Abraham Spector

Lens epithelial cell apoptosis is an early event in the development of UVB-induced cataract.

Epidemiological and experimental studies have revealed that exposure to UV can induce cataractogenesis. To investigate the mechanism of this induction, viability of the lens epithelial cells from UVB-treated rat lenses were examined. Irradiation of the cultured rat lenses with 8 J/s/m2 UVB for 60 min triggers lens epithelial cell apoptosis as determined by terminal deoxyribonucleotide transferase (TdT) labeling and DNA fragmentation assays. The apoptotic lens epithelial cells were initially found in the equatorial region and then quickly appeared in both equatorial and central regions. The percentage of apoptotic cells continuously increased during the postirradiation incubation. After a 5-h post-UVB incubation, more than 50% of the lens epithelial cells were apoptotic. By 24 h, all of the lens epithelial cells in the irradiated lenses were dead through apoptosis. Associated with this apoptotic process is a large upregulation of the proto-oncogene, c-fos. Opacification appears to follow the death of lens epithelial cells occurring first in the equatorial region and then in the central area. This is also true of classical cataract parameters such as non-protein thiol and wet weight, which are significantly modified only after appreciable epithelial cell apoptosis. Together, these results suggest that the rapid apoptotic death of the lens epithelial cells induced by UVB initiates cataract development.

The state of sulfhydryl groups in normal and cataractous human lens proteins. I. Nuclear region.

Abstract The level of water-insoluble proteins in the nuclear region of the human lens increases progressively with aging. Increases beyond the normal level occur with opacifications until more than 90% of the proteins in the nuclear region are water-insoluble in severe cataracts. The level of nuclear protein disulfide groups in cataract also is dramatically higher than that found in aged, normal lenses. More than 90% of the water-insoluble protein sulfhydryl groups are oxidized in advanced cataracts. Although water-soluble nuclear proteins of certain cataracts contain high levels of protein disulfide groups, the total amount of protein disulfides in the nuclear region that are soluble stay within the range of normal values. Part of the large increases from normal levels in acid-soluble sulfhydryl groups released by borohydride reduction of the insoluble fraction of advanced cataracts is probably due to an increased number of particular nondisulfide bonds susceptible to reductive cleavage. In spite of differences in the distribution of nuclear sulfhydryl and disulfide groups in aged, normal and cataractous lenses, the total amount of lens nuclear sulfhydryl plus disulfide groups remains reasonably constant.

A brief photochemically induced oxidative insult causes irreversible lens damage and cataract. II. Mechanism of action.

Using photochemically induced oxidative stress and rat lenses in organ culture with 4% O2 and 4 microM riboflavin, it has been found that the observed changes in lens parameters are, in most cases, irreversible. This has made possible the elucidation of the sequence of biological changes leading to cataract. The earliest detectable changes in lens cell biology are observed in the epithelial cell redox set point and at the DNA level in terms of DNA integrity and 3H-thymidine incorporation followed by decreased membrane transport and changes in gene expression. Significant modification in classical cataract parameters such as hydration, steady state non-protein thiol, glyceraldehyde-phosphate-dehydrogenase activity and transparency occur at later times. The data suggest a definitive pattern of lens breakdown resulting in opacity starting at the epithelial cell level and leading to subsequent fibre cell involvement.

The prevention of cataract caused by oxidative stress in cultured rat lenses. I. H2O2 and photochemically induced cataract

