their proteins do not appear to oxidize with age and in contrast to humans, they show no significant loss (373), while other rodent lenses display almost complete oxidation of protein cysteine residues perhaps related to the relatively low levels of glutathione and complement of antioxidant enzymes involved in

26 glutathione metabolism (261). Interestingly, this group showed that monkey and human lenses contained approximately one hundred times the levels of glutathione reductase

higher than other species.

Together with animal models, cell lines and whole lens explant culture systems

have been used to understand and recapitulate human cataractogenesis at the biochemical

level with mixed success. Immortalized human and mouse lens epithelial cell lines serve

to compensate for species differences in animal work, but these cells do not recapitulate

completely primary cells in all aspects (360). Nevertheless, new molecular models are

being developed that have impaired lenticular glutathione (GSH) synthesis (67) or

overexpression of the human sodium-dependent vitamin C transporter (SVCT2). They

are now better able to recapitulate human cataracts at the molecular level or result in

yellow pigmentation, respectively (99).

Some models have focused largely on diabetic or glycation-mediated cataract,

since diabetes is associated with a marked increase in cataract formation. The higher

incidence of cataract among diabetics has been clearly demonstrated by epidemiologic

exploration and study such as the Framingham Eye Study (94). Several theories have

arisen to explain the etiology of sugar cataracts and perhaps uncouple glycation from

normal metabolism, if possible. Cataract formation in diabetic lenses has been attributed

to elevated levels of reactive aldehydes, which lead to an osmotic imbalance (156)

through the polyol-osmotic pathway. Accordingly a pressure-generated influx of water is

thought to cause the lens to swell and form a fluid-filled pocket, where these

27 accumulations cause light-scattering. Models that explore perturbations in crucial cellular membranes (113) and membrane proteins, such as membrane intrinsic protein

(MIP) and the aquaporin water channels (1, 301), are implicated in cataractogenesis as

well. Epidemiologic studies looking at increased risk from sunlight exposure do show

increased risk for cortical and subcapsular, but not nuclear opacities (70).

The hyperosmotic theory implicates the reactive aldehyde detoxification system

aldose reductase as the primary factor responsible for the pathobiology seen in lenticular

opacification due to hyperglycemia or hypergalactosemia leading to osmotic imbalance

from sorbitol and galacitol through the NADPH-dependent reduction of glucose and galactose (156). In this regard, diabetic patients have been found to show differences in

lens hydration properties as compared to control age-matched normal lenses, with higher

total water content found in non-cataractous diabetic lenses (41). Sorbitol, which is one

osmotic model of cataract, will not be addressed further.

Models involving oxidative stress stem from evidence for a mode of causality that

comes from early studies of oxidative stress in cataractogenesis, wherein cataracts could

be produced in animals exposed to hyperbaric oxygen (293). In addition to oxidative

stress, carbonyl stress is a major target implicated in cataractogenesis and the two

stressors can work synergistically. Further, human lens crystallins undergo a number of

posttranslational modifications by reducing sugars and oxoaldehydes with age that lead to

protein pigmentation and crosslinking (216). These modifications are thought to

predispose lens crystallins toward formation of high molecular weight (HMW)

28 aggregates that scatter light and will be addressed later.

The sugar cataract model is not sufficient to account for all forms of cataract

regardless of whether or not they occur with advancing age. One problem is determining

the sources of the carbonyl stress involved. Evidence suggests some of these

modifications may originate from ascorbic acid oxidation and degradation products (36)

(226) (65, 236) especially since high ascorbic acid (ASA) concentrations are found in the

human lens, which is about 1-3mM. Ascorbic acid in reduced form is not a glycating

agent, however it can become highly reactive forming carbonyl compounds and reactive

agents in its oxidized and degraded forms, i.e., dehydroascorbic acid (DHA) and 2,3-

diketogulonic acid (2,3-DKG), which is the spontaneous delactonization product of DHA

(226).

Other reactive compounds, such as ascorbic acid degradation compounds can be

just as significant, namely, reactive aldehydes or alpha-dicarbonyl compounds and the

oxidation or degradation compounds of DHA. In order to investigate the biochemical

nature of the ascorbic acid degradation products and how they might be responsible for lens crystallin pigmentation in diabetes and aging, a model was necessary as well as the means to distinguish among the contributions to the process from glucose, glucose- derived oxoaldehydes, and ascorbic acid (Figure 1.2.). In this regard, we initiated metabolomic studies with 6-fluoro-6-deoxy-ascorbic acid (F-ASA) as a 19F Nuclear

Magnetic Resonance (NMR) spectroscopic tracer for the in vivo assessment of ascorbic

acid degradation products (199, 283). These studies revealed that glycemic stress

29 suppresses the uptake of F-ASA into the human lens epithelial cell line HLE B-3 and catalyzes the degradation of F-ASA (230, 283). We also showed that glutathione depletion strongly enhances F-DHA and F-DKG formation in HLE-B3 cells. Overall, similar observations were made in lenses from diabetic rats treated with F-ASA. These studies also revealed that both F-ASA and F-DHA were specifically transported into the human lens via a sodium-dependent uptake mechanism, most likely via the SVCT2 transporter.

Figure 1.2. 19F NMR Ascorbic acid, oxidation and degradation product tracers.

30 During the course of testing the specificity of the above findings in other rodent

cell lines with the ultimate purpose of overexpressing the human transporter in rodents,

we developed a transgenic animal model capable of taking up millimolar concentrations

of ASA into the lens. In doing so, we discovered that the uptake of ASA in rodent lenses

(0-100 micromolar) was markedly suppressed when compared to human lenses (1-3

millimolar). This finding held true for whole lens explants as well (232). The data above

strongly suggested a need for a humanized mouse model of cataractogenesis, one that

reflects physiologically relevant levels to humans or, even, increased levels of lenticular

ASA coupled with increased oxidative or photo-oxidative stress. Indeed, we now have developed such a mouse model of lenticular aging, which is in the testing stage for

compounds that inhibit these non-enzymatic processes (99).

1.2. Sources of damage to lens crystallins during aging

Since the aging humans lens has little to no protein turnover, it is susceptible to

accumulation of post-translational modifications and damage to its proteins and epithelial

cells as well (178). Lenticular protein damage can be largely attributed to post-

translational modifications. The natural order and chronological history of post-

translational modifications can be summarized in the following manner. First, there is a set of modifications that are developmentally regulated, as evidenced by the dramatic changes that are observed at an early age. These include phosphorylation, deamidation and enzyme mediated protein truncation (292, 324). While the phosphorylation status appears to be little changed once fiber-like cells are differentiated, deamidation is an

31 ongoing process that participates in lens aging (324) and is associated with enhanced risk

of crystallin aggregation (124). In contrast, senescence-related changes typically relate to

stochastic forms of damage, i.e. post-translational chemical modification of side-chain

residues resulting from oxidation, cleavage of the peptide backbone through reactive

oxygen species and covalent modifications of amino acid residues by low molecular

weight compounds.

Long-lived proteins undergo age-related postsynthetic modifications (PTM) that

destabilize them by altering their conformation, charge, and helicity, thereby enhancing their resistance toward proteolysis and propensity to aggregate (334). In that regard, the

lens proteins display numerous post-translational modifications with age that can affect

structure and function, including phosphorylation (175), acetylation (184, 260)

glutathionylation (72) ascorbylation (172), glycation (112, 321), general mixed disulfide

formation as well as truncation of C-terminus (325) or N-terminus (170) of some

crystallin subunits, and covalent binding of kynurenins to specific hydrophobic sites on

the crystallins (329). Acetylated alpha-A-crystallin increased during eye lens aging along

with phosphorylation of ser 122 and 148 (291). Further, racemization of aspartic acid,

deamidation of asparagines and increases in ornithine (ORN) content have been found to occur in the aged lens (296). Although all of these processes occur throughout life, they are by themselves unlikely to be sufficiently rapid to trigger cataract formation, per se, but are thought to predispose lens crystallins toward aggregation and formation of light

scattering high molecular aggregates. In this regard, modifications such as oxidation,

deamidation, or cleavage also result in incorrect protein–protein interactions and lead to

32 aggregation and precipitation.

1.2.1. Oxidative damage

Oxidative modification or oxidative stress is an important process that occurs in vivo during aging. It is considered one of the main causes of molecular damage to cellular and tissue structures and is considered key in the mechanism of senescence-related and stochastic forms of damage (9). It is the inevitable side effect of oxygen metabolism, which is the basis of the free radical theory of aging, and is widely believed to be the root cause of age-related deterioration of the lens and other tissues in the body. In this regard, early studies demonstrated that cataracts could be produced in animals receiving hyperbaric oxygen (HBO) exposure, which was further supported by similar in vitro studies (293). The role of oxygen in the formation of high-molecular-weight (HMW) lens protein aggregates during the development of human nuclear cataract was explored in guinea pigs exposed to HBO. The lens crystallin aggregate formation was investigated with dynamic light-scattering (DLS) and by HPLC analysis of water-insoluble proteins.

The results indicate that molecular oxygen in vivo can induce the cross-linking of guinea pig lens nuclear crystallins into large disulfide-bonded aggregates capable of scattering light. A similar process may be involved in the formation of human nuclear cataract

(306).

Alterations in the redox state of transition metals and the generation of oxidative stress through transition-metal-catalyzed oxidation reactions are important in the genesis

33 of Fenton-type reactions and the subsequent formation of free radical species. Simple exposure to high oxygen tension coupled with free metals, such as iron and copper can cause cellular oxidative stress. If reducing equivalents are in ample supply to combat this stress oxidation is held in check, largely accomplished through the nicotinic-adenine- dinucleotide monophosphate (NADPH)-dependent enzymatic oxidases and reductases, particularly thioredoxin/thioredoxin reductase system (143). Unfortunately, there are instances where endogenous defenses are taxed and overwhelmed. Such is the case for diabetes and aging, both of which have increased cataractogenesis. With age, and under conditions of oxidative stress from such sources as singlet oxygen, hydroxyl radical, hydrogen peroxide or other oxidants, the once significant pool of glutathione (GSH) can be diminished (118, 217, 256). Further, as the lens ages the de novo synthesis and the recycling system for GSH become less efficient (195, 263) which causes GSH concentrations to decline in various species (128, 195).

Purely oxidative modifications found in human lens crystallins include oxidized amino acids in the following order: Dopamine (DOPA) > o- and m-tyrosine > 3- hydroxyvaline, 5-hydroxyleucine > dityrosine (108)) methionine sulphoxide

(MetSOX)(375). Brunescent cataracts, in which the normally transparent lens becomes deeply brown and opaque, contain high levels of these hydroxylated amino acids. Most interestingly, these modifications can be duplicated by metal catalyzed oxidation (Fenton or Haber-Weiss type chemistry), but not by photo-oxidation (108). Indeed, we demonstrated that another source of oxidative stress damage includes diabetes-associated oxidative stress, which includes carboxymethyl-lysine (CML), a marker of glycoxidative

34 stress and a major modification in the aging human lens and combines oxidation

mechanisms with oxidative stress. Here, CML can, in fact, bind redox active Cu2+ and

lead to protein depolymerization in presence of hydrogen peroxide (H2O2)(290). This type of damage is found in aged and diabetic cataractous lenses.

The major proposed source of glycoxidative stress is glycation coupled with mitochondrial superoxide production, which is responsible for sorbitol pathway overactivation, and downstream activation of protein kinase C (PKC), eventually leading to changes in signal transduction and metabolism (24). PKC activation can have further deleterious downstream effects on ascorbic acid metabolism by inhibiting the Sodium- dependent Vitamin C transporter (182). However, the concept of sorbitol pathway overactivation is not fully supported by in vivo studies aimed at inhibition of the sorbitol pathway in peripheral nerve, retina, and lens by antioxidants and superoxide scavengers

(233). Nevertheless, in addition to the sorbitol pathway, diabetic hyperglycemia causes increased formation of glucose-derived AGEs. Moreover, these glycation-related modifications in human lens proteins spontaneously generate reactive oxygen species

(ROS)(117), when exposed to UV-A, and some can bind redox-active divalent metal cations (289). Here, superoxide anion formation was measured in both cataractous lens protein and calf lens proteins incubated in vitro with ASA in a process referred to as ascorbylation. The activity was found to increase in brunescent cataracts, in in vitro ascorbylated calf lenses, and was completely dependent on the presence of oxygen (187).

Superoxide anion formation has a role in lenticular aging and glycoxidative stress

35 and can be spontaneously generated in lenses during aging and cataract formation. When

the source of superoxide anion was explored in lens protein, preparations containing a

solution of purified N(epsilon)-fructosyl-lysine produced no detectable superoxide.

However, superoxide formation increased in brunescent lenses and with fructosyl-lysine- modified lens proteins. Copper-stimulated superoxide formation was found in glycated bovine serum albumin, but no stimulation was found with cataractous proteins (187). To

balance the involvement of superoxide against the detoxifying enzyme superoxide

dismutase (SOD) and reducing agent GSH, Linetsky and Ortwerth assayed for specific

compounds and found that catechol, hydroquinone, 3-OH kynurenine, and 3-OH anthranylic acid exhibited the greatest activity for superoxide generation, but had a very short half-life. Interestingly, the water-insoluble fractions of aged human lenses and

brunescent cataracts are capable of generating >100 nmol mg protein(-1) and >170 nmol

mg protein(-1) of superoxide anion respectively, which is likely due to the presence of

AGEs in human lens proteins, or redox active metal complexes with these AGEs (187).

Spontaneous generation of superoxide anion in vivo is believed to play a large role in the oxidation of sulphur-containing or other amino acids in the cataractogenesis of

human lenses. Oxidation of amino acids, such as threonine, and of ascorbic acid to DHA,

can lead to methylglyoxal formation, which will be addressed below, and contribute to

protein damage. Other modifications of an oxidative nature include methionine

sulfoxide, disulfide bonds and β-carbolines (83, 114, 243)whereby the latter are likely to

be photo-oxidation products as well (359).

36 In addition to reactive oxygen species, reactive nitrogen species (RNS) are also

implicated in deleterious lens protein modifications. Peroxynitrite reactions are known to produce nitrotyrosine, nitrotryptophan, dityrosine, non-disulfide covalent cross-linked aggregates, and protein degradation. The hydroxyl radicals produced by peroxynitrite can cause increased chain degradation (26). However, not all damage is equivalent when it comes to the structural proteins of the lens. For instance, alpha crystallin retains its chaperone activity despite modification by reaction with peroxynitrite (333), which is not

unique to nitrating species, as methylglyoxal (MGO)-mediated modifications to lens

alpha crystallins appear to augment chaperone function as well (225).

1.2.2. Photo-oxidative damage

The daily exposure to radiation from the sun is another source of damage to lens

proteins associated with structural changes and a gradual loss of transparency. Photo-

oxidation can inflict extensive damage to crystallins, especially when constant exposure

to high levels of irradiation, both UV and ionizing, provides an ideal environment for the

generation of reactive oxygen species in ocular tissues. The human lens has two primary

functions: to focus light on the retina and to prevent optical radiation between 295 and

400 nm from reaching the retina. It has been shown that high doses of UV damage DNA

and protein and thus one should not understate the importance of light-filtering capacity

because the retina is almost an order of magnitude more sensitive to damage by UV

radiation than by visible light (252). Ascorbic acid and the tryptophan metabolites

kynurenins, which absorb maximally at 260 nm, protect ocular tissues from wavelengths

37 of UV that would cross-link proteins and DNA.

Extensive damage can be inflicted upon crystallin proteins by photo-oxidation

from sunlight. The wavelengths of sunlight that could impact the eye in a deleterious

fashion are predominantly UV light wavelengths, which are 400 nm - 320 nm for UV-A

or long wave, (black light 320 - 400 nm), 315 nm - 280 nm for UV-B or medium wave

and below 280 nm for UV-C, short wave. Normally light of shorter wavelengths is

largely absorbed by the ozone layer. The lens is normally transparent to photons of lower energy, which pass through unobstructed to become incident on the retina (see table 1.1).

With age and molecular deterioration lower energy photons, in the UV spectral range 3.4-

3.1eV (365-400nm), are gradually absorbed (382). Using published data, this group calculated the flux of solar UV-A and UV-B that reach the cornea and lens.

Wavelength % Absorption % Absorption % Absorption % Absorption nm by Cornea by the by the lens by the Aqueous Vitreous < 280 nm 100 - - -

300 nm 92 6 2 -

320 nm 45 16 36 1

340 nm 37 14 84 1

360 nm 34 12 52 2

Table 1.1. Wavelengths of UV light incidenting on structures of the eye, protecting the retina from UV damage (317).

38 In Table 1.1., above, is the UV exposure to the eye, identifying the wavelengths of light incidenting through the vitreous and transmission absorption with each eye structure, adapted from Boettner and Volter and Slineg and Volbarscht (317).

Apparently, UV-B (290–320 nm) and UV-A (320–400 nm) can reach the cornea.

However, UV-A and some UV-B can reach the lens. Both UV-A and UV-B are known to produce reactive oxygen species in lens proteins (10, 186, 189, 190), and in lens (27,

309). With time, even lower energy photons become unable to penetrate and the lens absorption spectra reaches into the high-energy end and shorter wavelength range of the visible spectrum (28). Absorption of radiation at the blue end of the spectrum may result in the increasing yellowish color of the lens, which deepens, darkens and may become brunescent, i.e. the darkest classification of lens pigmentation (250).

Taken together, components of the eye appear to be easily damaged by low doses of UV irradiation. This includes sub-solar UV radiation that was shown to cause irreversible damage to rabbit corneal and lens epithelial cells, i.e. UV-A at 6.3 J/cm2

(273). This group also has shown that higher doses of UV also damage antioxidant enzymes and cell membranes. Irradiation of corneal and lens epithelial cells using a relatively low dose of UV decreased the activity of catalase to 30–50% of its original value, while the activities of glutathione peroxidase and superoxide dismutase did not decrease within experimental error (273).

When cultured rabbit lenses and epithelial cells were exposed to UV-A

39 irradiation, equivalent to several hours under the sun, significant damage and cell death occurred in corneal and lens epithelial cells with a concomitant increase in lipid peroxidation accompanied by a decrease in cell viability due to UV-A irradiation. Lipid peroxidation was assayed using the thiobarbituric acid reaction. Both UV-B and UV-A induced cell death in corneal epithelial cells. In addition, it was discovered that catalase in epithelial cells is much more susceptible to UV damage than superoxide dismutase and glutathione peroxidase.

When a human lens epithelial cell line was exposed to UV-B at low doses (2.5 mJ/cm2), cell death appeared to be primarily apoptotic and this damage was determined using the terminal deoxynucleotidyl transferase mediated dUTP-X nick end labeling

(TUNEL) assay followed by the single cell gel electrophoresis (comet) assay. At 10 mJ/cm2, cell death showed both apoptotic and necrotic characteristics (304). These researchers have shown that higher dose of UV also damage antioxidant enzymes and corneal and lens epithelial membranes, however, using a relatively low dose of UV decreased the catalase activity 30–50% without affecting the activities of glutathione peroxidase and superoxide dismutase.

Together with the formation of protein-attached fluorescent material, of both blue

(440 nm) and green (520 nm) emission spectra, results in increased filtering capacity of the lens due to a generalized yellowing of lens proteins, the creation of higher and lower molecular weight polypeptides, and an increase in the net negative charge of the crystallins. Often these changes can be explained by photo-oxidation. The numerous

40 chemical and photochemical processes that account for these changes include the

photochemical modification of tryptophan, the oxidation of lipids and the chemical

attachment of sugars or ascorbic acid through the Maillard reaction. Yu et al. scanned human lenses (cortical to nucleus) and found a decrease in fluorescence caused by 3-

hydroxykynurenine (3-HKG) with the formation of a new green fluorescent species in the

nucleus. They suggested that there is an age-related conversion of 3-HKG to the new

fluorophore (373).

Fluorescence in the eye can be either intrinsic (tryptophan-related) or extrinsic

(Chromophores, AGE fluorophores etc). These extrinsic fluorophores indicate changes

in the pathological state of the lens as a function of age. They are useful indicators of

redox potential, protein degradation lipofuscin accumulation and other physiological

parameters such as cross-linking (305). Fluorescence in the lens is correlated with UV-

mediated damage, particularly lipid peroxidation (155).

The loss of tryptophan fluorescence is believed to be largely due to its photo-

oxidation products that become directly attached to lens crystallins, namely, the O-

glucoside of 3-hydroxykynurenine to alpha-crystallin (86), hydroxytryptophan, N-

formylkynurenine, and kynurenine (102). These modifications may substantially

contribute to lens crystallin aging (353, 354). Unfortunately, the age-related

modifications that do occur to lens proteins and the surrounding epithelium can occur as a

direct consequence of covalent binding of these UV/visible filters to lens crystallins. β-

carbolines and another potential product from photo-oxidation, namely 4-

41 hydroxyquinoline, have been found in brunescent cataracts (197). UV-A filters decrease

with increasing age and increasing glycation. This allows some UV-A to be absorbed by the lens and lenticular AGEs, which have photosynthesizing activity (19, 21).

A synergistic effect of different carbonyl-containing lens metabolites, which form

AGEs, exists in conjunction with photo-oxidation. Zigler, Ortwerth and colleagues

demonstrated that extracts both from native or Maillard reaction-modified crystallins are

active as photosensitizers (238, 380) and this activity is implicated in lens photodamage

(21). In experiments designed to explore whether glycated proteins produce ROS, when

lens explants or cells were incubated with MGO, glyoxal (GO), ASA or fructose and

exposed to 200J/cm2 UV-A, the finding was glycated model proteins produced 2-3 fold

more singlet oxygen species compared to the unmodified protein and superoxide radical

formation was 30-80% higher than native proteins. The same group found that

ascorbylated proteins demonstrated the highest photosensitizing activity compared to all

other glycating agents studied and that lens proteins had a protective effect against UV-

A-induced cytotoxicity but this effect decreased with increasing photosensitizer activity.

These glycation-related photosensitizers could explain the rapid pathogenic changes seen

in human lens aging and under diabetic conditions.

It is important to note that the oxidation of ascorbic acid, mediated by UV-A

ambient sunlight on human lens chromophores, can feed forward the process and further

oxidize ASA and other substrates in the absence of oxygen and is capable of glycating

lens proteins (235). This glycation process was actually shown to involve incorporation

42 of uniformly labeled (U-14C) ascorbate into lens proteins in vitro. These data suggest that

UV-A light-induced oxidation of ASA, even in the absence of oxygen, can activate the photosensitizers that exist in the aged human lens, and that these oxidation products are the same as those formed in the presence of oxygen, apparently through as yet to be determined mechanism involving the Maillard reaction. The results described above also occur under the low oxygen tension known to exist in the human lens nucleus (235).

UV-A light can photo-bleach the yellow chromophores and yellow AGEs of aged human lens, namely the UV-A-absorbing AGEs OP-lysine and Argpyimidine (18). However, the physiologic significance must be weighed against the presence and activity of endogenous protective mechanisms. The ascorbate sensitizer, when irradiated together with tryptophan (Trp) at 365nm, resulted in photosensitizing activity of the AGEs that was optimal at 5% oxygen pressure, which is lenticular oxygen pressure. Further, the Trp oxidation rate increased with increasing photosensitized-AGEs content of the solution, when compared to lenticular riboflavin. When compared to Trp, ASA is more easily photodegraded and can protect Trp from oxidation up to 1mM. This led the investigators to conclude that in the aging lens, ascorbate has a significant UV-A protecting activity, but doesn’t impede some Trp residues from being photodegraded proportionally to the

AGE concentration of the lens (77).

1.2.3. Carbonyl damage

Several forms of carbonyl damage have been found in the lens. The sources of the stressors, propagators and mediators of this damage are not unequivocal. In other words,

43 the endproducts of the reaction mechanisms can come from multiple sources. For example, the oxoaldehydes that are implicated in carbonyl damage can come from glucose as well as DHA or methylglyoxal. In this regard, one important process and contributor to lens damage is carbonyl stress, namely the glycation of lens crystallins through the Maillard or “browning” reaction, which increases with advancing age. This reaction can begin with various reactive compounds or stressors, such as those arising from reducing sugars, lipids and lipid peroxidation products, intermediates of the glycolysis pathway or cytokine stress from inflammatory mediators. They form irreversible endproducts or AGEs. The specific alterations in lens proteins caused by glycation from carbonyl metabolites, such as fructose, glucose, galactose, MGO, GLX and ASA have been explored extensively in cataractous lenses (180). It is known damaging carbonyls are generated from glycolytic intermediates, i.e., reactive aldehydes such as glucose, fructose (208), or fructose 3-phosphate (168), other sugar phosphates

(322), and ribose (93). The damage can cause decreases in tryptophan related fluorescence and in the level of reduced protein sulfhydryl groups, which indicate protein conformational changes after reaction with these carbonyl compounds.

