Lens and eye toxicity research最新文献

Perfusion of the isolated rabbit corneal endothelium with 0.3 mM hydrogen peroxide (H2O2) caused an increased passive permeability to bicarbonate relative to control tissues. This was accompanied by a reduction in the active flux that resulted in a reduced net bicarbonate flux. Perfusion with 0.3 mM H2O2 resulted in a marked increase in the active and net flux of sodium beginning at two hours. By four hours the net sodium flux had increased by nine-fold over control values. Perfusion with 0.3 mM H2O2 resulted in a 16% and 30% increase in endothelial permeability to inulin and dextran, respectively. Suppression of catalase activity by in vivo pretreatment with intravenous 3-aminotriazole (3AT) did not result in an increased sensitivity of the corneal endothelium to 0.2 mM H2O2: both bicarbonate and sodium fluxes were normal. Inhibition of glutathione synthesis with intravitreal buthionine sulfoximine (BSO) increased the sensitivity of the corneal endothelium to 0.2 mM H2O2 only in the case of sodium flux, with a 4.8-fold increase in net sodium flux at 3 hours after initiation of perfusion. Bicarbonate fluxes were unaffected after BSO pretreatment. The data show that ionic and non-ionic fluxes are altered by H2O2, that pretreatment with 3AT has a minimal effect on ion fluxes while BSO markedly alters sodium flux without changing bicarbonate fluxes, and that sodium and bicarbonate movement are not locked in a symport.

Cell attachment to a suitable substratum is a precondition for the mitotic growth of nontransformed lens epithelial cells. Cultering of cells in suspension results in a strong decline of the DNA synthetic rate, whereas reattachment induces the reentrance into the cell cycle. Further studies revealed that not anchorage itself but cell flattening is prerequisite for the entrance of cells into the cycle. Flattened cells exert tension to the substratum via numerous filopodia. If the rigidity of the substratum is reduced by loosening of the collagen gel from the bottom of the petri dish, the gel becomes contracted by the traction forces of the cells and the cell shape becomes transformed from a flattened shape into a more spheroidal or longstretched one. This cell shape transition is connected with a decrease in RNA- and protein synthesis and a stop of DNA synthesis. During further experiments it was demonstrated that microfilaments are involved in gel contraction and cell shape alteration, respectively. Furthermore, intact microfilaments are needed for G0-G1-S-transition. Desintegration of microfilaments by cytochalasin is without influence on ongoing DNA synthesis but hinders strongly the entrance of cells into the S-phase. The survey gives some recent results on the molecular basis of cell substratum interactions as well as the structure and function of the cytoskeleton. The role of the cytoskeleton in cell shape-mediated growth regulation is discussed.

The present work discusses the role of certain coenzymes in the metabolic and the biophysical processes maintaining the nativity of lens components. It also analyses results of the levels of oxidized and reduced forms of nicotine amide coenzymes and glutathione in the lens folling insults by both physical and chemical cataractogenic agents. The role of flavine and flavine coenzymes in the maintainance of the biochemical and biophysical stability of the lens has also been discussed. Data concerning the enzymatic biosynthesis and degradation of the lens coenzymes has been presented.

Because the organogenesis and physiology of the lens are essentially similar in various mammals, an understanding of the etiology and pathogenesis of the formation of cataract in an animal model will enhance our knowledge of cataractogenesis in man. In this review, we summarize the background, etiology, and pathogenesis of cataracts that occur in rodents. The main advantages of using rodent mutants include the well-researched genetics of the animals and the comparative ease of breeding of large litters. Numerous rodent models of congenital and hereditary cataracts have been studied extensively. In mice, the models include the Cts strain, Fraser mouse, lens opacity gene (Lop) strain, Lop-2 and Lop-3 strains, Philly mouse, Nakano mouse, Nop strain, Deer mouse, Emory mouse, Swiss Webster strain, Balb/c-nct/nct mouse, and SAM-R/3 strain. The rat models include BUdR, ICR, Sprague-Dawley, and Wistar rats, the spontaneously hypertensive rat (SHR), the John Rapp inbred strain of Dahl salt-sensitive rat, as well as WBN/Kob, Royal College of Surgeons (RCS), and Brown-Norway rats. Other proposed models for the study of hereditary cataract include the degu and the guinea pig. Because of the ease of making clinical observations in vivo and the subsequent availability of the intact lens for laboratory analyses at different stages of cataract formation, these animals provide excellent models for clinicopathologic correlations, for monitoring of the natural history of the aging process and of metabolic defects, as well as for investigations on the effect of cataract-modulating agents and drugs, including the prospect of gene therapy.

We have investigated the effects of 0.02 and 0.2% thymoxamine hydrochloride on the isolated rabbit corneal endothelium. The corneal swelling rate, measured by specular microscopy, indicated that 0.02% thymoxamine caused a swelling rate equal to controls while a 0.2% concentration caused a significantly increased swelling rate (34.1 vs 10.3 microns/h; P less than 0.05). The data suggests that the maximum recommended intracameral concentration of thymoxamine be 0.02% in order to allow a 10-fold safety factor for the corneal endothelium.