H2O2 stress is shown to produce cataract in cultured rat lenses. The loss of transparency begins in the equatorial region within 24 hours and the entire superficial cortex is opaque by 96 hours. No involvement of the nuclear region is observed. However after an additional 48 hours, the nuclear region becomes opaque. The loss of transparency is accompanied by a large uptake of H2O which occurs gradually over the 96 hour period, complete loss of glyceraldehyde phosphate dehydrogenase (GPD) activity, almost complete loss of non-protein thiol and a slight decrease in protein thiol. Control lenses show no change other than the establishment of a new non-protein thiol base line approximately 60% lower than 0 time levels. The Alcon glutathione peroxidase type mimic, AL-3823A, completely eliminates almost all of the H2O2 induced effects and the lens remains transparent. Utilizing a more severe photochemical model than may be anticipated physiologically with 10 microM riboflavin and exposure to daylight fluorescent lamps, significant concentrations of superoxide and low levels of OH. are produced as well as extraordinarily high concentrations of H2O2 ranging from about 400 to 1000 microM. As with the H2O2 model, opacification begins at the equator but the cataract develops more rapidly, the lens being completely opaque by 68 hours. Hydration, GPD activity, non-protein and protein thiol all decrease more rapidly than in the H2O2 model. AL-3823A prevents loss of transparency until approximately 92 hours and markedly decreases changes in other parameters. At 92 hours, slight loss of transparency is observed. Catalase is somewhat less effective. AL-3823A is shown to also significantly decrease superoxide levels. The marked delay in the onset of changes in lens biochemistry and physiology in the severe photochemical stress model and the maintenance of normal parameters in the H2O2 model in the presence of AL-3823A suggests that such compounds may prevent cataract caused by oxidative stress under physiological conditions.

Isolation and characterization of an age-dependent polypeptide from human lens with non-tryptophan fluorescence

With aging of the human lens there is an increase in insoluble protein which occurs primarily in the nuclear region. The character of this material changes with age from a white precipitate isolated from young lenses to a predominantly heavy yellow material obtained from older tissue. An increase in protein-bound non-tryptophan fluorescence also occurs with aging. This fluorescence has a corrected absorption maximum of 325 nm and a corrected emission maximum of 415 nm. The fluorescence is primarily associated with the insoluble yellow protein of the nuclear region, particularly in older lenses. It is also found in the soluble proteins where it is predominately in the high molecular weight components. In both the soluble and insoluble fluorescence containing protein fractions this fluorescence could be detected only in a 43 000 dalton polypeptide. This polypeptide probably contains two C-terminal residues but no N-terminal groups. Ba(OH)2 or proteolytic hydrolysis of some of the fluorescent proteins produces a low molecular weight fluorescent component containing equimolar amounts of glycine and proline but no N-terminal groups or other amino acids. It is therefore concluded that the 43 000 dalton polypeptide is composed of two smaller polypeptides linked by a fluorescent component via their N-terminal groups, glycine and proline.

Hydrogen peroxide-induced DNA damage in bovine lens epithelial cells

The present investigation was undertaken to determine the types and extent of DNA damage resulting from incubation of primary cultures of bovine lens epithelial cells with hydrogen peroxide. Significant numbers of DNA single-strand breaks were detected by alkaline elution after exposure to as little as 25 microM H2O2 for 5 min at 37 degrees C. The extent of single-strand breakage was concentration dependent and linear from 25 to 200 microM H2O2. The observed single-strand breaks appear primarily due to the action of the hydroxyl radical via a Fenton reaction as both an iron chelator, 1,10-phenanthroline and OH. scavengers, including DMSO, KI and glycerol, significantly inhibited the DNA-damaging effect of H2O2. Diethyldithiocarbamate, an inhibitor of superoxide dismutase, further potentiated the DNA-damaging effects of H2O2, presumably by increasing the steady-state concentration of Fe2+. DNA-protein cross-linking was not observed. In addition, significant levels of 5,6-saturated thymine residues or pyrimidine dimers were not detected after modification of the alkaline elution methodology to allow the use of either E. coli endonuclease III or bacteriophage T4 endonuclease V, respectively. No double-strand breaks were detected after incubation of epithelial cell cultures with H2O2 concentrations of up to 400 microM for 10 min and subsequent neutral filter elution. Since, in vivo, the lens epithelium contains populations of both quiescent and dividing cells, the degree of susceptibility to oxidative damage was also studied in actively growing and plateau-phase cultures. Reduced levels of single-strand breakage were observed when plateau-phase cultures were compared to actively growing cells. In contrast, essentially no differences in repair rates were noted at equitoxic doses of H2O2. The above results suggest that lens epithelial cells may be particularly sensitive to oxidative damage and thus are a good model system in which to study the effects of oxidative stress.