Maillard reactions by alpha-oxoaldehydes, such as glyoxal and methylglyoxal, lipids and ascorbic acid and its oxidation and degradation compounds (19), lead to strongly enhanced protein carbonyl content. Argirova et al., made a comparison between modifications of lens proteins resulting from glycation with methylglyoxal, glyoxal, ascorbic acid, and fructose and found the alpha-oxoaldehydes are amongst the most reactive cross-linking and glycating agents known to lead to protein aggregation (20).

44 Protein carbonylation and glycation have been found in the lens epithelial cell fractions

of lenses from individuals with mature cataract (31). The highest levels were found in

senile cataracts from diabetic patients compared to non-diabetic senile cataract patients.

Balog’s group also reported extremely high protein carbonylation in mitochondrial

epithelial cells of heavy cigarette smokers (31). These results suggest that the glycation

and carbonylation of the human lens proteins play a role in cataractogenesis.

Sources of oxoaldehyde-mediated oxidation damage in lens proteins include the

oxidation of amino acids, such as threonine, and ascorbic acid to DHA. These can lead to methylglyoxal and glyoxal (GO) formation, which are precursors of AGEs in vivo. GO-

derived AGEs were found in human lens proteins. When bovine aortic cells were

cultured for seven days with 30 mM glucose, intracellular GO-AGEs did not accumulate

(280). According to these authors, the extracellular accumulation of AGEs from GO

underscores the importance of this mechanism in tissues. However, in the lens, GO-

AGEs are likely to be formed intracellularly. While this may appear as a contradiction, it

need not be. Accumulation of GO-derived AGEs intracellularly in dividing cells may not

represent physiologically relevant conditions.

Despite the similarity in chemical structures of alpha-oxoaldehydes and ascorbic

acid degradation products, incubation results obtained show that alterations in lens

proteins do not follow the specific reactivity of studied carbonyl compounds. For

example, significant loss in lactate dehydrogenase activity results from incubation with

methylglyoxal, followed by glyoxal and then ascorbic acid (19). When the lens

45 crystallins become modified they often exhibit increased autofluorescence. The autofluorescence of the lens changes with aging and in cataractous lenses. A blue-green

AF range 495nm-520nm AF is highly correlated with age. Lens transmission for blue- green light remains almost unchanged up to the 6th decade and then decreases rapidly with increasing inter-individual variation (305). Concentrations in human lens and serum proteins were measured and compared to pentosidine, a fluorescent AGE derived from pentose sugars, and was found to be two to three fold higher in diabetic serum proteins than in non-diabetic controls. Argpyrimidine concentrations were found to be seven fold higher in brunescent cataractous lenses than in aged non-cataractous lenses and even less in control non-cataractous lenses and the relative amount of Argpyrimidine was higher than pentosidine.

Other modifications include carbamylation, which resulted in high-molecular- weight crystallin aggregate formation from non-enzymatic modification of lens crystallins (40, 176) (374) and glutathione adducts (308), which are implicated in cataractogenesis. Carbamylation was proposed to result from high urea concentrations that may lead to high ammonium cyanate levels in the blood. For this to happen, it is suggested that cyanate must pass the blood-ocular or aqueous barrier and pass the ciliary epithelium to cause carbamylation of lens proteins. Since carbamylation of amino acids forms quantifiable stable adducts, they were explored in the lens and the data did not support this theory (308).

Glycation, as measured by glucitol-lysine, the reduced aldehyde form of

46 fructosyl-lysine, was significantly increased in lens from rats with streptozotocin-induced

diabetes (3.92 +/- 0.59 nmol/mg protein, n = 5), as compared to those with

streptozotocin-induced diabetes treated with insulin (2.94 +/- 0.36 nmol/mg protein, n =

4) and normal rats (1.23 +/- 0.22 nmol/mg protein, n = 5) (372). In relation to the lens, the hemoglobin A1c (HbA1c) level in plasma correlated with glycation levels in lens protein and paralleled the severity of diabetes in rats.

1.2.4. Crosslinking

Protein-protein cross-links are known to play an important role in determining the functional properties of food proteins, however, the same crosslinks have been found in tissues and occur in an age-dependent and disease-related manner. In that regard, several lines of evidence implicate cross-links along with AGEs in human cataractogenesis. We and other groups have identified age-related crosslinks in the lens. They include pentosidine, a lysine-arginine cross-link (64), pyrrolic crosslinks, and vesperlysine A

(LM1) among many other modifications identified in the lens. However, these are but only few relevant compounds so far been detected in vivo.

Recent work from Srivastava's laboratory has brought evidence for age-related accumulation of multimeric protein crosslinks in the human lens (313), the structures of which are currently unknown. However, among the most abundant physiologic crosslinks identified to date are the recently discovered crosslink glucosepane (42) , and malondialdehyde (MDA) crosslinks between lysine and arginine. Other lysine-arginine

47 protein crosslinks of the “odic” series, namely MODIC, GODIC, DODIC, derived from

MGO, GO (119) and the lysine (LYS) lysine imidazolium crosslinks methyl-glyoxal-

lysine-dimer (MOLD) and glyoxal-lysine-dimer (GOLD) are also found in the lens.

These MGO and GO-derived lysine dimers were present at lower concentrations

compared to MODIC and GODIC (234). The lens concentrations of GOLD and MOLD

correlated significantly with one another and also increased with lens age. GOLD and

MOLD were present at significantly higher concentrations than the fluorescent cross-

links pentosidine and dityrosine, identifying them as major Maillard reaction cross-links

in lens proteins. The presence of GOLD and MOLD and the unequivocal identification of the lysine-arginine crosslinks glucosepane, DODIC, MODIC, and GODIC in tissue proteins implicates methylglyoxal and glyoxal, either free or protein-bound, as important

precursors of protein crosslinks formed during Maillard reactions in vivo during aging and in disease. The formation of these crosslinks is through the alpha-dicarbonyl

compounds N(6)-(2,3-dihydroxy-5,6-dioxohexyl)-I-lysinate, 3-deoxyglucosone,

methylglyoxal and glyoxal and are redox active (171). Other lens crosslinks involving ascorbic acid, such as a cross-link between the (epsilon)-amino groups of two lysine residues and a five-carbon atom ring, assigned it the trivial name of (K2P). The K2P crosslink can be formed from oxidation products of ASA, which can act like glucose when oxidized and is highly reactive (172). Other lens crosslinks include LM-1

(vesperlysine A) (331), and dehydroalanine crosslinks (43)

Several physiologic crosslinks are also known to form in the proteins isolated from aged human lenses and brunescent cataracts, which exhibit extensive disulfide bond

48 formation. In this regard, the process can be coupled to oxidation as oxidation has been

shown to decrease gamma-cystathionase in experimental conditions (282). Diabetic rat

lenses similarly contain disulfide-bonded protein aggregates. These observations are

consistent with the known link between diabetes, glycation and oxidative damage, and suggest a role for reactive oxygen species (ROS) in this process.

Heterocyclic cross-links are another group of crosslinks that are likely to be found in lens tissue, since they readily form from the apparent spontaneous reaction of lysine and derivatives with allysine, which is an aldehyde formed from the oxidative deamination of lysine catalysed by the enzyme lysine oxidase (100). The epsilon-amino group of lysine residues in long-lived proteins oxidatively deaminates with age forming the carbonyl compound, allysine (alpha-amino adipic acid-delta-semialdehyde) (297), which can further oxidize into 2-aminoadipic acid (AAA). Our laboratory measured the oxidized product of allysine as its acid-stable product AAA. The extent to which this marker of lysine oxidation and the heterocyclic crosslinks occur in lens tissue has not been well studied, but is expected to be found in ongoing studies. While oxidative crosslinks are known to occur, and are important for Maillard reaction-mediated cross-linking via Strecker degradation together with allysine formation, non-oxidative forms of cross- linking may be more important to aging and diabetic complications in collagen (215).

The major glucose-derived non-UV active crosslink known to date is glucosepane, a lysyl-arginine crosslink that forms under non-oxidative conditions in collagen.

Besides glycation-related crosslinks, dehydroalanine crosslinks were identified in

49 cataractous, brunescent and aged lenses. These include histidinoalanine, lanthionine and lysinoalanine (185). Histidinoalanine and lanthionine were the most abundant dehydroalanine crosslinks in both water-soluble and water-insoluble lens proteins, with histidinoalanine levels 6.2-fold higher in water-soluble proteins of Indian origin than from normal lenses and 2.2-fold higher in water-insoluble fractions. Lanthionine levels were significantly higher in water-insoluble cataractous lenses when compared to non- cataractous lenses and unlike histidino-alanine, which accumulates in the water soluble fraction, this crosslink and reached 9-fold higher concentrations in the water-insoluble lens fraction. The lysinoalanine was found in cataractous lens proteins only and was the least abundant of the three (185).

1.2.5. Protein fragmentation, truncation and deamidation

A common and early post-translational modification of human crystallins is deamidation (170). Thus, it is believed that deamidation is a normal post-translational modification in the maturation of the human lens, which results in increased stability.

The spontaneous deamidation of both glutamine and asparagine has been shown to occur.

Moreover, deamidation of glutamine can also be catalyzed enzymatically by transglutaminase. In vitro studies show crystallins are a substrate of transglutaminase

(192, 355), which is present in the lens (138). Oxidative deamination is also an oxidation reaction, which occurs under aerobic conditions in most tissues, and occurs largely on glutamic acid residues since glutamic acid is the end product of many transamination reactions. Oxidative deamination is also known to cause the loss of the amino groups of

50 lysine and has been identified in oxidized proteins.

While enzymatic forms of this reaction are important in maintaining nitrogen

balance, oxidative deamination can also occur as an enzymatic reaction, where the

deamination of lysine can be catalyzed by the enzyme lysine oxidase (100). Enzymatic deamination is a similar process, wherein an amino acid is converted into the corresponding keto acid through the loss of an amine group that is replaced by a ketone group. These adducts accumulate in high levels in the plasma and are expected to be of significance to lens proteins. Taken together with non-enzymatic protein truncation, these modifications may be a significant source of damage in long-lived proteins that are not readily turned over. For example, the epsilon-amino group of lysine residues of long- lived proteins oxidatively deaminate with age forming the carbonyl compound, allysine, which can be reactive.

The deamination reaction resulting in the formation of the major carbonyl products of alpha-aminoadipic semialdehyde (AAS) and gamma-glutamic semialdehyde

(GGS) has been identified in oxidized lens proteins in vitro and in vivo (4) AAS is an

oxidative deamination product of lysine residues, while GGS results from the

deamination of arginine and proline residues. Akagawa et al., used model reactions of

benzylamine to elucidate oxidative deamination by glycoxidation. Glucose, 3-

deoxyglucosone (3-DG), and methylglyoxal (MGO) oxidatively deaminated benzylamine

to benzaldehyde in the presence of Cu2+ at a physiological pH and temperature but not

glyoxal and found 3-DG and MG were more effective oxidants than glucose. In this

51 study, it was determined that metal ions, pH, oxygen affected oxidative deamination,

which was greatest with Cu2+, accelerated at a higher pH and in the presence of oxygen.

The oxidative reaction was inhibited by EDTA, catalase, and dimethyl sulfoxide,

suggesting the participation of reactive oxygen species in this process. They proposed a

Strecker-type reaction and reactive oxygen species-mediated oxidation during

glycoxidation as a mechanism for oxidative deamination.

When the enzyme lysyl oxidase crosslinks collagen, for example, hydrogen

peroxide is released. However, enzymatic deamination reactions involving oxidases may

be attenuated by endogenous protective mechanisms. For example, H2O2 that is formed

in these reactions was completely decomposed by coexisting catalase activity in vivo (5).

Further, lysyl oxidase activity has been found to be greatly elevated in the presence of

H2O2, and is believed to be due to the oxygen produced by catalase, indicating that lysyl

oxidase is coupled with catalase in some tissues and protects against H2O2.

However, in the presence of metal and ascorbic acid, essential for some hydroxylation reactions, H2O2 can lead to hydroxyl radical formation by Fenton type

reactions (92). In this regard, hydroxyl radicals are also implicated in deamidation in

models involving metal-catalyzed oxidation reactions. In cell culture models, cells

grown in the presence of ascorbate were found to synthesize and accumulate significantly

less insoluble elastin than ascorbate-free cultures. Ascorbate was determined to be

important in hydroxylation reactions in aortic pulmonary smooth muscle cell cultures. In

the presence of ascorbate, elastin became incorporated into the extracellular matrix, and

52 was found to contain a slightly elevated content of hydroxyproline and lysine, and was

turned over more rapidly (92).

As pointed out above, due to their long life the crystallins, among other proteins,

undergo an unusually large number of modifications, of which deamidation is especially

prevalent. Age-related deamidations are difficult to model in vitro, but have been

identified by the presence of beta-aspartate, which occurs on asparagine (Asn) residues

via the succinimidyl intermediate, and results in the addition of a carbon to the

polypeptide backbone (116, 357). The structure of the C-terminal domain of human

gamma S-crystallin (255) shows that a beta-aspartate, identified at Asn 143, is a

consequence of deamidation in cataract (324). This is believed to be destabilizing if

occurring in the folded protein. A more global study of the deamidation sites of gamma

S-crystallin from nuclear cataractous lenses showed the overall level of beta-aspartate in

the human lens was correlated only with age and not with cataract (343).

When beta-crystallins undergo age-related modifications, a variety of molecular

masses and altered chemical properties result (376). The beta-B2-crystallin, the least

modified and most soluble of the beta-crystallins, was found to undergo extensive

truncation during aging (312). Zhang and colleagues identified, in vivo, deamidations among all beta-crystallins except betaB3, truncation of betaA3, betaB1 and betaA4, and oxidation of some methionine and tryptophan residues. Many of these modifications are known to occur before age 20 and found to increase modestly in lenses, aged from 20-87 years old (169). This may suggest they do not play a major role in destabilizing the

53 crystallins.

C-terminal truncated alpha-crystallins have been found in lenses of hereditary cataractous rats and included two truncated alpha B-crystallins and several truncated alpha A-crystallins believed to result from degradation by m-calpain and Lp82 (327).

However, not all truncation is destructive. For example, C-terminal lysine truncation of porcine alpha-B crystallin was shown to increase thermostability and enhance chaperone- like function (183). In contrast, molecular chaperone activity of lens alpha-crystallins is reduced by C-terminal truncation. Loss of alpha-A crystallin chaperone activity seems to be related to truncation of the C-terminal amino acid residues (332).

Several lens proteins undergo nonenzymatic cleavage at asparagine residues.

When such modification occurred to aquaporin 0 (AQP0), it was found to affect spatial and temporal aspects of AQP0 distribution (29). These reports indicate AQP0 undergoes modification, racemization and truncation within lens fiber cells with age. Further, this racemization/ isomerization also occurred on L-asparagine (Asp) residues to form D-Asp and D-iso-Asp. Aspartic acid racemization is one established method to determine the age of long-lived tissues, when no other information is available. Studies aimed at further characterizing the effect that C-truncation has on water permeability was examined in a model system. It was found that a 15% decrease in permeability, along with a 20 amino acid truncation occurred, which seemed to have a marked impact by impairing protein trafficking to the plasma membrane in Xenopus oocytes (30).

54 C-terminal truncation was enhanced in diabetes with carbonyl stress implicated in either enzymatic or nonenzymatic cleavage of peptide bonds between specific C-terminal amino acid residues (332). N and C-terminus truncation, and glycation crosslinking can affect crystallin structure (169). When the alpha B-crystallin was engineered with five amino acid residues deleted (C-terminal truncation), intrinsic chaperone activity decreased (326). In human alpha-B crystallin, the N146 residue undergoes truncation in vivo and several fragments containing this modification were found in normal and cataractous human lenses (312). Further, these modifications could occur through endopeptidase activity in vivo (342). Taken together, this process may contribute to the increased level of specific alpha crystallin fractions that are generally present in diabetic lenses and insolubility of lens crystallins.

1.2.6. Denaturation, solubility and conformational changes involving crystallins.

There are several reports in the literature that the crystallins, which can be classified as small heat shock proteins having chaperone-like function, are modified with age (152). In this regard, alpha-A crystallins, are known to be modified with advancing age, especially in the retina, which was found to decrease in abundance and underwent numerous modifications. These modifications result in changes in intrinsic protein charge as well as in truncation (discussed above). Crystallin modifications that can affect structure included acetylation, phosphorylation, deamidation, oxidation, racemization, N and C-terminus truncation, and glycation-mediated crosslinking that can affect their structure (169). C-terminal truncation, as discussed above, can cause intrinsic chaperone

55 activity to decrease in diabetic lenses, when compared with alpha-crystallins from the

normal rat lens. Several mutations are known to affect overall chaperone activity of

alpha-crystallins, which are remarkably reduced as well. The decreased chaperone

activity accompanying truncation of alpha-crystallins is believed to lead to insolubility of

many proteins in the mutant lenses, which likely leads to the progression of cataract

formation (327).

Human eye lens transparency requires solubility and stability of the crystallin

proteins over a lifetime. Aged crystallins accumulate a high degree of covalent damage,

including glutamine deamidation. Beta-B2 crystallin readily forms a homodimer in vitro.

The interacting residues across the monomer-monomer interface are conserved among

beta-crystallins. Similarly, human gamma D-crystallin is a two-domain beta-sheet

protein contained in the lens nucleus that interacts through side chain contacts with

glycine and are known to be critical for stability and folding of the N-terminal domain of the protein (104). Potential deamidation sites were determined and when the effects interface deamidation has on stability and folding was tested through single and double glutamine to glutamate substitutions. Equilibrium unfolding/refolding experiments performed with native and deamidation mutants indicate that interface deamidation decreases the thermodynamic stability of human gamma-D-crystallin and lowers the kinetic barrier to unfolding due to the introduction of a negative charge into the domain interface. Similarly, deamidation at critical sites destabilizes beta-B2 and may disrupt the function of beta-B2 in the lens. Such effects may be significant for cataract formation by inducing protein aggregation or insolubility.

The effects that specific truncations had on the structural properties of human betaA3-crystallin, compared to those with deletion mutants within the crystallin, showed all the mutant proteins exhibited fluorescence quenching and a red shift, which suggests that the truncations caused changes in the exposed hydrophobic patches (124). The cicular dichroism (CD) spectra showed that deletion of the N-terminal domain had a relatively weaker effect on the structural stability than deletion of the C-terminal domain and changes occurred in the microenvironment of the mutant proteins following truncations. This truncation led to higher-order aggregation compared to that in the wild- type (WT) protein and these results suggested that the N-terminal domain is relatively more stable than the C-terminal domain in beta-A3 crystallin. These results demonstrate the importance truncation has on lens protein structure and stability.

Damage to Crystallins by the Maillard Reaction

The Maillard reaction is the process by which various reactive compounds or

“stressors”, such as carbonyl stress from reducing sugars, lipids and lipid peroxidation products, intermediates of the glycolysis pathway or cytokine stress from inflammatory mediators react with proteins or other biomolecules. When this occurs in lens proteins, these age-dependent chemical modifications and crosslinks are major contributors to lens opacity. Some of the post-translational modifications, namely non-enzymatic glycation reactions, which involve carbohydrates, reactive aldehydes or reducing sugars and protein, have been strongly implicated with numerous age-related diseases. Evidence in

57 support of this process occurring in the lens has grown enormously ever since we first

hypothesized its existence (214). The scheme below summarizes the select pathways of

the Maillard reaction, some showing the source of the AGEs are diverse and Include

ASA (Figure 1.3).

Figure 1.3. Select pathways of the Maillard Reaction

Figure 1.3. is a scheme of the Maillard reaction in vivo, and the role of oxidative stress, which is simplified due to space constraints and due to limited knowledge of the actual pathways involved. It is readily apparent that advanced glycation endproducts

(AGEs) can originate from at least five sources, i.e., glucose, ascorbic acid, lipid

peroxidation (CML only), amino acids, inflammatory mediators and glycolytic

58 intermediates.

Various model reactions have shown that crystallin structure and function can be altered by glycation. Glycated crystallins undergo conformational changes (179), display enhanced protein aggregation and impaired chaperone function (66, 110, 246), form redox active species and bind redox active metal (220, 290) to name just a few.

Interestingly, Liang et al. concluded that HMW alpha-crystallin aggregates resulted from partial unfolding and disassembling-reassembling of low molecular weight (LMW) alpha-crystallin caused by posttranslational modification rather than chaperone complex formation (177). Not all modifications, however, are deleterious since CML formation was found to increase chaperone function of both αA, and αB crystallins (6), suggesting that the corresponding lysyl residues may play a protective role when carboxymethylated.

This, however, may apply only to proteins with no critical lysine residues in the active site.

1.3.1. Glycation by hexoses and pentoses

Carbohydrates with reactive aldehyde and ketone groups undergo nucleophilic attack by primary or secondary amines, sulfhydryl groups, etc., to form a Schiff base, which is the initial highly reversible reaction forming the Amadori product. The Schiff base and Amadori product can go on to form more stable Amadori products, which can also undergo further complex rearrangement, such as oxidation fragmentation and rearrangement and result in a mixture of compounds called the advanced glycation end

59 products (AGEs). Carbohydrates are not the only substrates that can undergo Maillard

reactions with proteins to form advanced glycation end products. Nevertheless, the

reducing sugars that were found to contribute to glycation reactions in the lens and

membrane include fructose (210), ribose (224, 253), galactose (111) and glucose (154).

Here, glycation by fructose may also play a role in cataract formation under conditions of

diabetes and aging; levels of fructose adducts in diabetic rat lens were 2.5 times that of

the control, and correlated with sorbitol levels. This was mainly due to enhanced

glycation of beta and gamma-crystallins by fructose under diabetic conditions. In a

diabetic lens the concentration of fructose significantly exceeds the concentration of

glucose, suggesting that the contribution of fructose-derived AGEs may be greater than

that of glucose-derived AGEs (377).

Incubation with both glucose and fructose showed that alpha and gamma

crystallins and some proteins of a mean molecular mass of 36-37 kilodalton (kDa) rapidly

incorporated sugars. After 6 days of incubation, more crystallins were glycated

compared with 3 days, in particular beta-crystallin. The gamma III beta-crystallin is the

most susceptible lens protein to glycation and the primary target of glucose is gamma-

IIIA crystallin in Bovine lens (371). The early glycation of gammaIII-crystallin by

glucose and fructose could result in structural alterations, leading to aggregation of

crystallin and eventually cataract formation.

In a study aimed at finding a correlation between the levels of plasma glucose and

degree of lens opacification, several groups found indications for a glycemic threshold in

60 forming glycation and glycoxidation-products and cataract (223). Further, in streptozotocin treated old rats having a broad range of plasma glucose levels, only lenses of moderately and severely diabetic rats developed cataracts whereas lenses of the mildly diabetic rats remained clear (320). This data supports the existence of a plasma glycemic threshold, above which incidence of diabetic cataract formation increases exponentially.

This threshold level seems to be at approximately 180 mg/dl or 10 mM plasma glucose.

Significant increase in the levels of glycation and glycoxidation products, mainly in cataract lenses, suggests that glycation and glycation-mediated oxidation play an important role in the development of diabetic cataracts, however, even subthreshold levels of glycation reactive intermediates are believed to glycate proteins over time.

Oxoaldehydes with mixed effects

Oxidation of amino acids (threonine) and ascorbic acid can all lead to methylglyoxal (MGO) formation, the alpha-dicarbonyl compound that can be produced in vivo by several metabolic pathways and the Maillard reaction and has a predilection for arginine residues. One AGE identified was an acid-stable blue fluorescent product called argpyrimidine (299, 302). Other oxoaldehydes, such as 3- and 1-deoxyosones from glucose and other sugars are likely present and possible precursors for the “odic” series of protein crosslinks (43). It is readily apparent from this scheme that oxidation of the precursor carbonyl agents is required for several AGE products, as indicated by “Ox”, whereby the oxidizing agent may vary from superoxide and hydrogen peroxide and metals, as in the case of the transition from the Amadori product to carboxymethyl-lysine

61 (CML)(222). MGO reacts rapidly with proteins to form advanced glycation end products

or AGEs. AGEs formed oxidatively, namely, pentosidine and N-(epsilon)-

carboxymethyl lysine (CML) or non-oxidatively (imidazolone) in human lenses and their

relationship to lens coloration, cataract type, and patients' diabetic state was examined in patient lenses (105). Pentosidine, CML, and imidazolone were increased in cataractous lenses when compared to controls. The highest AGE concentrations were found in mature cataracts, which increased with relative brunescence. The highest increase was due to imidazolone as compared to oxidatively formed AGEs. This data indicates a pivotal role for both processes in cataract formation (366).