Evidence supporting the role of non-enzymic post-translational modification of lens proteins in cataract is reported as presented at a meeting in Bydgoszcz, Poland in August 1990. Glycation and carbamylation have been studied intensively recently. Both produce modified proteins with properties similar to those of 'molten-globule' intermediates of protein folding and unfolding pathways.

Lanthanum nitrate (LN) and horseradish peroxidase (HRP) were used as tracers to study intercellular permeability as well as the existence and location of tight junctions and changes in them, if any, in the lens epithelium of Sprague Dawley rats. Thin sections of lenses taken from animals at birth and at three day intervals until the neonates were weaned at approximately 22 days were studied at the electron microscope level. At every age both tracers permeated the intercellular spaces of the anterior region epithelium and between the fiber cells of the equatorial region. The LN precipitates terminated near the apical part of the epithelium in the anterior polar region and were not seen at the epithelial-fiber interface or between fibers in this region. At the site of the tracer termination the intercellular space was found to be considerably constricted or completely sealed. The HRP precipitates, however, were present in these intercellular spaces and by using the HRP "washout" procedure could be washed out of the basolateral site of the intercellular spaces of the epithelium up to the point of constriction if a tight junction were present. The lens area occupied by epithelium with tight junctions was age related being very small in lenses from animals less than 24 hours old and becoming increasingly enlarged as the newborn aged. These findings further confirm the existence of a barrier in the form of tight junctions between epithelial cells in the central anterior region of the maturing rat lens. Moreover, our observations also provide information regarding the pattern of tight junction development and distribution during lens maturation.

The crystallin genes encode the major soluble proteins of the lens. Some of the crystallin genes are expressed exclusively in the lens while others are also expressed in different tissues. The two alpha-crystallin genes, alpha A and alpha B, differ in their tissue specificity. Transcription of the alpha A-crystallin gene occurs only in the lens, while the alpha B-crystallin gene is also expressed in other tissues, including heart, skeletal muscle, kidney, lung and brain. MIP (also called MP26), the major intrinsic protein of the lens fiber membranes, is also expressed exclusively in the lens. Correct expression of both alpha-crystallin and MIP are required for normal lens function. Here we review our studies on the molecular basis of expression of the alpha-crystallin and MIP genes in the lens. The 5' flanking sequences containing the initiation site of transcription of the alpha A-crystallin, alpha B-crystallin and MIP genes were fused to the bacterial chloramphenicol acetyltransferase (CAT) gene, and the expression of this reporter gene was studied in transient assays and transgenic mice. DNA sequences flanking the 5' end of the alpha A-crystallin gene contain regulatory elements responsible for the lens-specific expression and developmental regulation of the CAT gene in transgenic mice. Interestingly, although some of the murine alpha A-crystallin regulatory sequences are conserved in the human and chicken genes, different functional regulatory elements appear to control the expression of the murine and chicken alpha A-crystallin genes. The 5' flanking sequence of the alpha B-crystallin gene preferentially directs expression of the CAT gene to the lens and to skeletal muscle. Different regulatory elements of the alpha B-crystallin gene appear to be responsible for its transcription in various tissues. The 5' flanking sequence of the MIP gene also contains regulatory elements that direct expression of the CAT gene to lens cells; these sequences are not functional in transfected non-lens cells and are different from the cis regulatory elements controlling alpha-crystallin gene expression. The multiplicity of cis-regulatory elements controlling the transcription of these three genes indicates the complexity of the mechanisms that regulate gene expression in the lens.

The naphthalene cataract in the pigmented rabbit, in contrast to the corresponding model in the pigmented rat, is characterized by a rather unstable cataract development during the later stages. Some animals rapidly develop mature cataracts, others develop cortical cataracts with almost no further progression inspite of a continued naphthalene treatment. To get more insight into this phenomenon, a study with pigmented rabbits (8 weeks old) was designed and at different stages of cataract development lenses were separated in single layers with the Bonn freeze-sectioning device. The key enzymes of the carbohydrate metabolism and the redox potential were determined in those layers. The results of the biochemistry failed however to explain the prominent differences of cataract development in-vivo. We conclude from these findings that the naphthalene cataract model in the pigmented rabbit shall no longer be used in drug side-effect or efficacy studies.

Raman spectra from the lenses of rat and mouse were measured in situ at various conditions of lens aging and opacification. Laser Raman spectroscopy is a powerful nondestructive structural probe to provide us useful informations such as hydration or dehydration state, thiols and its oxidation or micro-environmental state of tryptophan or tyrosine residuse of the lens proteins. In this paper we summurized our Raman studies on various normal, aged and cataractous lenses. Lens dehydration was a common phenomena during normal aging. Regional distribution of lens water showed lens fiber aging from nucleus to cortex. On the other hand lens hydration was a common in the lens with opacification. Using regional water content as a diagnostic marker, the effect of aldose reductase inhibitor was monitored objectively. Lens slfhydryl (SH) groups were reduced with age and with opacification in general, and concomitant increase in disulfide (S-S) bonds was detected. In ICR/f rat lens the decrease in SH groups and simultaneous increase in S-S bonds were found even before lens opacity was manifest.