Ultraviolet light induced DNA damage and repair in bovine lens epithelial cells

DNA damage caused by UV-B and UV-A irradiation and the rate of repair of such damage was quantitated in bovine lens epithelial cell cultures using a modified alkaline elution methodology. Two enzymes, bacteriophage T4 endonuclease V, which cleaves at the site of pyrimidine dimers, and E. coli endonuclease III, which cleaves at the site of thymine glycols, were utilized. Pyrimidine dimers were not detected after UV-A irradiation of lens cultures with up to 400 J/m2. In contrast, after exposure to as little as 2 J/m2 of UV-B irradiation, large numbers of pyrimidine dimers were observed. At higher fluences, thymine glycols were also found. Significant levels of DNA-DNA crosslinking were suggested by reduced rates of elution of DNA from cells treated with both UV-B irradiation and H2O2 in comparison to treatment with H2O2 alone. Protein-DNA crosslinks, in contrast, were not observed. The rate of repair of UV-B induced DNA damage was quantitated by harvesting cells at various times after the UV-B exposure. Single-strand breaks were never observed immediately after UV-B exposure but appeared later during the repair phase. In contrast to the repair of H2O2 induced DNA damage, which is largely completed within 30 min of exposure, more than 50% of the UV-B light induced DNA damage remained unrepaired five hours after exposure. This difference between the rate of repair of H2O2 and UV-B induced DNA damage could provide valuable insights into the nature of DNA damaging agents in the lens environment and may reflect underlying differences in the potential for epithelial cell DNA mutation in response to various DNA damaging insults.

Non-tryptophan fluorescence associated with human lens protein; apparent complexity and isolation of bityrosine and anthranilic acid.

Abstract In order to characterize the age-related protein bound fluorescent compounds of human cataractous lenses, the insoluble yellow protein component was proteolytically digested and then fractionated using ion-exchange, gel filtration and high pressure liquid chromatography. A complex and heterogeneous mixture of fluorescent components was found to be associated with these lens proteins. In the course of this study 3,3′-bityrosine and anthranilic acid were structurally identified as constituents of the proteolytic hydrolysate. The latter compound appears to be a degradation product of an unstable precursor.

Glucosylation of human lens protein and cataractogenesis

Abstract Examination of glucosylation of lens protein was conducted utilizing tritiated BH4−. The overall results indicate that approximately 0.20 moles of tritium were incorporated per mole of protein. Similar results were obtained with normal and senile cataractous lenses with varying degrees of opacity. Furthermore no difference in the 3H incorporation was observed between soluble and insoluble protein fractions derived from these lenses. Investigation of selected polypeptides isolated from the senile cataracts gave comparable results. Protein isolated from diabetic lenses had only slightly higher levels of tritium incorporation, giving an average value of 0.27 moles per mole of protein. Analyses of the tritiated products indicate that approximately 50% of the incorporation is probably due to reduction of other types of compounds. These results suggest that glucosylation does not appear to be a primary factor in cataract formation.

The phosphorylation sites of the B2 chain of bovine α-crystallin

Abstract The B 2 chain of bovine lens α-crystallin is phosphorylated in a cAMP-dependent reaction. By analysis of 32 P-labelled chymotryptic peptides isolated from α-crystallin obtained from lenses labelled in organ culture, two phosphorylated B 2 chain fragments were found. Sequence analysis of the fragments gave the following results: Arg-Ala-Pro-Ser-Trp-Ile-Asp-Thr-Gly-Leu and Ser-Leu-Ser-Pro-Phe corresponding to residues 56 to 65 and 43 to 47, respectively. It is established by this work that B 1 is a phosphorylated post-translational product of B 2 . Both the A 2 and B 2 chains of α-crystallin are phosphorylated at a similar site with the sequence Arg-(X)-Pro-Ser. This is an unusual site for cAMP-phosphorylation since the phosphorylated serine is preceded by a proline residue. It may also be of significance that the other B 2 chain phosphorylation site even more radically differs from previously reported cAMP-dependent phosphorylation sites.