Except for the hydroimidazolones, most of the structures shown in the boxes

(Figure 1.3.) above have been found with immunological or chromatographic technique in the lens. The most recently described structures are the LYS-ARG and LYS-LYS crosslinks such as glucosepane and glycine amide (GOLA) respectively (43, 119). Both crosslinks have been found in the aging human lens. Finally, Ortwerth and colleagues recently described a novel hydroxypyridinum structure, “OP-lysine” that could be synthesized from glyceraldehyde and glycolaldehyde (16) It was found in relatively large quantities in the aging human lens. The compound is fluorescent, but is not a crosslink.

They also report a new LYS-LYS fluorescent crosslink, the origin of which is currently unknown.

When the concentrations of methylglyoxal-derived AGEs, the hydroimidazolones

MG-H1 and -H2, in soluble human lens proteins were compared with the concentrations

62 of other methylglyoxal-derived AGEs and pentosidine in lens, the fluorescent AGEs

argpyrimidine and pentosidine were assayed by fluorometric detection as were MG-H1 and -H2 after derivatization with 6-aminoquinolyl-N-hydroxysuccimidylcarbamate

(AQC): The concentrations of AGEs were increased in cataractous lenses in comparison with non-cataractous lenses: the increases were MG-H1 85%, MG-H2 122%, argpyrimidine 255%, and pentosidine 183% and the concentration of MG-H1 in human

lens protein correlated positively with donor age and the concentration of MG-H2 and

argpyrimidine (3). Methylglyoxal hydroimidazolones are quantitatively major AGEs of

human lens proteins. These substantial modifications of lens proteins may stimulate

further glycation, oxidation, and protein aggregation leading to the formation of cataract.

However, just as the argpyrimidine modifications improve alpha crystallin chaperone-like

function, the hydroimidazolones may be protective as well, but this would require further

investigation.

1.3.3. Ascorbic acid degradation products

Vitamin C (ascorbate) L-ascorbic acid has antiscorbutic properties. It is

reasonably stable in air but in solution can rapidly oxidize and exists, basically, in two

physiological (enolic and ketonic) forms. For the purpose of this review, we will discuss

mostly the redox reversible forms, namely L-ascorbic acid (ASA), the reduced form, and

dehydro-L-ascorbic acid (DHA), the oxidized form. It is important to note that low

reduction potentials enable ascorbate and the ascorbyl radical to react with and reduce

basically all physiologically relevant radicals and oxidants (57)(See Figure 1.4.).

63 Vitamin C can be synthesized chemically from glucose and sucrose and is produced in

plants from glucose, via the uronic pathway. For studies, we initiated using the 19F-

ascorbic acid, (F-ASA), we considered this fact as the basis for establishing an internal standard for quantitating ASA uptake 6F-6deoxyglucose. In most animals, but not all, the enzyme l-gulonolactone oxidase is functional and converts gulonolactone to ascorbic acid for immediate bioavailability. It is proposed that the cause of human inability to synthesize ascorbic acid is the due to the absence of the active enzyme, l-gulonolactone oxidase in the liver of various animals (53).

In plasma, ascorbic acid is the most important water-soluble antioxidant (106). In the aqueous humor and the lens, the major role of ascorbate is thought to be an antioxidant and perhaps a UV-B filter, with a contributing role in retinal protection.

During the process of quenching free radicals, ascorbate donates an electron, becoming the unstable intermediate ascorbyl radical that can be reversibly reduced back to ascorbate. Ascorbyl radical can donate a second electron and be converted to DHA (364)

(Figure 1.4.).

OH OH ASA O HO O HO O O + R + RH O OH O O AscH Asc DHA

Figure 1.4. Ascorbate reaction to form ascorbate radical and the dismutation into ascorbic acid and dehydroascorbic acid.

However, in the presence of photosensitizers such as riboflavin, ascorbate traps

64 reactive oxygen species such as superoxide (351) to form the ascorbyl radical (Asc-), which is stable and has low reactivity when ascorbate scavenges a reactive oxygen or nitrogen species. The ascorbyl radical readily dismutates to form ascorbate and dehydroascorbic acid, or can be reduced back to ascorbate by an NADH-dependent semidehydroascorbate reductase (339). It is the discovery that vitamin C can play a role in protein glycation, which is observed in cataract formation that makes DHA of major interest in disease. Cataract formation is believed to result largely from an oxidative insult, which decreases redox homeostasis and antioxidant defense of the lens, particularly reduced vitamin C and glutathione concentrations (338).

The experiment shown below illustrates the reactive nature of DHA, when compared to other reducing sugars. Here, rabbit lenses were incubated for 20 days with

10 mM carbonyl agents such as ascorbic acid, dehydroascorbic acid, D-ribose and D- glucose (Figure 1.5, courtesy of Dr. Zhenyu Dai).

10yr 75yr 70 yr Control ASA 10 mM DHA 10 mM Ribose 10 mM Glucose 10 mM human lenses (Brunescent)

Figure 1.5. DHA compared to reducing sugars and ASA incubated with rabbit lens explants best mimics the pigmentation of the aging lens.

Among all carbonyl agents, dehydroascorbic acid most readily results in the pigmentation in the aging human lens (left). This high reactivity of ascorbate led Ortwerth and

65 colleagues to propose that ascorbylation damage might be more important that glucose

mediated damage (171).

Zigler, Ortwerth and colleagues also showed that extracts from both native or

Maillard Reaction modified crystallins are active as photosensitizers (238, 380). It

follows that the aging lens constantly undergoes combined attacks from free radicals and

reactive carbonyl agents. Paradoxically, it can be argued that the lens serves two

evolutionary functions, i.e the lens is needed to search for food and spot predators, while

the old UV/VIS absorbing lens acts as an environmental entropy buffer to shield the

retina from photoxidative damage. The drawback is that the pigmentation process is

highly associated with the risk of cataractogenesis (69). Efforts to quantitate aging

effects on the vertebrate lens have been approximated by measuring the changes in the

absorption characteristics of the lens. The lenticular adsorption spectrum has already

been elucidated and is related largely to the fluorescence and the yellowing of lens

proteins.

Upon aging, the lens accumulates these fluorophores and brown products, mainly derived from the Maillard reaction between ascorbic acid, or vitamin C oxidation products, and lysine residues of crystallins (84). The oxidized form of ASA, or its

degradation products, can contribute to the same modifications found in cataractous

lenses (237). What is unclear is the percentage of compounds that come form glucose.

Several factors affecting ascorbate degradation include the presence of thiol compounds,

enzymes, such as reductases or amines, which can trap the dicarbonyl compounds or

66 form adducts, and conversely, the presence of oxidases, free redox-active metals, H2O2 or hydroxyl and superoxide free-radicals; all affect ascorbate levels and its redox state.

These yellow fluorophores were found to increase up to fifteen fold in aged human lenses

(65). The group demonstrated increases in five individual (330nm)-absorbing yellow chromophores and eight fluorescent peaks 350/450nm ex/em from the water-insoluble fraction of human lenses. These fluorophores increased in an age-dependent manner.

The similarity of the yellow chromophores isolated from human cataracts with those from ascorbic acid modified calf lens proteins was recently published (65). This led Ortwerth and colleagues to propose that ascorbic acid-mediated damage

(ascorbylation) might be more important than glucose-mediated damage (172). Further, the oxidized forms of ascorbic acid and other dioxo-compounds from this degradation pathway are several fold more reactive than glucose or fructose (74). Among proposed degradation products of DHA are xylosone, 3-deoxy-xylosone, L-threose, L-threosone and L-erythrulose (307). The latter compound is thought to be a major product of DHA, forming under anaerobic conditions.

Further, once ascorbylation-mediated damage occurs, the process can feed forward, which may explain the exponential increase in particular AGEs with age (187).

The ability to regenerate ASA due to glycation and loss of glutathione may increase ascorbate-mediated glycation (288). A Japanese group found hyperglycemia enhances the synthesis of 3-DG, where it has been shown to inactivate glutathione peroxidase in uremic serum and uremic erythrocytes (231). Treatment with an aldose reductase

67 inhibitor reduced 3-DG, which rapidly reacts with amines to form imidazolone, pyrraline, and N-epsilon-(carboxymethyl)lysine. The elevated 3-DG levels in uremic patients was suggested to promote the formation of imidazolone in erythrocytes, aortas, and dialysis- related amyloid deposits (231). While many experimental studies have shown a protective effect of vitamin C in age-related cataract and oxidative-damage, other studies have revealed contrasting roles for this nutrient. A pro-oxidant effect of vitamin C through metal-catalyzed oxidation and H2O2 generation has been suggested. In that regard, the relevance of 2,3 DKG in impaired lens homeostasis may involve the inactivation of the key antioxidants in the lens and intracellular enzymes such as glutathione peroxidase, a key enzyme in the detoxification of hydrogen peroxide (231).

Upon oxidation, vitamin C, along with glucose, contributes to protein glycation, favoring tryptophan oxidation, which results in fluorescent peptide crosslinks and protein insolubility. Vitamin C content appears to be a good indicator of cataract severity, suggesting that oxidation could take part in cataract progression becausee the formation of hydroxyl radicals in the human lens is related to the severity of nuclear cataract. (193).

One protective effect of caloric restriction appears to be inversely related to plasma vitamin C levels in rodents (344) and that maintenance of sufficient plasma vitamin C is needed to prevent oxidative damage in the lens. When the relationship between cataract and lenticular vitamin C was analysed in graded cataractous lens nuclei, lenticular vitamin C concentration significantly decreased with cataract severity, most notably in severe brown cataracts. However, the dehydroascorbic acid concentration was always low and stable as was the furosine concentration, perhaps owing to the maintenace of

68 reduced intacellular concnetrations of ASA. The fluorescence of insoluble advanced

glycation end products was significantly higher in severe cataracts than in milder ones.

The peptide tryptophan content was stable, but the tryptophan to tyrosine ratio decreased

and was highly correlated to the ascorbic acid concentration (330).

A modification, which implicates the role of ascorbic acid unequivocally is modification of lysine epsilon-amino group by DHA identified as 1-(5-ammonio-5-

carboxypentyl)-3-oxido-4-(hydroxymethyl) pyridinium (OP-lysine), which was isolated

from DHA modified calf lens protein (16). (See figure 1.6 below).

Figure 1.6. AGEs formed from Ascorbic acid or degradation products of DHA

Figure 1.6. AGEs formed from Ascorbic acid or degradation products of DHA.

This study by Simpson et al. (307), confirmed that L-erythrulose was an important

intermediate in protein modification by DHA and in vitro experiments suggested that L-

erythrulose could further transform to L-threose, L-erythrose and glycolaldehyde under physiological conditions. The present study revealed that the modification of epsilon- amino groups of lysine residues by DHA is a complex process that could involve a number of reactive carbonyl species. This study by Simpson et al. (307), confirmed that

L-erythrulose was an important intermediate in protein modification by DHA and in vitro experiments suggested that L-erythrulose could further transform to L-threose, L- erythrose and glycolaldehyde under physiological conditions. The present study revealed that the modification of epsilon-amino groups of lysine residues by DHA is a complex process that could involve a number of reactive carbonyl species.

A newly identified glycation product of lysine in the lens, the 3- hydroxypyridinium derivative of lysine, 2-ammonio-6-(3-oxidopyridinium-1- yl)hexanoate (K2P), was synthesized independently from 3-hydroxypyridine and methyl

2-[(tert-butoxycarbonyl)amino]-6-iodohexanoate (17). The experimental incubations showed that an anaerobic reaction mixture of N-alpha-tert-butoxycarbonyllysine, glycolaldehyde, and glyceraldehyde could produce the N-alpha-t-butoxycarbonyl

derivative of OP-lysine. It is a marker of aging and pathology of the lens, and its

formation could be considered as a potential cataract risk factor based on its concentration and its photochemical properties.

70 The irradiation of OP-lysine with UV-A under anaerobic conditions in the presence of ascorbate led to a photochemical bleaching of this compound. Some AGEs in the human lens such as OP-lysine, OP-phenethylamine (a phenethylamine analogue of

OP-lysine), and argpyrimidine are abundant AGEs, which can be transformed by UV-A light (18). In that regard, due to irradiation with UV-A light in the presence or absence of oxygen and ascorbic acid, both OP-lysine and argpyrimidine decreased 20% when irradiated with UV-A light in the absence of ascorbic acid. Under the same conditions,

OP-lysine was bleached 80% in the presence of ascorbic acid during irradiation experiments. In contrast, argpyrimidine UV-A light bleaching was not affected by the presence of ascorbic acid. Interestingly the major product of OP-phenethylamine after

UV-A irradiation in the presence of ascorbic acid was phenethylamine and L- dehydroascorbic acid (DHA).

Other lines of evidence implicate oxalate monoalkylamide as one of the AGEs formed from the Maillard reaction of ascorbate with proteins in vivo and suggest that ascorbate degradation and its binding to proteins are enhanced during lens aging and

cataract formation (227). Here, oxalate monoalkylamide was absent in most very young

lenses and increased in old and cataractous lenses, with the highest levels found in senile

brunescent lenses. Incubation experiments using bovine lens proteins confirmed that

oxalate monoalkylamide could form from the ascorbate degradation products 2,3-

diketogulonate and L-threose, implicating degradation products of ASA in this process.

Lipid peroxidation products

Various metabolic pathways and metabolites of arachidonic acid are found in the

lens, which is indicative of the presence of polyunsaturated fatty acids (PUFAs).

However, as a whole, the lens has low levels of unsaturated lipids with a non-uniform

distribution of these unsaturated lipids. The Ansari group (68) reported that the degree of

unsaturation decreases the more one dissects the lens regions from the exterior to the

interior regions. The lens epithelium in many species is known to have up to 20-fold

more unsaturated lipids per milligram protein than the cortex or nucleus. Since a high

degree of unsaturation occurs in the lens, it is not surprising that lipid-derived

modifications, particularly to the polyunsaturated fatty acids from biological membranes occurs, since they are particularly vulnerable to oxidation by free radicals and, thus, the lens accumulation of primary diene conjugates, acetodienes and lipid peroxidation (LPO) products can be found in the early stages of cataract, while in later stages there is a prevalence of fluorescent LPO end-products (25).

Following UV irradiation, corneal epithelial cell lipid peroxide levels were found

not to be significantly altered, implying that corneal antioxidant enzymes and other

antioxidants, such as reduced glutathione, were able to reduce the ROS generated by UV

irradiation under experimental conditions. However, UV-A irradiation of lens epithelial

cells resulted in a three-fold increase in lipid peroxides. One lipid peroxidation product,

malonaldedyde, may contribute to some of the UV-A-induced damage in lens epithelial

cells. The degree of lens opacity correlates with the level of the LPO fluorescent end-

product accumulation. Further, the injection of LPO products into the vitreous has been

72 shown to induce cataract and leads to the conclusion that peroxide damage of fiber cell

membranes may be one of many factors leading to the initial development of cataract.

LPO-mediated peroxide damage results in lipid peroxide production, which degrades to

form lipid-derived aldehydes (LDAs). These LDAs are implicated in pathogenic

processes of several diseases. LDAs, are stable and can diffuse from the initial site of

lipid peroxidation and propagate oxidative injury to surrounding cells and tissues. These aldehydes are electrophilic and react with nucleophiles, such as thiols and amines and free amino acids or form crosslinks. To date, the major LDAs found in disease include malonaldehyde (MDA), acrolein, and 4-hydroxynonenal (HNE). 4-HNE, an oxidation degradation product of unsaturated lipids, is a reactive alpha, beta unsaturated aldehyde, as is acrolein and others (352). Of the various LDAs, HNE and 4-hydroxyhexenal

(HHE), arising from the peroxidation of arachidonic acid and docosahexaenoic acid, respectively, are the most toxic and are generated in high concentrations. 4- hydroxynonenal (HNE), formed as a result of increased lipid peroxidation in oxidative stress, causes loss of lens transparency and is believed to involve perturbations in the ubiquitin-dependent lysosomal degradation of the HNE-modified proteins in lens epithelial cells (200). Hegde et al., demonstrated that the level of glycated protein and

MDA increased in the water-soluble fraction of diabetic lenses, while glutathione, undoubtedly the major antioxidant of the lens, decreased along with the level of adenosine triphosphate (ATP), an indicator of the overall metabolic state of the tissue

(135).

Protective mechanisms; Defense against lenticular damage

It is probably fair to say that no single modification resulting from glycation, oxidation, photo-oxidation or binding of kynurenine is expected, by itself, to impair protein function as observed during cataractogenesis. In that regard, most of the UV radiation never reaches the retina as it is absorbed both the aqueous humor and the epithelium by the low-molecular-weight tryptophan metabolites known as the kynurenines and through the maintained of a high concentration of ascorbic acid, all of which protect the lens from visible and select wavelengths of UV light. The lens also contains an armamentarium of antioxidant defenses, which includes many NAD+/H- dependent reductases and oxidases. However, these defenses can fail and keeping them intact may be one means to retard or prevent cataracts.

Endogenous protective mechanisms

Primarily, the first line of defense in the lens is accomplished largely through lens development and maintenance of structural integrity. Table 1.2. summarizes the endogenous protective mechanisms of the lens. As the lens develops, it maintains a very low oxygen tension, which is why the lens is sometimes referred to as being

“canned”(166). Thus, a low oxygen tension means less metabolic activity or the potential to generate reactive oxygen, or nitrogen, free radical species and is one structural mechanism through which the lens protects itself by minimizing photo-oxidative and oxidative stress.

Stress Source Defense Denaturant Stress Heat Chemical proteolytic Intrinsic Chaperone Function/Homology Carbonyl Stress Glycation Intermediates, NAD+/H Reductases and Lipids, Aldehydes, Oxidases Ascorbylation Products Oxidant Stress Ionizing Radiation, “Canning” (Having Low O2 ROS, RNS Tension), GSH, GPX, SOD Catalase and Ascorbic Acid UV Stress UV/VIS Irradiation Ascorbic Acid, Kynurenins

Table 1.2. Endogenous protective mechanisms for the lens and other eye structures.

The healthy lens protects itself from largely deleterious processes and oxidation through another ocular mechanism, namely by efficiently dissipating energy from incident light between 295 nm and 400 nm. It has been shown that between two and seventeen percent of solar UV would reach the eyes (276) and most of the incident light between 295 and 400nm is absorbed through the physical mechanisms for the protection of the lens and retina, which are mainly contained in the aqueous humor and include a role for proteins, tryptophan, tyrosine, ascorbic acid, uric acid and by the low-molecular-

weight tryptophan metabolites of the kynurenine pathway. The second important line of

defense is a battery of intrinsic repair enzymes that constantly dethiolate protein-thiol

mixed disufides or protein-protein disulfides induced by oxidative stress (193).

However, the effectiveness of this mechanism is unknown in the face of glycation involving sulfhydryls on lens proteins. Those important detoxifying enzymes are illustrated in (Figure 1.7.), adapted from Lou et al. (193) and others including protein disulfide isomerase (PDI) as well as small non-enzymatic antioxidant compounds.

Figure 1.7. Important antioxidant systems in the lens.

One endogenous mechanism for efficiently dissipating energy by nondestructive photophysical pathways, involves ascorbic acid, which protects the lens from reactive oxygen species (ROS) and UV/visible light. In that regard, the ascorbate concentration gradient in the anterior bovine eye is consistent with this notion; levels were highest in the corneal epithelium, with higher values (1.56 mg/g) in the central vs. (1.39 mg/g) the peripheral area, again compatible with the idea that ascorbate acts as an UV filter for internal eye structures (271). In separate studies, the ascorbate concentration in the aqueous humor was 0.21 mg/ml as compared to 0.0008 mg/ml in the serum. Further,

76 diurnal animals show a higher ascorbate concentration in the corneal epithelium, aqueous

humor and lens than nocturnal animals. Ascorbate levels in the corneal epithelium seem to vary in accordance with ambient radiation exposure of the respective species, just as in the aqueous humor. Again both phenomena are regarded as environmental adaptations, and are consistent with the notion that ascorbic acid, with the highest concentration in the corneal epithelium, is protective against photokeratitis and acts as an ultraviolet filter for internal eye structures (270).

Also, considered part of lens defenses are UV filters like the kynurenine, which are L-tryptophan metabolites that protect the lens from UV light between 295 and 400nm.

This is accomplished largely through the absorbance above the 288nm wavelength by the o-β-glucoside of 3-hydroxykynurenine (3-HKG) and other UV-absorbing substances.

The absorbance above 288nm is known to be low in the aqueous humor of fish, frogs, aquatic mammals and some ground-living birds (269). In that regard, the concentration of the kynurenine UV filter compounds have been found to decrease with age (32, 318).

Loss of cytoprotection is a key factor in cataractogenesis, which has been linked to increases in lens fluorescence, in particular, the amount of 3-HKG in the human lens.

However, and interestingly, an age-related modification in the human lens is the covalent attachment of 3-HKG to lens protein. With increasing age the absorption from 3-HKG at

365nm decreases, and the maximum shifts to 320nm. At the same time, the filtering capacity of the human lens actually increases due to a generalized yellowing of lens proteins. Looking at the ratio of absorbance of old lenses at 320nm to the absorbance of young lenses at 365nm, researchers have determined that the yellowing of the lens is a

77 result of chronological processes, namely chemical or photochemical modifications, and not due to biological aging per se (109).

Intrinsic chaperone function of crystallins

Mechanical mechanisms related to lens structure maintenance are of key importance to lens homeostasis and prevention of cataract. Included here is the intrinsic chaperone-like function of crystallins toward other crystallins (144) and enzymes (44), through which non-native proteins are efficiently captured and refolded. Indeed, age- related nuclear cataract can be characterized as a net loss of intrinsic alpha-crystallin chaperone and function, due to the paucity of defenses inherent in the oldest part of the lens. Similarly, age-related cortical cataract may be characterized as well by the absence of cytoprotection, which had been supplied by alpha-crystallin (13). Of considerable interest for the glycation field is the fact that a number of mutations involving arginine residues, such as R58H and R36S in γD crystallin are associated with dramatically lowered solubility of the protein (241). Similarly, the R116C mutation in αA crystallin is associated both with congenital cataract, polymerization, impaired chaperone activity

(303) and diminished protective ability against stress-induced lens epithelial cell apoptosis (12).

It is possible that modification of these critical arginine residues by oxoaldehydes derived from ascorbic acid (66), particularly ascorbic acid degradation products, methylglyoxal or other oxoaldehydes impair its chaperone function and perhaps mimic

78 the effect of mutations. Thus, ascorbic acid degradation products can impair lens integrity both via protein crosslinking as well as impaired chaperone function. Indeed, the most recent study by Derham supports this view (81). However, a recent study demonstrated that modification of specific arginine residues of alpha-crystallin by methylglyoxal improved its chaperone function (225). In contrast, our data suggests that ornithine (ORN) is formed in vivo and its AGEs arise in aging proteins, suggesting that arg-based AGEs likely serve as precursors of ORN. Ornithine, the non-protein amino acid, and decarbamidation product of arginine increased with age in acid hydrolysates of human skin collagen and lens crystallins (295). Carboxymethyl-ornithine, glycated ornithine ("furornithine"), increased with age in pmol/mg protein, from ~0 to 60 in lens and 0 to 180 in skin, respectively. Ornithine was found to increase from 1 to 15 nmol/mg protein from ages 10 to 90 years, whereas diethyl-ornithine increased from 0.5 to 15 and from 0 to 5 nmol/mg protein in lens and skin, respectively. The partial degradation of arginine modifications may reverse instability and lead to the formation of ornithine.

This mechanism would imply a secondary PTM that would restore a partial positive charge to the arginine residues, which were previously lost to modification and result in a stabilizing effect on non-native proteins, perhaps analogous to the stabilizing mechanisms of chaperone proteins.

Glutathione

A key aspect in the small molecular weight antioxidant, non-enzymatic, line of defenses involves high levels of glutathione (GSH), both in the aqueous humor and the

79 lens. The roles for GSH as a biological antioxidant, mixed disulfide bond forming agent, and enzyme cofactor are well understood (193, 194). Together with glutathione, vitamin

C, vitamin E and carotenoids are the other major lenticular antioxidants (193). With

Marjorie Lou, our group has recently demonstrated that thioltransferase can reduce

dehydroascorbic acid into ascorbate (ASA) with GSH as cofactor (101). In addition to its

role as a chemical and enzymatic reducing agent, it is likely that GSH can also act as a

trapping agent of reactive AGE precursors, based on the demonstration that N-

acetylcysteine inhibits AGE formation (368). In an in vitro system, inactivation of GSH

reductase by aldehydes led to the NADPH-independent reactions and to the formation of

non-fluorescent cross-linked products with an accompanying loss of histamine and lysine

residues. In the lens, sulfhydryl group oxidation of lens proteins can be due, in part, to a

decrease of reduced glutathione concentration in the lens, which is a second line of

defense.

With age, and under conditions of oxidative stress from such sources as hydrogen

peroxide or other oxidants, the once adequate pool of glutathione is ostensibly diminished

(193). Further, as the lens ages, the de novo synthesis and the recycling system for GSH

become less efficient (262) which causes GSH concentrations to decline in various

species (128, 193, 195). Since GSH concentration in diabetic lens is significantly

decreased and the glucose concentration can increase 10 fold and higher, the formation of

Amadori products of GSH with this monosaccharide may be favored under these

conditions and could contribute to a further lowering of glutathione levels and the

increase of oxidative stress observed in diabetic lens and feed forward oxidative stress-

80 mediated damage. Formation of H-1-Deoxyfructos-1-yl glutathione is the major

glycation product formed in the mixtures of GSH with glucose (188). Also, the same

group has found that glutathione reductase failed to recycle the disulfide bond within the

structure of the did-substituted form of GSSG and showed only 1% of the enzyme’s

original activity, but retained its ability to reduce the disulfide bond within the structure of N-1-Deoxy-fructos-1-yl GSSG at 57% of its original activity.

Protective enzymatic mechanisms for crystallin damage

Finally, the lens is equipped with a number of detoxifying enzymes, which is another means of self-protection. Detoxification occurs through a multitude of antioxidants, oxidation defense enzymes and by other means such as through NAD+/H-

dependent reductases, oxidases and dehydrogenases. Antioxidant enzymes such as

catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPX) have the ability

to reduce reactive oxygen species. Together with catalase, an important lenticular

antioxidant enzyme, contain a heme protein with one molecule of NADPH, which is

tightly bound to each of the four subunits of catalase in mammals (157), are important enzymatic antioxidant defense systems in the lens (Figure 1.7.).

The antioxidant homeostasis in the lens can be affected by diabetes, which causes increased polyol pathway activation, decrease in lens ATP and the subsequent formation of cataracts. Concomitantly, polyol pathway activation has been shown to also cause osmolyte loss. The major components lost were taurine and amino acids, which together

81 accounted for over 75% of the total osmolyte loss as well as a 4- to 5-fold increase in

crystallin leakage into the vitreous humour and a 4-fold increase in gamma-crystallin in

the aqueous humor. Since glutathione, ascorbate, taurine and cysteine have been

reported to have antioxidant activity, it appears that their loss may potentiate damage

occurring as a result of free radicals generated by non-enzymatic glycation by the

Maillard reaction. Amino acids also lost as a result of osmotic compensation, are estimated to be responsible for almost half of the antioxidant activity lost (158).

Antioxidant enzymes can be affected by UV irradiation. In corneal and lens epithelial cells UV irradiation decreased the activity of catalase to thirty to fifty percent of its original level, while glutathione peroxidase and superoxide dismutase were unaffected (273). Further, increases in UV absorption by the presence of additional lens chromophores makes catalase more sensitive to UV damage. In that regard, when catalase levels are decreased, it is possible that hydrogen peroxide generated by UV irradiation can be safely detoxified by glutathione peroxidase and other peroxidases, which makes GSH and GPX major lenticular detoxification mechanisms. Thus, even sub-solar UV radiation can cause irreversible damage to corneal and lens epithelial cells.

There are also enzymatic routes for the detoxification of carbonyls. Reactive lipid-derived aldehydes, such as HNE, are continuously generated in ocular tissues and their metabolic fate is largely unknown. Aldehyde dehydrogenase 3A1 (ALDH3A1) is a

NAD(P)+-dependent enzyme that is highly expressed in mammalian corneal epithelial cells and has been shown to protect against UV and 4-HNE-induced cellular damage,

82 mainly by metabolizing toxic lipid peroxidation aldehydes. Further, some detoxification

mechanisms for HNE are known to occur through enzymatic conjugation with

glutathione, which is catalyzed by glutathione S-transferases or nonenzymatic

conjugation to form GSH-HNE. HNE and GSH-HNE can also be detoxified through

reduction by aldehyde and ketone reductases and by alcohol dehydrogenase. The latter

leads to the formation of (1,4-dihydroxy- 2-noneneol) DHN and GSH-DHN, respectively;

and finally, HNE can be oxidized to 4-hydroxy-2-nonenoic acid (HNA) by aldehyde

dehydrogenase and carbonyl reductases (242).

Many aldehydes besides glucose are substrates of aldose reductase (AR), the first

enzyme in the polyol pathway. The endogenous carbonyl substrates of AR are glyoxal,

the simplest dicarbonyl, MGO, acrolein, acetol L-3-deoxyxylosone, D-3-deoxyglucosone,

L-xylosone, glucose D-glucosone, 4-hydroxynonenal and other reducing sugars. It also

detoxifies lipid peroxidation products and other reactive aldehydes associated with

diabetes (336). Growing evidence indicates that AR has a key role in oxidative stress.

MGO is a 2-oxoaldehyde and the preferred substrate of AR and of the

glutathione-dependent glyoxylase system. The glyoxylase enzymes are another efficient

means of detoxifying reactive compounds such as oxoaldehydes. The importance of these

enzymes in detoxifying oxoaldehydes is exemplified by the demonstration that chemical

inhibition of glyoxylase I leads to elevated methylglyoxal levels in intact cultured lenses

(300). Further, glutathione reductase can be inactivated by 4-HNE and other endogenous aldehydes (346) and by UV irradiation in the cornea and lens as well as in corneal and

83 lens epithelial cells (61, 62, 91, 363, 381, 382). When glutathione concentrations are

sufficient, MGO is converted to the hemithioacetal, the actual substrate of glyoxylase I.

At the same time the reduction of MGO by AR is increased, but the site of reduction shifts to the ketone from the carbonyl on the aldehyde group, in effect converting it from an AR to a ketone reductase (345).

Damage to crystallins in the aging lens is kept in check by a number of nonenzymatic mechanisms as well, such as antioxidant foods and compounds, which are known to detoxify reactive intermediates. For example, in the lens, several reactive compounds are known to be stabilized to sugar alcohols, e.g., incubation of lens homogenates with highly glycating agents like L-threose or L-erythrulose leads to formation of unreactive L-threitol (239, 307). Similarly, these carbonyl compounds can be detoxified through oxidation, as evidenced by the finding of threonic and erythronic acid in the aqueous humor (131).

Although the role of the carnitine system in the ocular tissues is not clearly understood, studies show that lenticular levels of acetyl-L-carnitine and L-carnitine (the combination of histidine and alanine) were the highest among ocular tissues, which is

depleted in streptozotocin-induced diabetic rats (320) (319). The results from this group

show that while L-carnitine did not have any effect on in vitro glycation of lens

crystallins, acetyl-L-carnitine and acetyl salicylic acid decreased crystallin glycation by

42% and 63%, respectively in a concentration-dependent manner. Inositol, another small

molecular weight compound of the lens, when reacted with glucose forms glucosyl

84 inositol. Inositol was shown to remove hydrogen peroxide from the reaction mixture and when reacted with arachidonic acid showed that they formed a conjugate. These observations indicate that the antioxidant activity of inositol could quench reactive oxygen, intermediates, and has an antiglycating property through scavenging glucose

(259).

Finally, vitamin derivatives and analogs have been used successfully to lower mean glycemia levels as well as inhibit AGE formation. In this regard, pyridoxamine, a vitamin B6 derivative, has been shown to inhibit formation of advanced glycation and lipoxidation products as well as inhibit the formation of MGO. Other vitamins, such as vitamin B1, have been show to affect diabetic retinopathy through activating transketolase enzyme activity and thus diverting excess metabolites toward the hexose monophosphate shunt (HMP) pathway. Further, glucose-6-phosphate dehydrogenase

(G6PDH) is an important lens enzyme, which is capable of diverting about 14% of the tissue glucose via the pentose pathway mechanism. The main function of such a pronounced activity of the enzyme is to support reductive biosyntheses, as well as to maintain a reducing environment in the tissue so as to prevent oxy-radical induced damage and consequent cataract formation (377). When this group incubated the enzyme with fructose (0-20mM) a significant loss of activity was noted. However, this loss in

activity was prevented by superoxide dismutase, catalase, mannitol and myoinositol.

Most interestingly, pyruvate at levels between 0.2 and 1.0 mM also offered substantial

protection. Hence, these results demonstrate the possibility of therapeutic prevention of

cataracts by pyruvate and other such keto acids, in diabetes or conditions involving

85 oxygen free radicals in their pathogenesis.

Taken together, the sum of deleterious protein modifications and the breakdown

of homeostasis during cataractogenesis are likely sufficient to explain the crystallin

instability that leads to the formation of light scattering aggregates during cataractogenesis. Any means to retard or delay any of the mechanisms of cataractogenesis, be they by drug intervention or dietary means, would likely have a positive impact on preventing blindness and improve quality of life for millions of individuals worldwide.

1.5. Ascorbic acid transport and biologic roles.

Vitamin C has an essential role in normal metabolic function and as a cofactor in several enzymatic processes. The emergence in vertebrates, some 360 million years ago, of L-gulono-γ-lactone oxidase, the enzyme needed for ascorbic acid biosynthesis, coincides with the exposure of terrestrial vertebrates to oxygen and the need to develop free radical scavengers (229). Ascorbic acid, along with the Dehydro-, Semidehyro- and

Monodehydro- forms of ascorbic acid oxidation and free radical species, respectively, has important intracellular redox properties that make this vitamin an important electron donor and acceptor for cells (127). Vitamin C is not only an important antioxidant that helps to protect cells against oxidative stress (ROS) and from RNS and has antioxidant properties that may inhibit formation of nitrosamines during digestion of protein and it is an important antioxidant involved in recycling oxidized tocopherol (vitamin E), which

86 protects the plasma membrane against lipid peroxidation (150, 202, 206)and in redox

reactions with glutathione, which is perhaps the singularly most important cellular

antioxidant. Ascorbate may provide a mechanism for extracellular detoxification of H2O2 by peroxidase-coupled reactions because, unlike superoxide and other radicals, ascorbate will not directly scavenge H2O2, but it is an avid secondary proton donor as a substrate

for peroxidases whereby it prevents H2O2 inhibition of peroxidase (205).

Ascorbate is essential in development and maintenance of connective tissues and

as a cofactor for prolyl- and lysyl-oxidases, involved in hydroxylation reactions in

collagen synthesis, hydroxylation reactions regulation of cholesterol biosynthesis, the

conversion of cholesterol to cholic acid and to bile acids. It is a cofactor in carnitine

synthesis in the activation of the B vitamin, folic acid and the conversion of folic acid to

tetrahydrofolic acid, the, the conversion of the amino acid, tryptophan to 5-

hydroxytryptophan, the neurotransmitter serotonin and in dopamine-beta hydroxylase to

convert dopamine to the neurotransmitter norepinephrine (149, 202). ASA aids in the

absorption and utilization of iron and in the adrenal gland for catecholamine synthesis

(115).

It is well-known that oxidative damage to biomolecules, such as protein, RNA,

DNA and lipids can be inhibited by antioxidants (9, 106, 107, 122) and these groups have

shown that vitamin C (ASA, DHA) is a powerful antioxidant and has synergistic effects

in preventing lipid peroxidation and/or damage to cells by lipid hydroperoxides in plasma

exposed to various types of oxidative stress. ASA is implicated in protecting DNA from

87 oxidative damage. Most of the oxidative damage is mediated through redox active metals, such as copper or, particularly iron, either free or bound play a large role in oxidative stress in vivo, but mitochondria are implicated as well. The studies on ascorbate and iron toxicity are mixed. ASA has been found not to increase oxidative stress induced by dietary iron in C3H mice (254). However, iron is a potent prooxidant that can induce lipid peroxidation and ascorbic acid, a potent antioxidant, has prooxidant effects in the presence of iron, in vitro. This group investigated whether ascorbic acid and iron co-supplementation in ascorbic acid-sufficient mice increases hepatic oxidative stress, but found that ASA does not further increase the oxidative stress induced by increased dietary iron, suggesting that a saturable process is involved in hepatic ascorbate-iron toxicity. The discovery of a fixation-resistant NADH ferricyanide reductase (146, 219) was the early ascorbate-mediated electron transfer activity attributed to this molecule. The associated reductase activity, identified as an ascorbate free radical

(mono or semi-dehydroascorbate) oxidoreductase is localized in the trans portion of the

Golgi, in transport vesicles and in membranes (218). Morre identifies it as an energy- producing enzyme (via proton gradient or membrane potential) and attributes a role for driving membrane translocations. In terms of free metal, bleomycin-detectable iron

(BDI) is one form of iron that is non-transferrin-bound, chelatable by bleomycin, and potentially redox-active (125, 126). It has been suggested that this free form of iron can contribute to oxidative damage in vivo (56, 98). In that regard, BDI has also been detected in various diseases.

Adequate intake of ASA leads to 50–100 micromolar concentrations in plasma

88 (150, 369). In patients with low plasma levels of ASA, vascular endothelial dysfunction

during atherosclerosis (58, 173, 369). Increased requirements have only been identified during periods of rapid growth, increased physical activity, pregnancy or hyperthyroidism. So it is no surprise that ASA has been championed as one of the most essential nutrients for human health.

Function of vitamin C transporters and putative substrate transport.

Specific non-overlapping transport proteins mediate the transport of the oxidized form of vitamin C, dehydroascorbic acid, and the reduced form, L-ascorbic acid, across biological membranes. The absorption of ASA occurs largely through two

Na+dependent vitamin C symporters (SVCT). SVCT1 is confined to epithelial systems of the intestine, kidney, and liver and is responsible for uptake of vitamin C from the intestinal lumen and results in plasma concentrations of approximately 50 to 100 uM, which are almost exclusively in the reduced form (134). Whereas uptake into most other metabolically active and specialized cells is served by the high affinity L-ascorbic acid transporter SVCT2, including smooth muscle, endothelium, neurons and tissue of the eye, lung, placenta, and a range of neuroendocrine, exocrine, and endothelial tissues, there has been debate over the presence of SVCT2 in lung and skeletal muscle possibly because some tissues express both transporters (75, 142, 204, 340). An SVCT2-knockout mouse reveals an obligatory requirement for SVCT2 as deletion of the svct2 gene in mice was embryonically lethal, and although many of the specific roles of this transporter remain unclear, ascorbate is clearly required for normal development of the lungs and

89 brain during pregnancy (134). SVCT1 is considered to be a low-affinity high-velocity cotransporter highly selective for L-ascorbic acid that is expressed in epithelial cells.

SVCT2 has slightly higher affinity (Kasa 0.5 of 10±100mM) and is expressed in lower abundance in most cells including endothelial cells (39) but belongs to a class of uracil permeases and thus may transport other substrates, such as D-glucose, uracil and intermediates of vitamin C metabolism, such as L-gulono-g-lactone excluded from most studies, which should be further explored. Golde et al., discovered a short form of human SVCT2 (hSVCT2-short) in which 345 bp including domains 5 and 6 and part of domain 4, which are deleted without a frame shift (198). In rats, ascorbate transport has been reported to decline with age (209).

The oxidized form of ascorbate, dehydroascorbic acid, is transported into a variety of cells by the facilitative glucose transporter Glut-1. Glut-1, Glut-3 and Glut-4 can transport dehydroascorbic acid, but GLUT1 and -3 have relatively low-affinity

(356)and the plasma levels are reported to be approximately 10 µM in the rat (161) and human (228), suggesting that Gluts may not transport significant quantities of ascorbic acid in vivo. Although the affinity of DHA for facilitative glucose transporters (Km =

93.4 µM) is reportedly greater than that of D-glucose, in the retina, the Km estimated for

D-glucose uptake in the rat blood retinal barrier was 7.81 mM (96). Agus et al. have reported that DHA crosses the blood–brain barrier (BBB) through GLUT1 at the luminal and abluminal side of BBB and accumulates as reduced ASA in the brain (2). Tissue uptake of dehydroascorbic acid can occur via GLUTs, particularly Glut 1, 3 and 4, but it is Glut 3 that has been shown to export DHA in neurons, cells which accumulate high

90 quantities of ascorbate (134) and recycled by enzymes that utilize reductants such as

glutathione and NADPH, present in most cells including vascular smooth muscle and

endothelium (142, 203).

Dehydroascorbate is present in small quantities in blood and is rapidly (6 min)

and irreversibly degraded in aqueous media at neutral pH. Cells known to take up DHA

are leukocytes and erythrocytes, which seem to be mediated by glucose transporters, as

such transport is blocked by glucose and are suggested to be important in recycling

ascorbate (205). Other cells known to scavenge DHA are astrocytes, which reportedly concentrate ASA up to 1 mM, but levels in neurons can reach up to 10 mM (121, 134,

145), however, concentrating against such a gradient cannot be mediated by GLUTs and

regulatory mechanisms that prevent ASA from being shuttled out may explain the high

concentrations in these cells. Nevertheless, DHA uptake through facilitative glucose

transporters is competitively inhibited by D-glucose, and the normal plasma D-glucose

concentration in most mammals is approximately 5 mM. The use of retinal cells, namely

TR-iBRB2, in experiments with D-Glucose, showed IC50 of 5.56 mM inhibited [14C]

DHA uptake in these cells.

The experiments above call into question the specificity of the transporters as it is unknown what substrates SVCT2 transports, other than vitamin C, but it is conceivable that it may transport multiple substrates, as SVCT2 reportedly belongs to a family of permeases (Xan_ur_permease, pfam00860). Members of this family have at least ten predicted transmembrane helices, which includes genes encoding a wide-specificity

91 purine permeases for diverse substrates, such as xanthine, uracil and vitamin C (82).

These proteins appear to be members of a new family of possible nucleobase transporters with significant sequence similarities with bacterial and Aspergillus nucleobase transporters. These early genes were cloned from human and mouse kidney and from

LLC-PK1 cells and revealed a novel conserved family that is homologous to bacterial and Aspergillus nucleobase transporters (123). However, many members of this family have not been well characterized molecularly or functionally and may transport other

substrates. Since SVCT2 may be a nucleobase transporter, it would be difficult to

conceive of a mechanism that would evolutionarily suppress this transporter for vitamin

C and not other substrates, as nucleobase transport is important for the metabolism of

nucleic acids and conserved in lower kingdoms (52, 141). SVCTs have been classified as

a putative uracil permease and nucleobase transporter. If this is true, uracil has crucial

role as a component of RNA and has many different derivatives, including UTP (uridine

triphosphate). Interestingly, this uracil derivative is used in carbohydrate metabolism by

acting as a coenzyme in the biosynthesis of critical sugars across many different

organisms. It can also be converted into ATP by donating one of its phosphate groups.

SVCT1 and SVCT2 cellular distribution.

Several groups originally cloned SVCT2 transcripts for the human gene,

approximately 7.5 kb. One clone, which was from human placental trophoblast cell line

cDNA, was identified as the human homolog of the rat SVCT2 (258). The transcripts

were identified in brain, placenta, heart, and liver. The function of SVCT2 was primarily

92 investigated by radio-tracer uptake studies in oocytes because the currents were relatively small, according to the study. Interestingly, the studies did not reveal any functional differences between the SVCT isoforms. The activities of SVCT1 and SVCT2 expressed in oocytes were consistent with those of several mammalian tissues, vesicles, isolated tissues or cultured cell lines (49, 337) (274).

SVCT2 was cloned from the HLE-B3 lens epithelial cell line (151). The open reading frame that encodes the SVCT2 protein is 1953 bp long, yields a 650 amino acid polypeptide having an unglycosylated molecular weight of 64.785kD. SVCT2 is an integral membrane protein with 12 membrane-spanning domains, which functions as a nucleobase transporter, a uracil permease and a vitamin C co-transporter. Using a

Xenopus laevis oocyte expression system, the apparent Km of hSVCT2 for ascorbate was

21.3 micromolar, which is consistent with plasma and tissue distribution of vitamin C

(75).

1.5.3. SVCT2 structural, functional domains and regulation of transport

Having discovered that the mouse and human SVCT2 proteins share ~95% sequence homology, we investigated sequence and structural differences to explain differences underlying the endogenous transport levels. Both proteins share the same regulator and sequence domains (details described in Chapter three). Most notable of these domains are PKC regulatory domains and ERK domains. In that regard, the PKC phosphorylation sites negatively regulate the transporter activity. In studies of SVCT1,

93 confocal microscopy and cell surface biotinylation suggested that there was a

corresponding reduction in the level of hSVCT1 transporter at the membrane surface

consistent with the notion that PKC is regulating the trafficking of hSVCT1 to the plasma

membrane. In contrast, no consistent change in the cell surface distribution of hSVCT2

was observed after Phorbol 12-myristate 13-acetate (PMA) treatment, which suggested

that the decrease in hSVCT2 transport activity results from a decrease in the translocation

capacity of the carrier (181).

The amino acid sequence of hSVCT1, hSVCT2 and mSVCT2 predicts that they

are glycoproteins containing N-linked oligosaccharides and that the human proteins exist in different molecular weight forms, having two potential N-glycosylation sites between the putative transmembrane 3 and 4 domains with an additional site between transmembrane 5 and 6 domains in hSVCT1, when expressed in COS-1 cells (181). The mouse SVCT2 has N-glycosylation sites as well, which will be addressed in chapter three. Interestingly, transfection studies by (181), demonstrated that the hSVCT1 human

protein, when overexpressed was predominately located intracellularly (88% of the total).

However, there are other potentially more significant differences noted between

the two proteins, namely, the prediction of a casein kinase II (CK-2) phosphorylation site

in the mouse by Prosite (cross-reference) which is a protein serine/threonine kinase

whose activity is independent of cyclic nucleotides and calcium. The pattern is found in most of the known physiologic substrates (90) and has the consensus pattern [ST]-x(2)-

[DE] [S or T is the phosphorylation site]. Pinna et al. identified the substrate specificity

94 of this enzyme, which is summarized as follows identified from the website

(http://scansite.mit.edu/cgi-bin/motifscan_seq): 1) Ser is favored over Thr under comparable

conditions, 2) an acidic residue (either Asp or Glu) must be present three residues from

the C-terminal of the phosphate acceptor site, 3) the rate of phosphorylation is increased

when additional acidic residues are found in positions +1, +2, +4 and +5 and most

physiologic substrates have at least one acidic residue in these positions, 4) Asp is preferred to Glu as the provider of acidic determinants, and 5) the occurrence of a basic residue at the N-terminal of the acceptor site decreases the phosphorylation rate, while an acid residue will increase it.

Since vitamin C is lost during physical exertion and under oxidative conditions, a redox-mediated mechanism for regulation of SVCT2 could explain lower levels in rodents. Due to finding in silico calcium binding sites in both proteins, the possibility arose as to whether a functional defect in transport possibly involved calcium binding. If there is a permeable gap junction, in the lens, then ascorbate may be concentrated with out possible efflux via gap junctions. Indeed, micromolar levels of intracellular calcium have been shown to reduced gap junctional permeability in lens cultures (73). Calcium- binding sites may be of considerable importance, though the mechanism may be cell- specific, since studies in pig endothelial cells demonstrated the ionophore A23187 or

ATP stimuli trigger an increase in cytosolic Ca2+ concentration and the release was inhibited by the inositol-selective phospholipase C inhibitor U73122 in pig coronary endothelial cells (PCEC), but pig coronary artery smooth muscle cells do not show the

Ca2+-mediated ASA release pathway (76). It has also been shown that substantial

95 secretion or release of ascorbate can occur in control cells during processing of primary cells, such as those in the above study, reminiscent of ascorbate depletion simply by isolation of tissues and cells, independent of oxidation and may represent leakage of ascorbate as seen with Leydig cells (213) and chromaffin cells, in which calcium stimulates ascorbate secretion (174). The calcium-mediated release of ascorbate led to increased nitric oxide (NO) production as well (46, 51, 54). Of interest here is the finding that ASA can protect NO from forming peroxynitrite, in vitro, but the ascorbate concentrations used in those experiments were 100-times higher than those present in the plasma (148).

Nevertheless, intracellular concentrations are closer to this value and our studies with the mouse and human transporters show both transporters have functional calcium binding domains. One caveat to interpreting comparative overexpression studies is the use of heterologous systems, such as those of mouse protein against background endogenous HEK-293 expression, which may complicate the findings. Indeed, support for this notion comes from very recent findings that SVCT2 expression seems to be regulated via AP-1 and NF-kB signaling (286), where transcriptional regulation of the svct2 gene could be positively or negatively modulated by the presence of oxidant or antioxidant compounds, respectively.

Studies with epidermal keratinocytes by Avigliano et al., involving the fra-1 gene, a Fos family member of transcription factors known to down-regulate activator protein-1

(AP-1) target genes, demonstrate that ascorbate mediates cellular responses aimed at

96 counteracting UV-mediated cell damage and ascorbate modulates AP-1 DNA-binding

activity most likely via Fra-1. Ascorbate inhibited both basal and UV-B-induced AP-1-

dependent transcription and also modulates UV-B-induced AP-1 activity by preventing

the phosphorylation and activation of the upstream c-Jun N- terminal kinase (JNK), thus

inhibiting phosphorylation of the endogenous c-Jun protein (60).

1.5.4. SVCT2 inhibition and secretion studies of ascorbate

Many biochemical and a few biomechanical mechanisms are known to inhibit or

activate uptake of ASA and DHA. Conditions that favor ASA in the literature include:

Dexamethasone, Na+ and PO4-, pH 7.4-7.6, and the bivalent cations Ca2+ and Zn2+.

Conversely, acorbic acid- 2-PO4-, Ni+ and high-density culture conditions decrease ASA

transport. As noted above, ASA uptake can be inhibited by flavanoids in some cells

(244). Included in this exploration of inhibitors are both species of ASA transporter with

attention to GLUTs, since it is not yet clear which tissues express which forms of the

SVCTs either, both or none and the studies illustrate co-transport and uniport modes of

the transport cycle. The SVCT1 and SVCT2 transporters can be regulated in the

following manner using various test compounds including: aspirin (acetylsalicylic acid),

xanthine, sulfinpyrazone and phlorizin, each evoked tiny outward currents in oocytes

expressing SVCT1 and also slightly inhibited SVCT1 or SVCT2-mediated L-14C-

ascorbic acid uptake (340). That is, they exhibited characteristics of weak blockers.

Phloretin evoked a sizeable outward current in oocytes expressing SVCT1 and

97 this effect is attributed to phloretin blocking the Na+ leak current. Phloretin is known to

block the inward current evoked by L-ascorbic acid and inhibit L-[14C]ascorbic acid

uptake (279) by SVCT1 or SVCT2 and which was a non-competitive inhibitor of

SVCT1. Phloretin also inhibited the Na+ leak current with an apparent Ki of 100 mM,

which indicates that phloretin may interact with SVCT1 at a single locus on the protein.

Evoked currents in both SVCT1 and SVCT2 showed a similar pattern of pH sensitivity,

indicating strong pH dependence for function and sensitivity to changes in extracellular

pH. Those at pH 5.5 were 50% smaller than those at pH 7.5 that can be partly restored by the addition of 5 mM excess ascorbate. It is postulated that pH sensitivity is probably a result of reduced binding affinities for L-ascorbic acid, rather than less available L- ascorbic acid in the deprotonated form, as more than 95% is in this form at pH5.5 (298).

Further, transport in UVECs has been shown to be significantly inhibited by TPA and

Phloretin.

Depending on the cell type studied, Phorbol 12-myristate 13-acetate (PMA),

100nM, can either inhibit ascorbate transport by 50% by acting on the protein kinases C sites (95) or activate it by production of superoxide and uptake via GLUTs (167) Protein kinases can alter the activity of transporters directly, by changing the substrate binding affinity or the translocation capacity, or indirectly, by altering the rate at which the carrier is inserted or removed from the plasma membrane. When metalloproteinase is activated, ascorbate can be consumed (95). In human neutrophils, neutrophilic NADPH oxidases can be inhibited by divalent cations and zinc. Free zinc inhibits transport of vitamin C in differentiated HL-60 cells during respiratory burst (167) and HL-60 cells were found to

98 accumulate large quantities of vitamin C (ascorbate) after activation of the NADPH

oxidases by phorbol esters (PMA) by generating superoxide and subsequent oxidation of

ascorbate to dehydroascorbate (DHA), which is preferentially taken up by those cells.

Hormones, such as adrenocorticotrophic hormone (ACTH), which is known to

stimulate ascorbic acid secretion into the adrenal vein and in the adrenal gland ascorbate uptake is blocked by ACTH. Glucocorticoids and prostaglandin (PG) F2 or PGE2 also deplete ascorbic acid in the corpus leuteum of the rat (22, 284). The non-steroidal hormones, glucocorticoids, have been known to block ascorbate uptake in the adrenal,

pituitary, retina, and pancreatic islet cells, albeit at high levels (275, 378) and may be one

mechanism whereby glucocorticoid-induced cataracts form. Tissue levels of ascorbic

acid can be rapidly depleted in response to hormones or simply by incubation. For

example, rapid depletion of ascorbate is induced in the corpus luteum by lutenizing

hormone (LH) and in the adrenal cortex by ACTH, which is the basis for bioassaying

these pituitary hormones. Years ago, inhibition of ascorbate uptake by rat luteal slices by

LH and progesterone was discovered, but uptake was not influenced by LH or

testosterone in rat Leydig cells (213).

ASA transport explored in collagen synthesizing and secreting 3T6 fibroblasts,

was a sodium-dependent and active and saturable process requiring ATP (Km of 112 uM and Vmax of 158 pmol/min/mg protein). Further, studies revealed that a serum- containing a heat-labile factor, when coupled with endotoxin in the reaction, inhibited

99 ASA transport (8). Later it was determined that activation of serum complement (at the

level of C3 or beyond) generates an inhibitor of ascorbate transport (240).

In non-scorbutic animals ASA is synthesized largely in the liver, but the kidney is known as a site of some ascorbate production. Plasma, liver and kidney ASA can be significantly lowered by streptozotocin (STZ) treatment in rats and decreased with increasing duration of diabetes. Hepatic ASA regeneration is decreased in diabetic (Db) rats despite increased gene expression of ASA regenerating enzymes. At the same time urinary excretion of ASA was increased with increasing urinary volume, which seemed to eventually normalize in this study (153). This data suggests that impaired hepatic and renal regeneration as well as increased urinary excretion and impaired hepatic biosynthesis of ASA can contribute to the decreases in plasma and tissues of STZ- induced diabetic rats.

The question remains as to whether the low levels of ASA in rodent and other nocturnal animal lenses is due to inhibition of uptake or efflux mechanisms, particularly via glucose transporters. However, only Glut 3 has been identified to export DHA in neurons, cells that are known to accumulate high quantities of ascorbate (134). Inhibition of transport activity is commonly observed when modulation of the activity of other carriers by PKC has been investigated. In that regard, Na+-glucose cotransporters, a mouse taurine carrier, a human dopamine transporter and SVCTs all exhibited diminished activity upon exposure to PMA, interestingly ascorbate is required during dopamine hydroxylation. Moreover, the regulation of some glucose transporters occurs by kinases

100 as well (139) and in binding associated with the human dopamine transporter (379).

Ascorbate is secreted in vivo by various tissues. In that regard, the nature and

regulation of ascorbic acid uptake and depletion in the rat CL and luteal cells was

investigated by Musicki, who found agents that stimulated ascorbate secretion were LH,

ionomycin, cytochalasin B, and generators of reactive oxygen species (221). While

PGF2α is known to activate protein kinase C and increase intracellular calcium levels in

luteal cells, this mechanism did not appear to be important as a mediator of inhibition of ascorbate uptake, whereas cytochalasin B, an agent that causes cytoskeletal disruption did. Taken together, the findings indicate that the secretion of ascorbic acid induced by

PGF2 alpha, and possibly LH, may be mediated by calcium, reactive oxygen, and the

authors emphasize the importance of intact cytoskeletal elements to prevent ascorbate

secretion, as the mechanism of inhibition of ascorbate uptake by PGF2 appears to be

attributable to disruption of cytoskeletal elements (247). Further, luteal cell ascorbate

uptake was not influenced by the absence or presence of glucose and ascorbate uptake

was unaffected by LH, PGE2, glucose, bromo-cAMP, progesterone, phorbol ester,

ionomycin, hydrogen peroxide (H2O2), or aminoglutethimide. Basal release of ascorbic

acid was energy-dependent, as secretion was blocked by a mitochondrial uncoupler and

lowered temperature.

In addition to issues around ascorbate efflux, activation and inhibition, it is

important to consider the fact cells can transport ascorbate via three distinct mechanisms,

and maybe more. In skeletal muscle, vitamin C not only enhances carnitine biosynthesis

but also protects cells against ROS generation induced by physical exercise. The ability

101 to take up both ascorbic and dehydroascorbic acid from the extracellular environment

recycle ascorbate and, maintain high cellular stores was explored in mouse C2C12 and rat

L6C5 muscle cell lines, which exhibit different sensitivity to oxidative stress and GSH

metabolism (285). This group found that both SVCT2 and SVCT1 was expressed in

L6C5 myoblast cells, but at very low levels only in proliferating L6C5 cells and the L6C5 cells are more efficient in ascorbic acid transport than C2C12 myoblasts. The C2C12 cells are more efficient in dehydroascorbic acid transport and ascorbyl free radical

/dehydroascorbic acid reduction. The myotubes showed increased SVCT2 expression and thioredoxin reductase-mediated dehydroascorbic acid reduction, perhaps due to

responses to oxidative stress, and whose induction was induced by glutathione depletion.

The authors conclude that SVCT2 and NADPH-thioredoxin-dependent DHA reduction is

part of an inducible system activated in response to oxidative stress (285).

The transport in other tissues has been studied as well. Skin is one organ that can

receive a tremendous amount of UV-induced damage. Steiling et al., looked at skin

distribution of SVCT2, the kinetics and the effect of UVB-induced oxidative stress (314).

They identified two sodium-dependent vitamin C transporter isoforms (SVCT1 and

SVCT2) in skin, but their roles had not yet been elucidated. The expression and function

of SVCT1 was primarily found in the epidermis expressed by keratinocytes, whereas

SVCT2 expression was in the epidermis and dermis in keratinocytes, fibroblasts, and

endothelial cells. Interestingly, in keratinocytes, SVCT1 was found to be responsible for

vitamin C transport, although SVCT2 gene expression was higher. On UV-B irradiation,

SVCT1 mRNA expression in murine skin declined significantly in a time- and dose-

102 dependent manner, whereas the SVCT2 mRNA levels did not change. The effect on kerotinocytes by UV-B irradiation was determined to be mediated by SVCT1 and was accompanied by reduced ascorbic acid transport.

The data concluded that in skin, the two vitamin C transporter isoforms fulfill specific functions, namely, SVCT1 is responsible for epidermal ascorbic acid supply, but

SVCT2 was responsible for ASA transport in the dermal compartment. The presence of polarized localization of vitamin C transporters, SVCT1 and SVCT2, in epithelial cells was determined by Boyera et al., who examined the mRNA levels of SVCT1 and

SVCT2, in the cultured intestinal cells suggested which suggested an apical presence of

SVCT1 but the function of SVCT2 was not known. They used enterocytes form heterozygous SVCT2-knockout mice, which had lower sodium-dependent vitamin C accumulation compared to those from the wild type and determined that SVCT2 appears to be functional in enterocytes of mice. Testing for redundant function of SVCTs by constructing and expressing the Enhanced Green Fluorescent Protein (EGFP) EGFP- tagged SVCTs in intestinal Caco-2 and kidney MDCK cells. They found in confluent epithelial cells, SVCT1 protein expressed predominantly on the apical membrane and

SVCT2, was localized to the basolateral surface. The SVCT1 expression was more functional having transport activity from the apical membrane, while SVCT2 expression

only increased the uptake under the conditions where the basolateral membrane was

exposed. This differential epithelial membrane distribution and function suggests non- redundant functions of these two isoforms (50).

103 In the following sections, we undertake step to determine whether the differential uptake of ASA between lens epithelial cells of mice or humans could be explained by a structural or functional defect in those cells. Having determined that the human cells contained the high affinity SVCT2 isoform of the gene, we embarked on experiments aimed and determining mechanisms of suppression, from expression of the protein to post ribosomal and post translational regulation for possible explanations for this suppression.

104 CHAPTER 2

Evidence of suppressed sodium-dependent Vitamin C transport in mouse compared

to human lens epithelial cells

2.1. INTRODUCTION

Human lens crystallins undergo a number of posttranslational modifications by

reducing sugars and oxoaldehydes with age that lead to protein pigmentation and

crosslinking (216). These modifications are thought to predispose lens crystallins toward

formation of high molecular weight aggregates that scatter light, i.e. cataractogenesis.

Evidence suggests some of these modifications may originate from ascorbic acid

degradation products comes from Ortwerth’s group when examining the similarity of the yellow chromophores isolated from human cataracts with those from ascorbic acid-

modified calf lens and other early work (36, 226). Ascorbic acid in reduced form is not a

glycation agent, but becomes highly reactive with carbonyl agents in its oxidized form,

i.e. dehydroascorbic (DHA) and 2,3-diketogulonic acid (2,3-DKG), i.e. the spontaneous

delactonization product of DHA (226). Among proposed degradation products are

xylosone, 3-deoxy-xylosone, L-threose, L-threosone and L-erythrulose (307). The latter

compound is thought to be a major product of DHA forming under anaerobic conditions.

In order to investigate the biochemical nature of the ascorbic acid degradation

products and how they might be responsible for lens crystallin pigmentation in diabetes

and aging, metabolomic studies were initiated with 6-fluoro-6-deoxy-ascorbic acid (F-

105 ASA) as an 19F-Nuclear Magnetic Resonance Spectroscopy (NMR) tracer for the in vivo

assessment of ascorbic acid degradation products (199, 283). These studies revealed that glycemic stress suppresses the uptake of F-ASA into the human lens epithelial cell line

HLE B-3 and catalyzes the degradation of F-ASA degradation compounds (230, 283).

They also showed that glutathione depletion strongly enhanced F-DHA and F-DKG formation in HLE B-3 cells. Overall, similar observations were made in lenses from diabetic rats treated with F-ASA. These studies also revealed that both F-ASA and F-

DHA were specifically transported into the human cells via a sodium dependent uptake mechanism, most likely the SVCT2 transporter (151). While native DHA is taken up by glucose transporters, namely GLUT1 or GLUT3 (278) the inability of F-DHA to form the bicyclic structure of native DHA likely explained why it was also taken up by the same mechanism as F-ASA.

In the course of testing the specificity of the above findings to other rodent cell lines, and with the ultimate purpose in mind of developing transgenic mice expressing high levels of ascorbic acid in the lens, we made the surprising discovery that ASA uptake was severely impaired in lens epithelial cells of rodents when compared to those of human origin. These data are presented, including the feasibility of overcoming the uptake defect by overexpression of the human SVCT2 transporter in cell lines transfected

with appropriate plasmids.

2.2. METHODS

106 2.2.1. Materials

Most reagents were from Sigma (St. Louis, MO) and of the highest grade available. 6-deoxy-6-fluoro-L-ascorbic acid (F-ASA) was synthesized as described (199).

6-deoxy-6-fluoro-L-dehydroascorbic acid (F-DHA) was freshly prepared from F-ASA by bromination on ice as previously described. All phosphate buffers were treated with

Chelex-100 resin overnight in order to remove heavy metal ions. Dithiothreitol (0.1 mM) or N-acetyl cysteine (20 mM) were added to prevent ASA oxidation. Cell transfections were performed using lipofectamine (Invitrogen, Carlsbad California). Culture reagents were from Sigma (St. Louis, MO), Geneticin selection reagent (Gibco, Grand Island New

York). All other media products were from Mediatech (Herndon, VA) unless otherwise specified.

Cell culture

Human lens epithelial cells, HLE-B3, a gift from Dr. Usha Andley, were cultured according to protocol previously described (11). The cultures were maintained at 37ºC in humidified air containing 5% CO2. Medium was changed twice per week, unless otherwise noted. For subculture, cells were washed and detached with 0.25% trypsin/EDTA (Mediatech). Standard lab protocols, for periodic monitoring of cells were utilized. JAR cells (ATCC# HTB-144), a human choriocarcinoma cell line from placenta, were cultured in RPMI1640 medium (Mediatech) supplemented with heat inactivated 10 % FBS, 2mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 100

107 U/ml penicillin and 100 µg/ml streptomycin. The 17EM15 and 21EM15 mouse lens epithelial cell lines (264), were cultured in Dulbecco’s modified Eagle’s medium

(DMEM, Mediatech) supplemented with heat-inactivated 10 % FBS, 2mM L-glutamine,

100 U/ml penicillin and 100 µg/ml streptomycin. The experiments with HLE B3 or

Mouse LE and JAR cells were performed at passage up to 22-23 and passage 44-46 for rabbit LE. The previously described mouse and rabbit lens epithelial cell lines (264) were cultured in a standardized medium composed of cultured in Dulbecco’s modified

Eagle’s medium (DMEM, Mediatech), containing 4.5 g/L D-Glucose, supplemented with

15 % heat-inactivated FBS, 2mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. For uptake experiments, HLE-B3 cells were cultured for 6 days after seeding at density of 1x105 cells per cm2. The medium was changed every 2 days. For hyperglycemic conditions, cells were cultured with medium containing 50 mM D-glucose or 50 mM D-galactose for 6 days. To explore the role of Na+ in uptake of F-ASA and F-

DHA by HLE-B3 Cells uptake experiments were performed in Krebs buffer containing a total of 135 mM Na+ concentration or sodium-free conditions, where sodium was replaced with equimolar potassium chloride and the pH adjusted to 7.4 with KOH. The uptake experiments were started by exchanging the medium with buffer or media containing 1 mM F-ASA or 1 mM F-DHA and 20% FBS/EMEM.

2.2.3. Lens explant culture

Lenses were obtained from 10, 6 month-old, C57Bl/6 male mice, weighing about

30 g each. The animals were euthanized by CO2 narcosis in accordance with IACUC,

IOVS and ARVO rules governing the ethical and appropriate treatment of experimental

108 animals. The eyes were extracted under sterile conditions and the 20 lenses were

carefully removed and immediately transferred to sterile pre-warmed TC-199 media

(Sigma). After standard transfection protocol, the lenses with intact epithelia were removed from the eye and incubated in fresh culture media in 12-well culture plates based on the method of Spector (311). Unless otherwise mentioned, each well contained

1 ml of medium 199 (TC-199) containing Earle's salts (without phenol red) (Sigma No.

M3769) supplemented with HEPES 25 mM pH 6.8, glutamine 100 mg l-1 and 0.9 g/ml

NaHCO3. The final medium had a pH of 7.3 and an osmolarity of ~300. Lens cultures were maintained at 37 °C in a water-jacketed humidified incubator with an atmosphere of

95% air/5% CO2. Streptomycin (200 U ml-1) and penicillin (200 U ml-1) were added to

prevent bacterial contamination. All media preparations were sterile filtered through a

0.22 µm filter (Gelman Sciences, Ann Arbor, MI, U.S.A.). Only plastic or Teflon-coated

instruments were used to handle lenses as metal instruments could cause damage.

2.2.4. Measurement of ascorbic acid transport

Transport assays were performed in either transport buffer with sodium (in mM:

140 NaCl, 4.2 NaHCO3, 5 KCl, 1.3 CaCl2, 0.5 MgCl2, 0.36 NaHPO4, 0.44 NaH2PO4 and

10 HEPES, pH 7.4) or without sodium (in mM: 4.2 KHCO3, 145 KCl, 1.3 CaCl2, 0.5

MgCl2, 0.36 K2HPO4, 0.44 KH2PO4 and 10 HEPES, pH 7.4) at 37°C, as described by

Liang (181). Each experiment was performed in triplicate or with more replicates in the

case of lens explants. Briefly, each well was washed with 1 ml of transport assay buffer

after aspiration of the culture medium. The uptake reactions began with the addition 0.5 ml buffer containing L-14C ascorbic acid (17.0 mCi/mmol; Amersham Pharmacia

109 Biotech) final concentration 100 µM ASA (specific activity 3.06 mCi/ mol) in 12-well

plates. The addition of 0.1 mM dithiothreitol or 10mM N-acetyl cysteine was made to

the transport buffer to prevent the oxidation of L-ascorbic acid and the buffer was added

to the cell monolayer and uptake was determined at 37°C under standard culture

conditions for up to 2 hours. The buffer was aspirated, and the cell monolayer

immediately washed with 1 ml of ice-cold buffer two times. The washing procedure took

less than 10 seconds for each well. Representative wells were used to enumerate cell

number either by hand or with a Coulter counter and the remaining cells were then lysed

by addition of 0.5 ml of 1 N NaOH and 1% triton X-100, and the amount of L-14C

ascorbic acid was quantified by liquid scintillation spectrometry and expressed per 106 cells (see method below).

2.2.5. Determination of 14C-ASA and 14C-DHA cellular uptake

For uptake experiments, HLE-B3, 17EM15, 21EM15, N/N 1003A (264) or

transfected cells were cultured for 6 days after seeding at density of 1x105 per cm2. The

medium was changed every 2 days. For hyperglycemic conditions, cells were cultured

with medium containing 25 mM D-glucose or 5.5 mM D-glucose for euglycemic

conditions. 14C-ASA uptake was studied by exchanging the medium for 100 µM 14C-

ASA (specific activity 3.06 mCi/ mol) in either transport buffer with or without sodium or in Krebs buffer. After 2 hours, the cells were collected by trypsinization and counted, then lysed with 150 µl of 1M sodium hydroxide in 1% triton X-100 at room temperature for 10 minutes and 14C-ASA was counted by scintillation spectrometry in a scintillation

110 counter (Beckman Coulter, Fullerton, California).

19F-NMR spectroscopy For NMR experiments Fourier transformed 19F 750 MHz

NMR spectra were obtained as previously described (283). The addition of 100 µM 6-

deoxy-6-fluoro-D-glucose in D2O immediately before the measurement served as an internal standard for calibration and quantitation. At the indicated time, the cells were collected by trypsinization and homogenized with a Kontes pellet pestle (Kontes Glass,

Vineland, NJ). The lysate was mixed on ice with a final concentration of 4% metaphosphoric acid/PBS(-) containing 19F-D-glucose. The supernatants of the turbid solutions were obtained by centrifugation at 15,000 rpm for 20 minutes at 4°C. The supernatants were stored at –80°C until assayed by NMR.

111 Figure 2.1. F-ASA and F-DHA are both taken up into cells by a sodium-dependent mechanism.

All uptake experiments included samples from the media, which confirmed the

stability 19F-ASA over the duration of the experiment and indicated that the compound

taken up was in the reduced form Figure 2.1. All uptake experiments included samples

from the media, which confirmed the stability 19F-ASA over the duration of the

experiment and indicated that the compound taken up was in the reduced form Figure 2.1.

2.2.6. Inhibition of glutathione synthesis

To inhibit glutathione synthesis, the cells were treated with 100 mM of L- buthionine-[S,R]-sulfoximine overnight, i.e. at a concentration that did not result in cytotoxicity as determined by the MTT assay ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, Roche, Mannheim, Germany) (data not presented). Intracellular

GSH levels were determined with a glutathione assay kit (Calbiochem, San Diego, CA)

according to the manufacture’s instructions. Briefly, the cells (4 x 106 cells) were

collected by trypsinization and homogenized in 500 µl of 5% metaphosphoric acid with a

Kontes pellet pestle. The acid-precipitated protein was pelleted by centrifugation at

3,000 x g for 10 minutes at 4°C. The supernatant was used for measurement of glutathione content.

2.2.7. Expression constructs and cellular transfection methods

112 Human SVCT2 (hSVCT2) cDNA in pcDNA3.1 topo/His/V5 vector was previously described by Liang et al. (181). The hSVCT2 cDNA sequences were amplified by PCR using the sense and antisense primers which contain SmaI and XbaI recognition sites (underlined sequences) respectively to facilitate cloning: (sense)

5'GCGCCCCGGGATGATGGGTATTGGTAAGAA3' and (antisense)

5'GCGCTCTAGACTATCCCGTGGCCTGGGAGT 3'. The PCR fragment was digested with SmaI and XbaI and inserted into the SmaI-XbaI digested lens-specific denaA promoter vector provided by Dr. Lixing W. Reneker (268). The hSVCT2 coding sequences in the resulting plasmid, named denaA-hSVCT2, were confirmed by DNA sequencing analysis.

In order to rescue the suppression of ascorbic acid transport in mouse epithelial cells, 17EM15 cells or lens explants were transfected either with hSVCT2 in pcDNA3.1, empty vector (Invitrogen, Carlsbad, California), or co-transfected with either denaA- hSVCT2 and pEGFP-N1 (Clontech, Palo Alto, California), or mock transfected with pEGFP-N1, using lipofectamine reagent (Invitrogen, Carlsbad, California). Briefly, cells were grown to 90-95% confluence in 12-well Falcon culture plates (Becton Dickinson,

Franklin Lakes, New Jersey) and rinsed with serum free Opti-MEM media without antibiotics (Invitrogen, Carlsbad, California). Lipofectamine reagent, 4 µl per 100 µl

Opti-MEM and 1.6 µg plasmid DNA per 100 µl Opti-MEM, was prepared and gently mixed after 5 minutes incubation at room temperature and directly added to each well and cultured for 3 hours at 37C. Fresh serum-containing media was added directly to each well and cells were cultured overnight. For stable transfection, cells were selected with

113 Geneticin (Life Technologies, Carlsbad California) at a concentration of 800 µg/ml for up

to one week. For transient transfection experiments, cells were transfected as described

and cultured in DMEM with 15% FBS and 5.5 mM D-glucose and 10mM N-acetyl

cysteine for 24 hours prior to transport assays. Uptake experiments were performed

directly in 12-well plates having given sufficient time for expression to take place.

2.2.8. Transient transfection of lens explants

To confirm the feasibility of overexpressing hSVCT2 in native lens epithelium, whole mouse lenses from animals six months in age were transiently transfected according to the same protocol used in cell culture experiments. Briefly, the lenses were

removed from the eyes of ten animals and were equally divided into two groups. The first

group of lenses was co-transfected as previously described with the denaA-hSVCT2

construct and pEGFP-N1 (n=6) and the second group was mock transfected with pEGF-

N1 (n=9), which served as a control. Transfections were performed in serum-free Opti-

MEM without antibiotics for 3 hours, after which the medium was exchanged for fresh,

sterile TC 199 medium (Gibco, Grand Island New York). The lenses were cultured for 18

hours in TC Medium 199, supplemented with antibiotics and 10mM N-acetyl cysteine.

Damaged lenses were found to rapidly lose transparency and were excluded from the

experiments. Lens viability was also checked by assessing protein leakage in incubation

media after 1 hr of incubation as described by Tumminia (341). After the lenses had

sufficient time to recover and the success of the lens harvest was determined, viable non-

opaque lenses from each group were transferred to fresh Krebs ringer buffer (Sigma) and

114 14C-ASA (specific activity 3.06 mCi/mmol), where uptake was performed for 2 hours.

Once the transport assay was performed, lenses were quickly rinsed in Krebs buffer 4

times and transferred to individual scintillation vials, where they were crushed in 500 µl distilled water and filled with 4 ml of scintillation cocktail. 14C-ASA dpm was determined

in individual lenses using a Beckman LS 6000SC scintillation counter.

2.2.9. Statistical Methods

Statistical significance was assessed by analysis of variance (ANOVA) using

Excel software. Values were expressed as mean ± S.D.

2.3. RESULTS

2.3.1. Comparative effects of glycemic stress on F-ASA and F-DHA uptake, degradation

and GSH homeostasis in HLE B-3, 17EM15 and JAR cells

Using F-ASA as an SVCT2 probe, the data in Figure 2.3. show that F-ASA

uptake was ~ 10 fold lower in the mouse Emory lens epithelial cell (17EM15) cells

compared to the human lens epithelial cell (HLE B-3) and the chorionicarcinoma (JAR)

cells, both of which are of human origin are known to transport vitamin. There was a

moderate inhibition of F-ASA uptake when the HLE B-3 cells were exposed to high

glucose or galactose levels, or when treated with BSO (p < 0.01).

115

Figure 2.2. F-ASA and F-DHA uptake is via SVCT2 and no other mechanism.

Surprisingly, the JAR cell line was completely resistant to these manipulations, implying thereby a strong ability of such cancer cells to resist oxidant stress. Indeed, since we hypothesized this as a mechanism for cancer cells to evade or resist their oxidative environment, it was shown that ASA can suppress superoxide-mediated chemotherapeutic mechanisms (365), which would be once such instance where mass doses of ascorbate may be contra-indicated due to drug interaction and interference effects with chemotherapeutic agents utilizing this mechanism. In mouse cells, glycemic stress or BSO did not further depress the already very low ascorbate levels, thereby, implying the existence of a “safety” mechanism to preserve a minimal concentration of ascorbic acid for cell survival.

116 Interestingly, glutathione levels were overall quite resistant to glycemic stress in

the HLE B-3 and JAR cells, but decreased by 50% both in the buthionine sulfoximine

(BSO)-treated and glucose stressed mouse cells (Table 2.1.), possibly reflecting the quasi

absence of the free radical scavenging ability of ascorbic acid.

Cellular glutathione concentration (nmol/106 cells)

Stressor HLE B-3 17EM15 JAR

Control 20.83 ± 0.08 18.70 ± 2.31 17.46 ± 1.34 50 mM 19.32 ± 0.09 9.02 ± 2.03** 23.33 ± 4.70 D-galactose 50 mM 15.64 ± 0.46 9.47 ± 0.85** 21.14 ± 3.06 D-glucose 100 µM BSO 1.30 ± 0.01* 9.86 ± 0.35** 7.05 ± 0.59*

Table 2.1. Intracellular glutathione levels under glycemic stress and BSO treatment. JAR, 17EM15 and HLE-B3cells were treated with 50 mM D-galactose or D-glucose for 6 days, or with 100 µM BSO for 24 hours. Data represent means ±S.D, n=3.The increase in GSH concentration in the medium of glucose treated cells reflects increased leakage, while its marked decrease in BSO treated cells suggests dramatic oxidation, both intracellularly and in the medium. Statistical significance between control and each treatment was calculated using ANOVA (*, p<0.01), (**, p<0.05).

This was accompanied by the appearance of a compound of yet unknown

structure at –212.4 ppm, (Figure 2.3.), which appears to be a marker of GSH-dependent

F-ASA oxidation. As previously reported, this compound does not form from F-DHA,

and is therefore not observed when F-DHA uptake is studied. Most importantly, this level

of GSH was sufficient to regenerate F-ASA from F-DHA to the same level in sugar stressed cells, albeit to very low levels compared to the human cells lines. Thus, lower levels of GSH might be fully adequate to regenerate low levels of ascorbic acid. In

117 support of this notion, the absence of a detectable increase in F-DHA and F-diketogulonic acid (F-DKG) in the mouse cells is also likely related to the low baseline levels of F-

ASA, since both F-DKG and F-DHA are observed in the human cells that contain much higher levels of ascorbic acid.

F-ASA F-DKG F-DHA F-Unknown 6-F-6-DeoxyGlucose

-211.9 ppm -213.8 ppm -216.6ppm –212.14 ppm -218.4 ppm

Table 2.2. Chemical shift of fluorocompounds of F-ASA, oxidation and degradation products.

118

Figure 2.3. Comparative effect of lens epithelial cell type and glycemic stress on intracellular concentrations of F-ASA and formation of F-ascorbic acid degradation products upon incubation with freshly prepared 1 mM F-ascorbic acid (left panel) or 1 mM F-dehydroascorbic acid (right panel) for 24 hours and after 6 days of culture in 50 mM D-galactose or D-glucose or 100µM BSO for 24 hours. A,D: HLE-B3 cells ; B,E : 17EM15 cells ; C,F: JAR cells. F-ASA concentrations are highly significantly suppressed in 17EM15 compared to HLE B-3 cells (p < 0.001 for all comparisons between human and mouse cells) Legend: Unknown peak –212.14, F-ASA =-211.9ppm, F-DKG=- 213.8ppm, F-DHA=-216.6ppm, Internal standard=-218.4ppm.

2.3.2. Confirmation that ascorbic acid uptake is suppressed in mouse LE cells

The above experiments were performed with fluorine-labeled ascorbic acid,

which allegedly can only be taken up by SCVT2 and not other transporters, such as the

glucose transporters GLUT1 or GLUT3 (277, 278). Thus, it could be argued that deficiency in SCVT2 transport could be compensated for by upregulation of GLUTs.

This led us to repeat and expand the above experiments with 14C-labeled (“native”) ASA

that also can be taken up in the form of 14C-DHA. In addition, we tested other mouse

cells, such as the 21EM15 cell line, to ensure that the observed differences were not an

idiosyncrasy of the transformation. Further, as we became aware that the mouse cell

culture medium contained much higher levels of glucose than that used for the human

HLE B-3 cell line, i.e. 25mM instead of 5.5mMm, we decided to test different culture conditions to ensure the reproducibility of the data. For the data in Figure 2.4., the human and mice (Emory Mouse lens epithelial) cell lines were cultured in their usual medium containing 5.5 and 25 mM glucose, respectively. This was necessary, as the

119 mouse cells would not grow at low (5.5 mM) glucose concentration. However, the HLE

B-3 cell line grew perfectly well under the higher glucose conditions. In contrast, the

actual uptake experiments were performed under identical conditions, i.e. at 5.5 mM

glucose concentration, which also was present in either “transport” or Krebs buffer.

The data show that under all experimental conditions, total 14C-ASA uptake was suppressed in the mouse 17EM15 and 21EM15 cells compared to the human cell lines

(p<0.0001) (Figure 2.4 A). Furthermore, the uptake was further suppressed by high glucose. Moreover, Figure 2.4. A shows that whatever the experimental conditions in the transport buffer, i.e. presence or absence of high glucose, or when the buffer is switched from Krebs to Ringer’s buffer (data not presented), uptake into HLE B-3 cells was again several fold higher than into the 17EM15 cells.

In figure 2.4. B, in which the cells were cultured in a sodium-free medium in the presence of a low glucose concentration (i.e. 5.5 mM), shows that the percent of total

14C-ASA uptake that was Na+-dependent was ~36%, 63% and 52% for HLE B-3,

17EM15 and 21EM15 cells respectively. This suggests that about 60% of ASA uptake

into mouse LECs is sodium-dependent, as indicated by results obtained with the sodium-

dependent vitamin C transporter (SVCT2), while it is only 40% in the human cells.

SVCT2 activity was reduced by one half, in HLE B-3 and 21EM15 cells when they were cultured in high glucose (i.e. 25 mM). No such effect was observed in the 17EM15 cells,

in line with the data in Figure 2.4. B, which shows little suppression by high glucose

levels. In summary, the data so far confirm the overall suppression of ascorbic acid

120 uptake in mouse vs. human epithelial cells and show that elevated glucose concentration

decreases the uptake by ~50%.

A Effect of Glucose Concentration

HLE B-3 17EM15 21EM15 600

500

400

300

200

100 14C ASA pmol/E^6 Cells

0 Low Glucose High Glucose

B 14C-ASA Na+-dependent Uptake

100

50 Percent of Total

0 HLE B-3 17EM15 21EM15 5.5 mM Glucose 25 mM Glucose

Figure 2.4. A.) Effect of high (25 mM) vs. low (5.5 mM) glucose concentration on 14C- ASA uptake into mouse 17EM15, 21EM15 and human HLE-B3 cells. Figure 2.4. B.) Percentage of sodium dependent 14C-ASA uptake determined in regular “transport” buffer vs. sodium-free buffer under conditions of high or low glucose.(Each represent means ± S.D. of triplicate experiments).

121

2.3.3. Effect of overexpression of the human SVCT2 transporter into mouse 17EM15

cells

The data obtained with F-ASA suggest that a large part of the uptake was SVCT2- dependent. In contrast, the data with 14C-ASA in absence of Na+ showed that only about

one half of total uptake of native ASA was SVCT2-dependent. Irrespective of these

results, the question emerged as to whether the uptake defect into mouse cells could be overcome by overexpression of the human SVCT2 transporter. This is an important question for our ultimate goal of creating a humanized mouse model of lens protein aging

based on ascorbylation reactions. As a preamble toward that goal, two constructs were

prepared consisting of hSVCT2 packaged into a potent CMV promoter driven plasmid

(182), and a plasmid expressing the same hSVCT2 sequence driven by a lens-specific

denaA promoter (denaA-hSVCT2) (268). These constructs were used to transfect

17EM15 cells and whole lens explants.

To exclude the possibility of an idiosyncratic phenomenon with the 17EM15 cell

line, we compared 14C-ASA uptake in the transfected mouse 17EM15 cells to other

rodent cells, i.e. the 21EM15 mouse lens epithelial cell line, the N/N3000A rabbit lens

epithelial cell and the non-transfected 17EM15 cell line (Fig. 2.5). When compared to

other rodent cells, approximately a two-fold increase in 14C-ASA uptake was observed at

2 hours with the pcDNA 3.1construct. The percentage of Na+-dependent uptake, relative

to total uptake (SVCT2), increased from 60 to 80% (not shown).

122

Figure 2.5. Comparative effect of 14C-ASA uptake into 17EM15 mouse cells transfected with pcDNA/hSVCT2 plasmid vs. mock transfection, and uptake into mouse 21EM15 and rabbit N/N 1003A cells at 2 hours. Comparative effect of 14C-ASA uptake into 17EM15 mouse cells transfected with pcDNA/hSVCT2 plasmid (x=186.62±25.14) vs. mock transfection(x=36.86±12.31), and uptake into mouse 21EM15 (x=44.41±13.84) and rabbit N/N 1003A cells (x=63.76±12.36) at 2 hours. (n=3 for all figures).

The above data, while confirming the ability of hSVCT2 to increase 14C-ASA

uptake by the sodium-dependent mechanism, revealed a relative minor effect of SVCT2.

For this reason, we also transfected the cells with a lens specific enhancer-promoter construct consisting of the chick lens delta crystallin enhancer fused to the mouse α- crystallin promoter. GFP was included as a reporter and in the mock transfection. The data revealed a 4-5 fold increased uptake that was almost entirely suppressed by removing Na+ from the medium (Figure 2.6.).

123

Figure 2.6. Effect of transfection of the chick lens delta crystallin enhancer mouse alpha crystallin promoter construct denaA-hSVCT2 co-transfected with GFP reporter construct on 14C-ASA uptake by 17EM15 mouse lens epithelial cells in presence or absence of sodium. The uptake into the transfected cells with sodium (x=122.48±10.71) was significantly higher than that in mock transfected cells (x=29.52±6.01) or in cells transfected with denaA-hSVCT2 cultured in the absence of Na+ (x=5.35±0.38) at (p<0.0001).

2.3.4. Effect of overexpression of the human SVCT2 transporter into cultured mouse

lenses

One caveat to working with cell lines is that they are not necessarily equivalent to

primary cells. Further, mouse primary lens cells are very difficult to culture outside of the

lens capsule and, to date, only few have been able to demonstrate primary cells amenable

to proliferation without the lens capsule itself (196, 287). Therefore, we have attempted to transfect whole lens explants (Figure 2.7.).

124

Figure 2.7. Effect of 24 hr transfection of cultured whole mouse lens explants with denaA-hSVCT2 construct co-transfected with GFP reporter (n=7) (x=58.38±7.5) on 14C- ASA uptake. The effect is significantly higher in the presence of the denaA-hSVCT2 transporter at than mock-transfected cells (n=10) (x=40.18±13.29) (p<0.001).

Here, 20 lenses were excised from 10 six month-old animals under sterile

conditions and immediately transferred to sterile pre-warmed TC-199 media. The lenses

with intact epithelia were removed from the eye and incubated in fresh culture media in

12-well culture plates for one hour prior to any transfection experiments. Damaged lenses, which quickly turned opaque, were discarded. Transfection of denaA-hSVCT2 construct alone or together with a GFP was carried out with lipofectamine reagent for 4 hours and allowed to express the transfected protein for 18 hours in TC-199 media.

Viable lenses were subject to a two-hour 14C-ASA uptake assay, which demonstrated a

33% correction in impaired uptake in the lens explant through transient transfection.

2.4. DISCUSSION

125 Landmark studies by Varma and colleagues showed that vitamin C is a potent

anticataract agent in experimental animal models (137, 349, 350) and is likely to be a first

line of defense against UV light mediated phototoxicity to the lens in species exposed to

daylight. The discovery that ascorbic acid concentrations in rodent lenses are close to

zero to 100 uM (133, 348) is presumably linked to the nocturnal behavior of rodents. The

absence of UV light and photooxidative stress in nocturnal animals along with low levels

of ocular ASA, led us to hypothesize that there may be evolutionary suppression of some

form of vitamin C uptake mechanism (350). Further, ascorbate in the guinea pig lens

seemed to show a dependence on drinking water supplementation and were suggestive of

a passive transport process in rodents (211). However, our data suggest that the high

affinity sodium dependent transport mechanism of lens epithelial cells is reduced

approximately ten fold in mouse compared to the human lens, in which ascorbic acid

concentration is in the 1-3 mM range (37, 347).

The ASA uptake suppression into mouse cells could be improved by transient

transfection with the human transporter, hSVCT2. Throughout these experiments,

however, we observed that experimental conditions needed to be controlled much more

rigorously when working with the rodent epithelial cells and 14C-ascorbic acid than when

working with F-ASA and other cells. Also, we found the ability of the pcDNA 3.1CMV-

driven hSVCT2 construct to upregulate ASA transport was relatively modest in mouse

LE cells compared to Chinese Hamster Ovary (CHO) cells (not shown) or the lens

specific denaA-SVCT2 promoter enhancer construct. The composition of the medium,

126 the Na+ concentration, the passage number of the cells and degree of confluence all

tended to influence the outcome of the data.

Several biological factors may explain experimental variations in ascorbic acid uptake in cultured lens epithelial cells. First, in human HLE B-3 cells, svct2 gene expression has been found to be upregulated by the chemical oxidant tert- butylhydroperoxide (TBH) treatment, which causes an up-regulation of svct2 gene expression and transport of ASA as compared with untreated controls (38, 151). The use

of dithiothreitol or N-acetyl cysteine in the transport buffer seemed to diminish transient

variable increases in ascorbic acid uptake due to chemical stress. While this possibility

may explain experimental variations in ASA uptake, it is unlikely to explain an apparent

constitutive defect.

Other conditions that affect ASA uptake are variations in glucose concentration,

as reported in this manuscript. Here, a key mechanism might involve high glucose

mediated down-regulation of SVCT activity by protein kinase C (PKC) mediated

phosphorylation (79, 162). Such a mechanism could explain suppressed ASA uptake in

the diabetic state that is associated with activation of PKC (162). In that regard, both

human and mouse species have putative PKC-dependent phosphorylation sites according

to the consensus pattern (ST)-x-(RK), which have been linked to suppression of activity

if phosphorylated (78, 151). In support of such a mechanism, we found high glucose

levels in HLE B-3 and 21EM15 cells suppressed the sodium-dependent 14C uptake,

although the effect was not apparent in 17EM15 cells. However, several other ascorbic

127 acid transporters have been described, which makes the interpretation of the data more

complex in any experiment performed with 14C-ASA, as GLUT 1 and 3 sodium-

independent transporters can transport DHA. Further, the existence in humans of a

dominant negative form of SVCT2 was discovered, which inhibited transport of ascorbic

acid (198).

In silico data demonstrates that both the human and mouse SVCT2 transporters

have multiple potential PKC phosphorylation sites, with the human having one more

potential site than the mouse. However, the mouse has numerous other phosphorylation-

competent sites, with largely unknown regulatory effects. How a chronic PKC-mediated

hyperphosphorylation could explain a constitutive mechanism of SVCT2 suppression in

mouse LE cells would be difficult to conceive. To our knowledge, the only mechanism

of chronic activation in the literature involves increases in PKC directly due to

Diacylglycerol (DAG), Inositol-3-Phosphate (IP3), glucose or lipids. Nonetheless, in

vivo, the Vmax and Km of the phosphorylation reaction is enhanced by the presence of

additional basic residues at either the N- or C-terminal of the target amino acid. In fact,

the mouse has a mutation in the first PKC phosphorylation site at the N-terminus. Also,

natural compounds, such as retinol, vitamin E and vitamin C itself, are important

regulators of PKC activity and show an ability to modulate PKC activation (55, 59).

Pseudophosphorylation, i.e., mutations at serine or threonine sites into glutamic acid in the mouse SVCT2 message could be another mechanism. However, while the comparative alignment of mouse and human SVCT2 reveals no such mutation, three new

128 serine residues appear in the mouse protein at sites 30, 338, 589 and 592 using the human sequence as a reference (Figure 2.8.).

Human MMGIGKNTTSKSMEAGSSTEGKYEDEAKHPAFFTLPVVINGGATSSGEQDNEDTELMAIY 60 Mouse -MGIGKNTASKSVEAGGSTEGKYEEEAKHSNFFTLPVVINGGATSSGEQDNEDTELMAIY 59 Human TTENGIAEKSSLAETLDSTGSLDPQRSDMIYTIEDVPPWYLCIFLGLQHYLTCFSGTIAV 120 Mouse TTENGIAEKSSLAETLDSTGSLDPQRSDMIYTIEDVPPWYLCIFLGLQHYLTCFSGTIAV 119 Human PFLLADAMCVGYDQWATSQLIGTIFFCVGITTLLQTTFGCRLPLFQASAFAFLAPARAIL 180 Mouse PFLLADAMCVGDDQWATSQLIGTIFFCVGITTLLQTTFGCRLPLFQASAFAFLAPARAIL 179 Human SLDKWKCNTTDVSVANGTAELLHTEHIWYPRIREIQGAIIMSSLIEVVIGLLGLPGALLK 240 Mouse SLDKWKCNTTEITVANGTAELL--EHIWHPRIQEIQGAIIMSSLIEVVIGLLGLPGALLR 237

Human YIGPLTITPTVALIGLSGFQAAGERAGKHWGIAMLTIFLVLLFSQYARNVKFPLPIYKSK 300 Mouse YIGPLTITPTVALIGLSGFQAAGERAGKHWGIAMLTIFLVLLFSQYARNVKFPLPIYKSK 297 Human KGWTAYKLQLFKMFPIILAILVSWLLCFIFTVTDVFPPDSTKYGFYARTDARQGVLLVAP 360 Mouse KGWTAYKFQLFKMFPIILAILVSWLLCFIFTVTDVFPSNSTDYGYYARTDARKGVLLVAP 357 Human WFKVPYPFQWGLPTVSAAGVIGMLSAVVASIIESIGDYYACARLSCAPPPPIHAINRGIF 420 Mouse WFKVPYPFQWGMPTVSAAGVIGMLSAVVASIIESIGDYYACARLSCAPPPPIHAINRGIF 417 Human VEGLSCVLDGIFGTGNGSTSSSPNIGVLGITKVGSRRVIQCGAALMLALGMIGKFSALFA 480 Mouse VEGLSCVLDGIFGTGNGSTSSSPNIGVLGITKVGSRRVIQYGAALMLGLGMVGKFSALFA 477 Human SLPDPVLGALFCTLFGMITAVGLSNLQFIDLNSSRNLFVLGFSIFFGLVLPSYLRQNPLV 540 Mouse SLPDPVLGALFCTLFGMITAVGLSNLQFIDLNSSRNLFVLGFSIFFGLVLPSYLRQNPLV 537 Human TGITGIDQVLNVLLTTAMFVGGCVAFILDNTIPGTPEERGIRKWKKGVGKGNKSLDGMES 600 Mouse TGITGIDQILNVLLTTAMFVGGCVAFILDNTIPGTPEERGIKKWKKGVSKGSKSLDGMES 597 Human YNLPFGMNIIKKYRCFSYLPISPTFVGYTWKGLRKSDNSRSSDEDSQATG 650 Mouse YNLPFGMNIIKKYRCFSYLPISPTFAGYTWKGFGKSENSRSSDKDSQATV 647

Figure 2.8. Sequence alignment of mouse vs. human SVCT2 reveals >95% sequence identity. The mutations between sequences that might affect the affinity of the mouse compared to the human transporter are highlighted.

Approximately 25 other mutations could affect mouse SVCT2 activity. However, there is, so far, no evidence that rodent SVCT2 in tissues other than the lens is severely suppressed, except for a gender effects in spleen of male mice in wild-type Slc23a2 mice only(164). That is not to say that in rodent tissues the ascorbic acid concentration do not vary, indeed, it is the corneal epithelium, which is known to have the highest concentration of ASA (11mM) (251). Ascorbate concentrations are high in tissues surrounding the aqueous humor, next to the adrenal gland. Thus, an explanation must be sought that is associated with a selective loss of activity in the lens. Preliminary studies with qPCR (not shown) indicate that an SCVT2 transcript can be amplified from

17EM15 cells with little quantitative difference being found, which raises the question of

129 how the transcript would be linked to the impaired ASA uptake. Data from Lutsenko et al.(198) identified an alternative spliced molecular isoform of SVCT2, which may play a role in the suppression of vitamin C uptake into rodent lenses.

The observation that vitamin C uptake is suppressed does not mean that rodent lenses are altogether unable to take up ascorbic acid. Mody describes in the rat,

Ascorbate in the Rat Lens is dependent on dietary intake (211). The lens ascorbate concentration in the Mody study increased linearly with dietary ascorbate intake and they concluded this uptake did not result in cataract development in the rat, which may also implicate the pool of available ascorbate as a limiting factor in rodent lens uptake or the existence a blood aqueous barrier if the SVCT2 was functional, due to the rapid uptake and concentration of ASA in tissues independent of Glut transporters. In that regard,

Dimattio did a number of studies investigating the effects of glucose on ascorbic acid uptake (87, 88). In those experiments, uptake of radiolabeled ASA and DHA from blood into aqueous humor, lens epithelium and lens (cortex) compartments were studied in male Sprague-Dawley rats with pulse chase kinetic experiments utilizing 3-O-methyl-D- glucose and L-glucose as a passive internal control. That data indicated that ASA enters aqueous humor at rates similar to L-glucose and likely via simple passive diffusion. In contrast, an active uptake of ASA by lens epithelium occurred more than 21 times faster than that of L-glucose. Concentrations in lens epithelium were found to be more than twice that of aqueous humor within only 7 min of delivering a 14C-ASA bolus into the systemic circulation. However, very little ASA was found to continue past the epithelium to the interior lens cortex compartment and no special uptake of ASA was

130 noted in the fiber cells, while the control, 3-O-methyl-D-glucose (glucose analog), readily

moved past the epithelium into the fiber cells at rates faster than the passive L-glucose, which suggests facilitated diffusion in the transport of that compound.

However, the Dimattio data will have to be reconciled, somehow, with the fact that rodent lenticular vitamin C concentration is close to zero. In that regard, it is not

impossible for some transport defect may also exist at the blood aqueous barrier, as

suggested by Dimattio himself (89). The data suggest that even in a nocturnal species,

with relatively low circulating levels of ascorbic acid in plasma and aqueous humor,

special mechanisms exist for moving ascorbic acid into intraocular or other tissues.

However, the active transport occurs only in the epithelial cells, while the interior fiber

cells appear to have no specialized uptake mechanism for this molecule, perhaps owing to

a loss of requisite machinery that is know to occur in fiber cell maturation.

2.5. SUMMARY

In summary, the feasibility of overcoming, at least in part, the vitamin C uptake

defect into rodent lenses by overexpressing the hSVCT2 under the control of a lens

specific promoter opens the possibility of creating a humanized mouse model of ascorbic

transport and homeostasis for studies of the ascorbic lenticular redox state and

ascorbylation reactions in the aging lens. However, considerable more work is needed to

precisely pinpoint the nature of the putative vitamin C uptake defect in the mouse lens.

131 CHAPTER 3

Vitamin C Uptake in Mouse and Human Lens Epithelial Cells: A Comparative

Study of the Role of the Sodium-Dependent Vitamin C Transporter 2 (SVCT2)

3.1. INTRODUCTION

Daily exposure to solar radiation is a potent source of damage to lens crystallin proteins that leads to structural changes and to a gradual loss of transparency. Photo- oxidation can inflict extensive damage to the proteins of the eye, especially when constant exposure to high levels of irradiation, both UV and ionizing, provide an ideal environment for the generation of reactive oxygen species in ocular tissues (212). The consequence of such damage is well known for the lens, as it results in increased light scattering and cataractogenesis. One proposed defense thought to protect the lens and retina from harmful photodamage and aid visual acuity by reducing chromatic aberrations is through dissipating UV radiation between 254 and 400 nm.

For diurnal animals, particularly primates, this is accomplished largely through

UV filters in ocular tissues and most abundant in the cornea. These filters include vitamin

C, which absorbs maximally at 260 nm and a class of small ninhydrin-positive compounds, including tryptophan metabolites that absorb between 295 and 400 nm, including the o-b-glucoside of 3-hydroxykynurenine (3-HKG), absorbs UV light above

132 288nm(47). The absorbance above 288nm is known to be low in the aqueous humor of fish, frogs, aquatic mammals and some ground-living birds (269). However, these compounds have not been identified in most laboratory animals in spite of the existence of an intact kynurenine pathway in these animals, except, interestingly, in the guinea pig.

Nevertheless, ascorbate is likely to be a first line of defense against UV-mediated phototoxicity, especially to DNA, and potentially explaining why millimolar levels are found in ocular tissues of diurnal animals. However, there is a double-edged sword to this protective mechanism because ascorbic acid, when oxidized, can lead to post- translational modification of proteins (307). In this regard, it is well known that human lens crystallins undergo a number of posttranslational modifications by reducing sugars and oxoaldehydes with age that lead to protein pigmentation and crosslinking (214).

These modifications are also thought to predispose lens crystallins toward formation of high molecular weight aggregates that scatter light and contribute to cataractogenesis.

Evidence now suggests that some of these modifications may originate from ascorbic acid degradation products (226). The relevance of this finding is important when we consider that human lenses contain relatively high levels of ascorbic acid, which becomes highly reactive if oxidized to dehydroascorbic (DHA) and 2,3-diketogulonic acid (2,3-

DKG), the spontaneous delactonization product of DHA (226). Among the proposed degradation products are xylosone, 3-deoxy-xylosone, L-threose, L-threosone and L- erythrulose (307). The latter compound is thought to be a major product of DHA forming under anaerobic conditions.

133 Comparative studies in biology are undertaken in order to better understand the

mechanisms in humans and for genetic differences between the species. Previously, we

reported that rodents lens epithelial cells have low lenticular levels of vitamin C (ASA) as

compared to humans (232) potentially provide an explanation for previous reports that

demonstrated ASA is low in ocular tissues of nocturnal animals, high in diurnal animals

and only reach intermediate levels in animals that span both. We have undertaken

several studies in the hope of uncovering an explanation for the differential uptake

between rodents and humans as far as the transporters are concerned and how this might

be further used to understand lens crystallin pigmentation in diabetes and aging. In

particular we have now performed comparative studies on the biochemical and functional

studies on the human and the mouse SVCT2.

3.2. MATERIALS AND METHODS

3.2.1. Materials

Most reagents were purchased from Sigma (St. Louis, MO, U.S.A) and of the highest grade available. Radiolabeled chemicals were purchased from GE /Amersham

(GE Healthcare Life Sciences/Amersham, Piscataway, NJ). All other reagents were of

commercial or analytical grade requiring no further purification, unless otherwise

specified. All phosphate buffers were treated with Chelex-100 resin overnight in order to

remove heavy metal ions. Either, dithiothreitol (0.1 mM) or N-acetyl cysteine (10 mM)

was added to prevent ASA oxidation in studies involving a time course with endogenous

134 cells. Cell transfections were performed using lipofectamine (Invitrogen, Carlsbad

California) or Polyethylimine (PEI) (Polyscience, Warrington, PA). Culture serum was from Fisher (St. Louis, MO) or other culture reagents, such as Hygromycin or Geneticin selection reagent were from Gibco (Gibco, Grand Island New York). All other media products were from Mediatech (Herndon, VA) unless otherwise specified.

3.2.2. Cell culture

Human lens epithelial cells, HLE-B3 and mouse17EM15 cells were cultured according to protocols previously described (232). The 17EM15 mouse lens epithelial cell line (265) was cultured in Dulbecco’s modified Eagle’s medium (DMEM,

Mediatech) supplemented with heat-inactivated 10 % FBS, 2mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. Experiments with HLE B3 or mouse LECs were performed at passages under 16, except when exploring the effect of late passage on function. CHO CK1 cells were cultured in standardized media as previously described for mouse cells. The HEK-293 a human embryonic kidney cell lines (ATCC# CRL-1573)

were cultured in a standardized Dulbecco’s modified Eagle’s medium (DMEM,

Mediatech), containing 4.5 g/L D-glucose, supplemented with 10 % heat-inactivated

FBS, 2mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. The M17

neuroblastoma cells were cultured in MEM with 10% FBS and antibiotics and all cells

cultures were maintained at 37ºC in humidified air containing 5% CO2. Medium was

changed twice per week, unless otherwise noted. For subculture, cells were washed with

135 PBS without calcium and magnesium and detached with 0.25% trypsin/EDTA

(Mediatech). Standard protocols for cell work were utilized.

Prior to uptake experiments, cultured cells were plated in 6-well Falcon plates

(Becton Dickinson, Franklin Lakes, NJ) and incubated in fresh culture media based on

the method previously described. After standard transfection protocol and unless

otherwise mentioned, each well contained 1 ml of medium. All specialized media and

transfection preparations were sterile filtered through a 0.22 µm filter (Gelman Sciences,

Ann Arbor, MI, U.S.A.). For uptake experiments, HLE-B3 and 17EM15 cells were cultured for up to 5 days after seeding at density of 1x105 cells per cm2.

3.2.3. RNA Isolation, RT-PCR and qPCR Analysis

Total RNA was isolated from cultured cells directly in 10cm dishes with Trizol

(Invitrogen) according to manufacturer’s procedure described. The purity was estimated

by spectrophotometric determination of the 260- to 280-nm absorption ratio. Purified

RNA was immediately assayed and the remainder stored at -70°C until further analyzed by qPCR. For RT-PCR, oligo dT’s, were used to make a cDNA library. In parallel, gene- specific primers, along with the respective species specific GAPDH primers, which all were generated from published sequences of human and mouse SVCT2, were also used.

All PCR primers were synthesized by Invitrogen Life Technologies. Primer sequences for qPCR products were as follows: for mSVCT2, 5'-TCCTTTGCTCACACCCTTCT-3’ lower primer (position 1747) and 5’-GGAATCGACCAAATCCTGAA -3 upper primer

136 (position 1607) and the PCR product length 150 bp and GAPDH product length 148 bp;

Human SVCT2 lower primer ‘5’-TTTCAGGTCAACCTCCT-3’ upper primer 5’-

CCTTGTCATTTG ACC CT GT-3’ The PCR product length was 143 bp and GAPDH

product length was 105 bp. Reverse transcriptase reaction was carried out with 10 µl

total RNA (800 ng of early and late passage of HLE-B3, and 800 ng of 17EM15) in a 50

µl reaction with Taq Man Gold RT-PCR kit using 0.75 units of reverse transcriptase

(Applied Biosystems). Amplification conditions were 10 minutes at 25°C, 30 minutes at

48°C, 5 minutes at 95°C. Synthesized complementary DNA (cDNA) was amplified by

PCR in 50 µl reactions with SYBR Green PCR Master Mix (Applied Biosystems) with supplied reagents according to manufacturers instructions. The amplification conditions were 20 s at 94°C, 30 s at the annealing temperature (Ta) specific for each primer pair, and 30 s plus 1 s/cycle for n number of cycles. (Ta = 47°C, for all primers). The specificity of the PCR products was confirmed by dye terminator sequencing with an ABI

PRISM cycle sequencing kit (Perkin-Elmer, Foster City, CA). DNA (10 µl) was electrophoresed on a 0.8 or 1.2% agarose gels containing 0.5 µg/ml SYBR Safe DNA stain. After electrophoresis, DNA was visualized by ultraviolet illumination and photographed. The relative amount of mRNA was determined by comparing the total intensity of each sample against the linear portion of the standard curve with the ABI

7500 system. Samples were analyzed in triplicate, and SVCT2 mRNA levels are expressed relative to GAPDH. The data analysis of SVCT2 mRNA levels was determined in duplicate from four separate experiments and is presented as means ± S.D.

Student's t-test was used to determine differences between groups. Values with P <= 0.05 were considered significant.

137

3.2.4. Construction of human and mouse SVCT2 expression vectors

The human (AF164142) and mouse (BC050823) sodium-dependent vitamin C

transporter 2 (SVCT2)/solute carrier family 23 (nucleobase transporters) member 2

cDNAs were cloned into pcDNA3.1 V5 His and pCEp4 vectors (Invitrogen, Carlsbad,

CA) using standard procedures as described in(281). Briefly, human and mouse SVCT2

cDNAs were amplified by PCR with Platinum® SuperMix High FidelityTM DNA

polymerase (Invitrogen, Carlsbad, CA) and primers (Integrated DNA Technologies, Inc.

Coralville, IA) or (Invitrogen, Carlsbad, CA) when using IMG clones (Open Biosystems,

Huntsville, AL) and from RT-PCR products from 17EM15 mouse lens epithelial cells as

the template cDNA. The following primer pairs were used for human SVCT2 cDNA

amplification for cloning in the pCEP4β vector 5’-GCGCAAGCTTATGATGGGTAT

TGGTAAGAATACCACATCC-3’ (forward) and 5’-GCG GCCGCCTATCCCGTG

GCCTGGGAGTCTTCATC-3’ (reverse), and the following primers were used for mouse

SVCT2 cDNA amplification (forward) 5’-GCGCAAGCTTATGATGGGTATCGGCA

AGAACACGGCATCCAAGTCA-3’ and 5’-GCGCGCGGCCGCTATCCCGTGGCCTG

GGAGTCT-3’ (reverse) and the following pairs were used in pcDNA3.1-V5-His vectors

for human SVCT2 5’-GCGGAATTATGATGGGTATTGGTA AGA–3 (forward) and 5’-

AGTCTCTAGATCCCGTGGCCTG GGAG–3’ (reverse) and mouse SVCT2 5’-

GCGATATC ATGATGGCAATCTACA CCACGAGA-3’ (forward) and 5'-

GCAGATCTTACTGTGGCCTGGGAATCTTTGTC-3’ (reverse). The resulting PCR

products were digested with NotI and HindIII (Roche, Indianapolis, IN) and inserted into pCEP4β vectors. For the pcDNA3.1 vectors the restriction enzymes were APA1 and

138 HindIII. The hSVCT2 and mSVCT2 coding sequences in the resulting plasmids, named

respectively as hSVCT2 and mSVCT2 for pcDNA vectors and hSVCT2-pCEP and

mSVCT2-pCEP for the pCEP4β vectors. All of the constructs were confirmed by DNA

sequencing analysis (Biotic Solutions, Inc. NY, NY) and by Western blot.

Figure 3.1. Vectors used in cloning mouse and human SVCT2.

3.2.5. Western blotting

Cell lysates were electrophoresed on 8% Tris Glycine gels and transferred to

PVDF membranes (Millipore) according to standard PAGE methods. The peptide

139 encoded by cloned SVCT2 was identical to that previously reported and new peptides were probed by Western blotting with either the human SVCT2 antibody from ADI

(Alpha Diagnostic Intl. San Antonio, Texas) or V5 antibody for pcDNA 3.1-V5-His constructs (Invitrogen).

3.2.6. Cellular transfection methods

In order to compare the transporter function of ascorbic acid transport from mouse epithelial cells vs. human LECs, the HEK-293, M17, CHO and COS-1 cell lines were transfected either with mSVCT2 or hSVCT2 in pcDNA3.1 constructs or mSVCT2-pCEP or hSVCT2-pCEP in pCEP4β constructs, δenαA-hSVCT2 or co-transfected with pEGFP-N1 (Clontech, Palo Alto, California), or mock transfected with the empty vector

(Invitrogen, Carlsbad, California), using lipofectamine reagents (Invitrogen, Carlsbad,

California) or polyethylenimine (PEI). PEI is a mixture of molecular weight polymers and required purification (48). It has been shown that short linear (<10 Kda) PEI is best as a transfection reagent and has low cytotoxicity and works well with plasmid DNA.

Transfection experiments carried out using lens-specific δenαA -hSVCT2 vector were from our work previously described (99). When LipofectAMINE2000 (Invitrogen, CA,

U.S.A.) was used for transfection manufacturers recommendations was followed.

Briefly, cells were grown to 90-95% confluence in 12-well Falcon culture plates (Becton

Dickinson, Franklin Lakes, New Jersey) and when transfected with lipofectamine, were rinsed with serum-free Opti-MEM Briefly, for lipofectamine treatment, cells were grown to 90-95% confluence in 12-well Falcon culture plates (Becton Dickinson, Franklin

140 Lakes, New Jersey) and when transfected with lipofectamine, were rinsed with serum-

free Opti-MEM media without antibiotics (Invitrogen, Carlsbad, California) according to

manufacturers recommendations, which was 4 µl per 100 µl Opti-MEM to 1.6 µg DNA

per 100 µl Opti-MEM, was prepared and gently mixed after 5 minutes incubation at room

temperature then directly added to each well and cultured for 3 hours at 37C. Post

transfection, cells transfected with lipofectamine were cultured in fresh antibiotic-free

serum-containing media was added directly to each well and cells were cultured

overnight Figure 3.2. shows the transfection efficiency when constructs were co-

transfected with GFP.

Figure 3.2. Transfection efficiency with GFP co-transfected as a reporter construct.

141 In most experiments, the linear polyelectrolyte, polyethylimine (PEI, M.w.

¼25000) as comparative studies revealed the transfection efficiency of PEI was superior

to other reagents, so PEI was used in all subsequent experiments due to higher transfection efficiency. PEI was prepared with 100 mg of PEI dissolved in 1.8 ml H20, final pH 6 adjusted with HCl and filtered the through 10KMWCO Amicon YM-10 filters

(Millipore, Woburn Ma). 2 ug of DNA was mixed with 0.5 ml serum free medium and 4 ul of PEI mixed with 0.5 ml serum free medium incubated 5 min at RT and were combined and mixed by inverting the tube and incubated 10 min at RT. PEI/DNA

complexes were added drop-wise on cells and incubated 4-6 hours. Cells transfected

with PEI were transfected in serum-containing media, as serum has no effect, once

complexes are formed. There was no need to change the medium. In all experiments, the

transfected cells were plated in 6-well plates for uptake. For transient transfection

experiments, cells were transfected as described above after having given sufficient time

for expression to take place, usually 24-48 hours. For stable transfection, cells were

selected with Hygromycin or Geneticin (Life Technologies, Carlsbad California) depending on the construct at a concentration of 500 µg/ml for up to four weeks for

HEK-293T and M17 and CHO cell lines.

3.2.7. Transport assay with L -14C-ascorbic acid

Transport assays were performed in Krebs buffer either with calcium (in mM: 140

NaCl, 4.2 NaHCO3, 5 KCl, 1.1 CaCl2, 0.5 MgCl2, 0.36 NaHPO4, 0.44 NaH2PO4 and 10

HEPES, pH 7.4) or without calcium at 37°C, as adapted from or described Liang (182).

L-[14C]ascorbic acid (14C-ASA) uptake was studied by exchanging the medium for 50

142 14 µM C1-ASA (specific activity 3.06 mCi/ mol) in all experiments unless otherwise

noted. Each experiment was performed in triplicate or more replicates. Briefly, each

well was washed with 1 ml of pre-warmed transport assay buffer after aspiration of the

culture medium. The uptake experiments were started with the addition of 1 ml buffer

containing 14C-ASA (17.0 mCi/mmol;) final concentration 50 µM ASA (specific activity

3.06 mCi/ mol) in 6-well falcon plates. The buffer was added to the cell monolayer and

uptake was determined at 37°C under standard culture conditions for up to 30 minutes but no more than 2 hours. Sub-confluent cells were used in uptake experiments due to

the inhibitory effect over-confluence can have on ASA uptake. The transport buffer was

removed, and the cell monolayer immediately washed one time with cold buffer

containing 0.01mM HgCl2 and rinsed subsequently with 1 ml of ice-cold radiolabel-free

transport buffer two times. The washing procedure took less than 10 seconds for each

well. Representative wells were used to enumerate cell number either by hemocytometer or a Coulter Counter and the cells in the remaining wells were lysed by addition of 200 µl

of 1M sodium hydroxide with 1% triton X-100 at RT. Use of this lysis buffer usually left undetectable amounts of radiation after the first wash. Each plate was washed two times with 0.5 ml of PBS. Washes and lysate were transferred to individual scintillation vials,

14 and filled with 4 ml of scintillation cocktail. The amount of L-[ C1 ]ascorbic acid dpm

was determined by liquid scintillation spectrometry using a Beckman LS 6000SC

scintillation counter (Beckman Coulter, Fullerton, California) quantified and expressed in

picomole per 106 cells. For non-transfected cultured cells, 0.1 mM dithiothreitol or

10mM N-acetyl cysteine was added to the transport buffer to prevent the oxidation of

ascorbic acid and to minimize oxidant stress in culture. For time course and kinetic

143 studies, 100 uM ASA was used as a saturating concentration. For any experiment

involving high glucose, cells were preconditioned in low glucose 24 hours before the

experiment to eliminate any potential effect of carbonyl stress on the system. For cell

counts, representative cells were collected by trypsinization and counted as described.

3.2.8. UV-exposure on Mouse Lens Epithelial Cells

17EM15 mouse lens epithelial cells were exposed to various doses of UV-B and

UV-C in a Stratalinker 2400 (Agilent/Stratagene, La Jolla, CA) to establish a killing curve for mouse cells (data not shown). The Stratalinker is a UV crosslinker, which can crosslink DNA and takes 25–50 seconds RNA also serves as a UV source. Each

Stratalinker UV crosslinker is equipped with (254nm) low-pressure mercury discharge bulbs, which emit most of their radiation at 254 nm, with weaker spectral lines in the

UVB, UVA and visible spectra according to information obtained from the International

Agency for Research on Cancer (IARC) at the 1992 meeting, and have an internal photodetector designed to compensate for the natural shift in power output of aging ultraviolet bulbs. A killing curve was conducted with exposures times ranging from 0 seconds to 2 minutes to determine the sublethal dose, which was determined experimentally to be (178µW/cm2) at a distance of 10cm from the source for 30 seconds to calculate the total irradiance. We used 20-second exposures in our system after culture media was removed from cells in 10 cm2 culture dishes.

3.2.9. Statistical Methods

144

Statistical significance was assessed by analysis of variance (ANOVA) using

Excel software. Values were expressed as mean ± S.D.

3.3. RESULTS

3.3.1. Comparative expression of SVCT2 in mouse and human lens epithelial cells

We previously reported that vitamin C uptake was relatively suppressed in mouse vs. human lens epithelial cells. In order to verify these results, we incubated mouse

17EM15 cells and human HLE B-3 cells with 100 uM 14C-ascorbic acid over a 15 hour period. As shown in (Figure 3.3. A.), ascorbic acid uptake was at all time points increased in human vs. mouse lens epithelial cells. Hypothesizing that transcriptional mechanisms were responsible for the downregulation of SVCT2, we determined mRNA levels by RT-PCR followed by qPCR in the cell extracts using PCR probes that were specific for the cDNA and not genomic DNA of the mouse and human transporters.

However, we found no differences in transcript levels in HLE-B3 cells at either passage

16 or 26 compared to mouse 17EM15 cells at passage 16 when expressed as a ratio of concomitantly amplified GAPDH transcripts (Figure 3.3. B.). Using a commercially available antibody against the human SVCT2 (hSVCT2) we were able to detect immunoreactivity in the lens epithelial but not in Chinese Hamster Ovary (CHO) cell extracts (Figure 3.3. C.). However, we could not reliably conclude that protein level was actually lower in mouse vs. human cells because crosspecies comparison of the specificity of the antibody affected immunoreactivity, as exemplified by lack a complete

145 lack of detection in CHO cells. Similarly, we concluded that any experiment attempting

to immunoprecipitate mouse vs. human SVCT2 for quantitation purpose would be

difficult to the structural and functional differences between the mouse and human

transporters.

Figure 3.3.A.

1400 1200 1000 800 HLE B3 600 17EM 400 200 0 0 5 10 15 20 Time Hours

Figure 3.3. B.

qPCR SVCT2

2.5 Ratio 2 1.5 1 0.5 HLE p12 HLE p26 17Em p16

146 Figure 3.3. C.

75 50 37 CHO HLE 17EM

Figure 3.3. A.) Time course of 14C-ASA uptake with non-transfected mouse and human lens epithelial cells. B) Comparative effect of lens epithelial cell type and passage on mRNA levels by qPCR (p < NS) for all comparisons (n=4). Passage 12 HLE-B3, Passage 26 HLE-B3, Passage 16 17EM15 Data are expressed as mean ± Standard Deviation. C) Mouse and human SVCT2 is immunologically detectable but not CHO cells in western blots with commercial antibodies to human SVCT2.

3.3.2. Comparison of Sequence and Structural Motifs, In Silico

Comparative sequence analysis using NCBI/BLAST search algorithms revealed

that the mouse and human genes share greater than 95% homology in DNA sequence. A

high stringency motif scan of human and mouse SVCT2 demonstrate several potential

motifs with 100% conserved identity (http://scansite.mit.edu/cgi-bin/motifscan_seq).

When we scanned for homologous motifs, we found no sequence motif differences, but

found common structural motifs (Table 3.1).

Sequence or Structural Description Functional Pattern Species Motif Site Description MOD_OFUCOSY Site for O- C.{3,5}[ST]C Human/Mouse attachment of Fucosylation a fucose site residue to serine (extracellular)

147 MOD_GSK3_1 Site GSK3 ...([ST])...[ST] Human/Mouse recognized by phosphorylati GSK3 for on site Ser/Thr Phosphorylati on MYRISTYL N- GIgkNT Human/Mouse myristoylatio GSteGK Mouse n site GGatSS Human/Mouse GAtsSG Human/Mouse 14 human vs 14 mouse GLpgAL Human/Mouse GMlsAV Human/Mouse GIfgTG Human/Mouse GTgnGS Human/Mouse GNgsTS Human/Mouse GStsSS Human/Mouse GAlfCT Human/Mouse GMitAV Human/Mouse GGcvAF Human/Mouse GVskGS/GVgkGN Human/Mouse GLrkSD Human ASN_ ASN is the N- NTTS Human GLYCOSYLATION glycosylatio glycosylation NTTD/NTTE Human/Mouse n site site NGTA Human/Mouse NSTD Mouse 6 human vs 5 mouse NGST Human/Mouse NSSR Human/Mouse NKSL Human PKC_PHOSPHO_SITE Protein TsK Human kinase C SkK Human/Mouse 6 human vs 5 mouse (PKC) StK/SdK Human/Mouse phosphorylati SrR Human/Mouse on site SsR Human/Mouse TwK Human/Mouse CK2_PHOSPHO_SITE Casein kinase SstE Human II SsgE Human/Mouse 13 human vs 11mouse phosphorylati SlaE Human/Mouse on site TieD Human/Mouse SliE Human/Mouse TvtD Human/Mouse SiiE Human/Mouse SigD Human/Mouse SlpD Human/Mouse TgiD Human/Mouse TpeE Human/Mouse SsdE Human SdeD/SdkD Human/Mouse LEUCINE_ZIPPER Leucine LievvigLlglpgaLlryi Human/Mouse zipper Pattern gpL

Table 3.1 common structural and functional motifs between human and mouse SVCT2.

148 The only difference that could be found was a slight variation in the relative frequency of

these motifs, with the mouse having fewer occurrences in all instances. They include a

basophilic serine/threonine kinase group (Baso_ST_kin) member in the intracellular domain and distal to the N-terminus of the protein at residue T117, which contains a

PKC-ε binding motif with the sequence YLTCFSGTIAVPFLL (gene card designation

PRKCE) and three kinase binding site group members (Kin _bind) that are all on the C- terminal end of both proteins.

Two members are PDK1 binding motifs (gene card designation PDKPK1) at residues S615, which is intracellular, with the sequence IKKYRCFSYLPISPT and T627, which is extracellular, with the sequence SPTFAGYTWKGFGKS. The last kinase binding site group member is an ERK D-domain (gene card designation MAPK1) at residue 1619 with the sequence RCFSYLPISPTFAGY that is also intracellular. These predictions are purely speculative and are used with caution, since they are based on the assumption that the peptide library data is correct and sufficient to predict a site.

Nevertheless, both species share other important functional residues, such as myristonylation and N(Asparagine) -glycosylation sites, which should be glycosylated in a similar manner when overexpressed in the various cell systems. We verified the above data using the clone sequence from mouse ear epithelial cells, which revealed absence of truncation or alternative splicing sequences that would have explained potential differences in SVCT2 function and using primers for the human SVCT3 the short isoform full length primers in the Lutsenko paper. Primer pairs for hsvct2 short were

AAT2-300 (GGG GTC ACA GCA CTA CCT G) and AAT2-R900 (GGA TGG CCA

149 GGA TGA TAG) and we could not identify such an isoform from the mouse cultured

cells.

Comparative functional studies of mSVCT2 and hSVCT2

To test whether the mouse transcript was functional, we cloned the complementary DNA encoding both the human and mouse SVCT2, inserted these into several expression constructs and conducted transfection experiments in HEK 293 cells.

When the transporters were inserted in pCEP4β, a variation of pCEP4 vector particularly

disfavored for stable transfection due to non-integration into the host genome because of

its epifocal nature (35), the resulting ASA uptake was hardly increased, whereby no

differences were observed between mouse and human transporters (p values <0.008

compared to controls) (Figure 3.4. A). Since pcDNA seemed to offer better expression

data (Figure 3.4. A), the mouse transporter was subcloned into pcDNA and used for

back-to-back comparison with hSVCT2. As shown in Figure 3.4. B, a seven-fold

increase in ASA uptake was noted for both transporters vs. the Mock transfected, with no

differences observed between the mouse X =362.17 , 88.26 and the human X=352.68 ±

62.5, Mock X=36.57626488 ± 11.19 (p<0.001 for mSVCT2, (p<0.003 for hSVCT2).

Immunoblotting withV5 antibody revealed similar expression levels in the cell membrane extract (Figure 3.4. B. insert).

150

Figure 3.4. A.) Effect of transient transfection of HEK-293 with mSVCT2-pCEP and hSVCT2-pCEP, hSVCT2-pcDNA 3.1-V5-His constructs relative to control on 14C-ASA uptake (n=3).

Figure 3.4. B.) Effect of 48 hr transient transfection of HEK-293T with pcDNA 3.1-V5- His constructs containing mouse and human SVCT2 (p<0.001) relative to mock transfection on 2 hour 14C-ASA uptake, hSVCT2-pcDNA (p<0.001) and mSVCT2- pcDNA (p<0.039) (Inset) is the Western immunoblot with anti-V5 antibody for the data.

151 To strengthen the above findings we explored the kinetics of mouse and human

ASA transporters in a time course with transfected HEK-293 cells (Figure 3.4. C.). Both mouse and human SVCT2 had similar slopes, whereby the mouse transporter was as good if not better than the human transporter, when transfected transiently. Having discovered several motifs in common between the two proteins, we explored biochemical aspects of putative calcium dependent motifs for possible explanation underlying the differences in uptake.

1200 y = 1801.2x - 47.582 1000 Human 800 Mouse

600

400 y = 1240.5x - 25.709 200

0 0 0.2 0.4 0.6 0.8 Hours

Figure 3.4. C. Effect of transient transfection of hSVCT2-pcDNA (NS) and mSVCT2- pcDNA (p<0.001) relative to control (p<0.01) of HEK-293 cells with and without calcium in the transport buffer on 14C-ASA uptake.

This reasoning is based on the recent report by Godoy et al. (120) suggesting that calcium plays an important regulatory role on SVCT2 function and early work indicated a strong probability. Using pcDNA constructs we could indeed confirm that presence of calcium

152 enhanced ASA uptake. The effect was in fact significant (p<0.01) for mouse SVCT2 but

the findings with the human SVCT2 transporter were not significant in this experiment

and this is not unequivocal (Figure 3.5.). From these experiments we conclude that the

mouse and human SVCT2 genes, when overexpressed in HEK-293 cells were able to

increase ASA uptake to a similar level, suggesting there are no major species differences

in SVCT2 sequence that can explain the relative suppression of ASA uptake in mouse vs.

human lens epithelial cells.

1000 *p<0.001 900 * p

400 *p<0.01 300 C ASA/E^6 Cells/30min 200

14 100 0 pmol mSVCT2 mSVCT2 hSVCT2 hSVCT2 Mock - Moc k -Ca +Ca -Ca +Ca Ca +Ca

Figure 3.5. Effect of transient transfection of hSVCT2-pcDNA (NS) and mSVCT2- pcDNA (p<0.001) relative to control (p<0.01) of HEK-293 cells with and without calcium in the transport buffer on 14C-ASA uptake.

3.3.4. Effect of Overexpression of the human vs. mouse Transporters into Various Cell

Lines

153 The results obtained with HEK-293 cells raise the question of whether some cell

specific effects would mask functional differences between the mouse and the human

transporter. For this reason we also used other cell lines, which were made stable

through integrating the DNA into the cells with geneticin for SVCT2-pcDNA constructs

or Hygromycin for use with pCEP-SVCT2, which is an episomal vector and not stably

integrated with these constructs. Clones from stably transfected CHO cells with both

transporters using both the pCEP4B and pcDNA vectors. However, here again, no

enhanced activity of the human vs. the mouse transporter could be documented (Figure

3.6. A.).

Figure 3.6. A). 14C-ASA uptake in stable cell lines A) SVCT2 constructs and pCEP constructs CHO cells hSVCT2 (p<0.001) mSVCT2 (p<0.039).

In fact, the trend toward higher activity of the mouse vs. the human SVCT2 in the pcDNA transfection experiments was also observed in the similarly transfected neuroblastoma M17 cell line (Figure 3.6. B.).

154

Figure 3.6. B) M17 neuroblastoma cells transfected with humanSVCT2-pcDNA (p<0.05) or mouse SVCT2-pcDNA (p<0.0001).

3.3.5. Effect of UV Exposure on ASA Uptake in Endogenous Mouse Lens Epithelial

Cells

Regardless of the system we have used in the hope of revealing functional

differences between the mouse and the human transporters, the data do not support

superiority of hSVCT2 over mSVCT2, suggesting that the relative suppression of ASA

uptake by the mouse vs. human LECs must be complex, possibly related to regulatory rather than structural features of the transporter or perhaps due to differences in the

enhancer or promoter sequences. Indeed, overexpression studies comparing the denA

vector, pCEP and pcDNA-CMV-driven vectors clearly show a significant difference

between the two promoters (Figure 3.7.).

155

Figure 3.7. Overexpression in HEK-293 cells using the dena construct, pCEP constructs or pcDNA constructs. Dena-hSVCT2 uptake was significantly increased over the CMV- driven expression systems.

On the other hand ASA has an antioxidant and protective role compound against

UV-photo-oxidation in ocular tissues (370) and may offer a place to explore in understanding, in part, the complex mechanism of differential uptake. In that regard, UV light has been shown to increase ASA uptake in bovine LECs (71). For this reason we explored the effect of sub-lethal doses of UV on ASA uptake in endogenous mouse lens epithelial cells. As in the study from Corti et al., a significant increase in ASA uptake in cells exposed to UV was observed, which was saturable within two hours and was consistent with an inducible activation of the endogenous transporter and unlikely due to protein synthesis in the short uptake period. However, recruitment to the membrane could be another explanation for the observation. Further longer time points showed the cells were viable, but the uptake was somewhat suppressed when the cells reached confluence (Figure 3.8.).

156

UVC-exposed Mouse Lens Epithelial Cells

120 100 80

60 40 20

14C ASA pmol/E^6 Cells 0 2Hr no UVC 2Hr with UVC

Time Course UV-exposed 17EM15 Cells 120

100

60 40 20

0 0.0 0.3 1.0 2.0 12.0 Hours

Figure 3.8. A. Effect of UV irradiation on 14C-ASA uptake at two hours in mouse lens epithelial cells 17EM15 vs. non treated cells (p<0.002) and 3.8. B. Time course of 14C- ASA uptake in UV-exposed 17EM15 cells.

4.1. DISCUSSION

UV protective mechanisms in the lens has been well-reviewed (85) and the notion that nocturnal animals evolved as physiological knockouts of the SVCT2 because of an absence of elevated ascorbic acid concentration in ocular tissues compared to diurnal animals, including the human, is a fascinating problem to understand. There currently are

157 two hypotheses to explain the presence of high levels of vitamin C in the lens of diurnal

animals. The first, stipulates that vitamin C acts largely as a UV-C filter absorbing

maximally at 260 nm. In the cornea, which absorbs all wavelengths of radiation below

297 nm, vitamin C can dissipate some UVB (290- to 315-nm)(266). Indeed studies by

Varma and colleagues showed that vitamin C is a potent anti-cataract agent and

antioxidant in the mouse lens in experimental animal models (137), and is likely to be a

first line of defense against UV light mediated phototoxicity to the lens (350). The

second hypothesis stipulates that nocturnal animals, some of which are known to have a

slight UV pole, meaning they can see some wavelengths of UV and do so because of

fluorescent cues and perhaps food that may fluoresce at night, i.e. weak light that would

be otherwise is quenched by high levels of UV filter (63). Thus, the ability to suppress

the uptake or synthesis of UV filters in the rodent eye could be have evolved as a

beneficial trait. More recently, the view that ascorbic acid is a potent UVB or UVA filter

is being met with some challenge and more emphasis may be placed on the kynurenines,

as UV-A filters. This can be explained by the action spectra of ASA, which absorbs

maximally at 260 nm would more readily afford protection from UV-C, which has an

action spectra of 254 nm. While the relative role of each of these as protectors of the

retina may be debatable, the absence of both ascorbic acid and the kynurenines from the rodent lens is clearly linked to their nocturnal life cycle. The loss of either of the UV-

protective class of compounds does not mean that the animals do not have functional or

effective pathways to dissipate UV or take up ASA altogether. Strong evidence in

support of such a link comes from the comparison of the Common Spiny mouse (Acomys

cahirinus) with the Golden Spiny mouse (Acomys russatus), one of two mice of the same

158 species that lives in the desert and forages during day time and has, like the human lens,

very high lenticular vitamin C levels (163).

Multiple mechanisms are conceivable by which nocturnal animals have adapted to

suppress lenticular vitamin C levels. First, it is conceivable that very low ascorbic acid

levels reach the lens due to some permeability problem at the blood-aqueous barrier.

However, DiMattio showed that intervenous (IV) injections of radiolabeled ascorbic acid

to rats was associated with rapid and saturable uptake of ASA. Thus, even though the absolute lenticular concentrations reported in the studies were low compared to the human lens, and studies by Mody et. al concluded that ascorbate could pass the aqueous

and accumulate in the lens in a dose-dependent manner (211) they would certainly be

sufficient to activate SVCT2 and ASA transport, if the latter is fully functional and

expressed in epithelial cells.

An alternative mechanism for the low lenticular ASA levels might conceivably be

explained by the presence of a rapid ASA or possibly, though unlikely, DHA efflux mechanism. However, it has been shown that DHA enters cells via Na+-independent

glucose transporters (GLUT) and is rapidly converted to ascorbate, unless glutathione

concentrations are diminished or depleted intracellularly. In that regard, it is known that

DHA is transported outside some cell by GLUT transporters, e.g. GLUT3 or GLUT2 in

liver (15) and if intracellular oxidation were to form DHA, then transporters that are

asymmetrical in structure, which implies GLUT 2, but not GLUT 1, would favor efflux of DHA as it does for glucose. However, most cells have GLUT 1 and GLUT 3 and some have GLUT4, with the former possible having efflux capacity. Ascorbate upon

159 stimulation that leads to increase in Ca2+ concentration was reported in that A23187 and

ATP elicit release of ascorbate release from Pig Coronary Endothelial Cell (PCEC).

However, GLUT transport 1 and 3 transporters are potently inhibited by flavanoids, such as quercetin and up to 50% by Myricetin in Jurkat cells (244), which further complicates the question of Ascorbate homeostasis in any animal or system where ASA is explored.

Further, it was found that Na+ removal inhibited DHA uptake by smooth-muscle cells cultured from pig coronary artery and the inhibition by Na+ removal was paradoxical since 2-deoxyglucose and cytochalasin B inhibited this suggesting intracellular acidosis from the Na+H+-exchange played a role, because inhibitors ethyl isopropyl amiloride and cariporide also decreased the uptake, by lowering cytosolic pH, so the inability to obtain ascorbate extracellularly, could imply a hypoxia-induced acidosis during ischemia and reperfusion or in culture when the cells become over confluent. In the mouse

17EM15 cells it was observed that lenticules or “lentoid bodies” form, which seem to result from both low serum and over confluence.

Whether similar reverse SVCT2 transporters exist for ASA itself are not known or whether other transporters may do this. However, there are channels that are implicated in the efflux of ascorbate when conditions of osmolarity are perturbed. More likely favorable for ascorbate efflux would imply a role for the existence of an anionic pump could equally be conceivable in the lens along with a role of calcium and the sodium/potassium exchangers involved in lenticular ascorbate homeostasis. Evidence against the presence of an efficient ASA or DHA extrusion mechanism from the rodent lens is suggested by our studies in the hSVCT2 transgenic mouse in which both ASA and

160 DHA levels were highly elevated (99), however, the efficiency of this abundantly

overexpressed protein to transport ASA efficiently is quite clear and whether this

overactive transporter may outpace any endogenous efflux mechanisms. Nevertheless,

careful studies on the ASA/DHA efflux form the rodent lens will be needed to fully

address this question.

Finally, since a high affinity transporter is an absolute requirement in order to take up ASA against a concentration gradient ranging from micromolar blood levels to millimolar lenticular levels, it would be very difficult to explain the potent suppression mechanism without considering a dysfunctional or underexpressed SVCT2 itself. Amid several possible mechanisms, we have considered in this study structural differences between the human and rodent transporters. However, we need to also consider the relative size, number or surface transporters available at any one time and possible perturbations in cell volume of the mouse epithelial cells, which are interestingly smaller than the human LEC counterparts. Using several different approaches, we came to the conclusion that there is no intrinsic difference between the human and the mouse transporters when their function was tested by overexpression in HEK-293, CHO of M17 cell lines. Importantly, the mouse SVCT2 we used was cloned from a mouse endoderm- derived tissue and was not a truncated isoform, as was reported by Lutsenko et al in

HeCat cells, was observed (198).

Our results thus exclude major structure-function differences between the mouse and the human transporters. However, they do not exclude more subtle effects in those

161 domains that differ between the two transporters, such as the glycosylation sites and PKC

sites that might lead to postsynthetic modification and such as decreased affinity of the

mouse (79). Somehow, however, the 95% sequence homology and presence of only minor sequence differences between the two transporters argue against a mechanism

linked to differences in amino acid sequence.

Nevertheless, we do not know all of the substrates that the SVCT2 can transport.

In that regard the transporter has been classified as a nucleobase transporter, which may

offer clues as to its regulation. The above conclusions shrink the list of possible

mechanisms to explain the lower ASA uptake in mouse vs. human LECs. If indeed the

SVCT2 transcript levels are identical in HLE-B3 and mouse 17EM17 lens epithelial cells, then one is left with differences in protein expression at the plasma membrane. The mRNA levels of SVCT2 were determined in many rodent tissues (38). Since mRNA levels are not decreased, mechanisms that enhance post-ribosomal expression should be considered along with promoter and enhancer differences between the two genes.

5.1. CONCLUSIONS AND FUTURE DIRECTIONS

In summary, the above studies have shown that the uptake of vitamin C in rodent lens epithelial cells is very low compared to human epithelial cells. No major molecular or functional differences between the rodent and human SVCT2 were found as an explanation for the differential uptake. However, there are numerous issues that will have to be reconciled in terms of bioavailability of ascorbate in rodent lenses from the

162 liver to the plasma and aqueous humor to the lens. Not enough of these details have been

provided to adequately address ascorbate homeostasis in rodents and animals that could

become scorbutic. In particular, we have not addressed adequately the possibility of

GLUT-mediated efflux of DHA out of the cells. In any event, there are compounds known to stimulate ascorbate efflux, such as a calcium ionophore, ATP, UTP and other

compounds. Further, we do not know which substrates may be transported by the SVCTs other than ascorbate as they are nucleobase permease/transporter family members.

Nevertheless, there must be efficient mechanisms for efflux of ascorbate, especially in cells that scavenge DHA and reduce and recycle it for bioavailability for other cells, particularly the brain. Cells, such as those of the eye (an avascular environment) require a sophisticated transport system, because the eye does not rely on simply utilizing conventional mechanisms for moving solutes. If this is true, the eye, which is not only lacking a vasculature, is immune privileged and would require specialized carrier proteins and bioflocculants, as well as rudimentary immune surveillance among other systems, to function in a similar fashion as do other cells. Basically, there must be analogous systems to deal with the same basic functions as any other tissue that is has a blood barrier or relies on ion flux, such as the myocardium, for example, which also relies heavily on ion flux for optimal contractility. Further, there must be a mechanism of efflux from epithelial cells into the lens, because these lens fiber cells are not contiguous with the aqueous, they require trophic and other support via the epithelium. So for these cells to receive and concentrate ascorbate, they would require a functional transporter, because passive transport will not explain the gradients that exist in the lens and epithelia.

Indeed, as Dimattio’s, experiments show, a barrier to movement of ascorbate but not O-

163 methyl glucose exists in the lens. Perhaps there are apical and baso-lateral differences in these cells that may explain differential uptake and complex regulation we are beginning to uncover in the lens and one must use the right system to approach the problem.

The lens epithelium takes up the ascorbate, as do other cells. Moreover there are some cells that scavenge DHA and reduce it, once it is intracellular. So there must be an equally efficient mechanism for recycling and releasing ascorbate, because it cannot just accumulate without limiting factors or a mechanism for efflux or consumption. Since there are no known mechanisms for the consumption of ascorbate in cells that recycle the molecule and there is a mechanism of efflux in endothelial cells upon swelling or osmotic changes, the latter is more likely (367). Reverse transport does not seem to be a plausible mechanism for cells. But it gets more complex. From recent work, an anion channel that is volume-sensitive has been discovered in cells (367).

Perhaps signaling through G-protein coupled receptors would provide the lens with a finely tuned system to regulate ion transport. Regardless, the first line of defense of oxidation is most likely ASA together with GSH. The first question that one considers in light of damage, is that not all damage is created equally. In other words, there appear to be a set of modifications that are on one hand damaging and on the other protective, such as argpyrimidine modifications to alpha crystallins. Next is the accumulation of pigments, chromophores and fluorophores in lens tissue. While these compounds, can cause the loss of refractive capacity in the lens, they can conversely have a photo- protective nature, at the same time, by increasing the barrier to UV-mediated damage to

164 the retina. Nevertheless, some chromophores in lens proteins act as photo-sensitizers, which only complicate the issue. When exploring whether glycated proteins produce

ROS, when lens explants or cells were incubated with MGO, GO, ASA or fructose and exposed to 200J/cm2 UVA, the finding was glycated model proteins produced 2-3 fold more singlet oxygen species compared to the unmodified protein and superoxide radical formation was discovered, so what we don’t know is which compounds or AGEs act as photosensitizers and what can be done to inhibit the process. We have determined that ascorbic acid can damage the lens and contribute to the accumulation of AGEs modifications and crosslinks, while there has not been much attention paid to the other small molecular antioxidants, such as other photosensitizers like riboflavin (328), that may also add to the pigmentation of the lens with age. A systematic review of these compounds should be explored.

Since the methylglyoxal hydroimidazolones are quantitatively major AGEs of human lens proteins and these substantial modifications may stimulate further glycation, oxidation, and protein aggregation, leading to the formation of cataract, we also must consider alternative roles for these compounds in tissue, such as signaling molecules and complex interaction these modifications may have on protein, e.g., the argpyrimidine modifications improve alpha crystallin chaperone-like function, the hydroimidazolones may be protective as well, but this would require further investigation.

It seems to one that the UV-protective compounds in the lens, cornea and humors, should be characterized as to the exact wavelengths of light they dissipate. For example,

165 ascorbic acid is the most important water-soluble antioxidant in the aqueous humor and the lens, however the question remains as to the role of the kynurenines or other small

molecules in relation to ASA. In other words, where do these molecules fit into this

protective system and can they also contribute to modifications to crystallins with age or

exposure to photooxidation. The major role of ascorbate is thought to be an antioxidant,

but also a UV-B filter. However ASA absorbs maximally at 260 nm, making it more of a

UV-C filter, which helps protect the retina from DNA damage, as DNA has an action

spectra of 254 nm, optimally. The lens has a well-designed system of defense against

oxidation (23).

The primary defenses to neutralize oxidative species are non-enzymatic (e.g.,

glutathione, vitamin C, vitamin E and carotenoids) and enzymatic defenses, such as

superoxide dismutase, glutathione peroxidase and catalase require energy and reducing

equivalents, so the NADPH-dependent thioredoxin/thioredoxin reductase and GSH-

dependent thioltransferase system (143) and even protein disulfide isomerase (PDI).

Undoubtedly all may be critical in maintaining the lens in a reduced state, cleave protein–

thiol mixed disulﬁde bonds that are formed through oxidation of lens proteins and

maintain lens integrity. Perhaps more attention should be paid to intervention of

lenticular damage through increasing reducing equivalents in the lens. Nevertheless, lens

cells are known to contain enzymes that can degrade damaged proteins through

proteolysis (140, 358) or repair damaged nucleic acids (14, 309). Taken together, the

sum of deleterious protein modifications and the breakdown of homeostasis during

cataractogenesis are likely sufficient to explain the crystallin instability that leads to the

166 formation of light scattering aggregates and, thus, cataractogenesis. Any means to retard or delay any of the mechanisms of cataractogenesis, be they by drug intervention or dietary means, would likely have a positive impact on preventing blindness and improve quality of life for millions of individuals worldwide.

167

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