Protective Effect of the Thioltransferase Gene on In Vivo UVR-300 nm–Induced Cataract

Abstract

Purpose.: To determine the protection factor (PF) for glutaredoxin-1 (Grx1) with regard to UVR-induced cataract by comparison of in vivo ultraviolet radiation (UVR) lens toxicity between double knockout Grx1 ^−/− and Grx1 ^+/+ mice.

Methods.: Twenty Grx1 ^+/+ mice and 20 Grx1 ^−/− mice were unilaterally exposed in vivo to UVR for 15 minutes. Groups of four animals each received 0.0, 2.1, 2.9, 3.6, and 4.1 kJ/m² UVR-300 nm. At 48 hours after UVR exposure, light-scattering in the exposed and contralateral nonexposed lenses was measured quantitatively. Macroscopic lens changes were documented with dark-field illumination photography.

Results.: UVR-300 nm induced subcapsular and cortical cataract in Grx1 ^−/− and Grx1 ^+/+ mice. In both Grx1 ^−/− and Grx1 ^+/+, the light-scattering intensified with increased in vivo exposure doses of UVR-300 nm. The intensity of forward light-scattering was higher in the lenses of Grx1 ^−/− mice than in the lenses of Grx1 ^+/+ mice. The threshold dose for in vivo UVR-300 nm–induced cataract, expressed as MTD_2.3:16, was 3.8 in the Grx1 ^+/+ group and 3.0 in the Grx1 ^−/− group, resulting in a PF of 1.3.

Conclusions.: The PF is an objective relative measure of protective properties. The Grx1 gene is associated with an in vivo PF of 1.3. This result signifies that the presence of the gene allows a 1.3 times longer in vivo exposure to UVR, at equivalent irradiance, than the absence of the gene before early-onset, UVR-induced cataract occurs. This finding indicates the important role of the Grx1 gene in the oxidation defense system of the lens.

The current study was an exploration of the impact of the glutaredoxin gene on oxidative stress induced by in vivo exposure to UVR. 

It has been demonstrated epidemiologically that gene expression is associated with development of cortical ¹ and nuclear cataract, ² with heritability accounting for 53% to 58% of cortical and 48% of nuclear cataracts. It has also been found that cataractous lenses, compared with clear lenses, globally demonstrate decreased expression of genes for oxidative stress defense, structural proteins, chaperones, and cell cycle control proteins. ³  

The redox balance in the lens is important for maintenance of lens transparency. ^{4 –6} Reactive oxygen species (ROS), induced by oxidative stress, are thought to be important in the development of age-related cataract. ^{7 –9} Further, there is strong epidemiologic evidence of an association between cortical cataract and exposure to UVR from the sun. ^{10 –14} In addition, experimental data indicate that in vivo exposure to UVR causes oxidative stress associated with short-delay onset of increased light-scattering in the lens. ^{5,6,15 –20}  

In a lens exposed to oxidative stress, ROS attack thiol groups in proteins and induce disulfide bridges between or within proteins, PSSP, or between proteins and thiol containing small molecular compounds such as glutathione (G), PSSG, and cysteine (C), PSSC. ^{21 –23} Oxidation induced disulfide bridges may cause change in structure of functionally important proteins, leading to reduction or loss of function (e.g., inactivation of enzymes). Further, the cross-linking of proteins may cause aggregation of proteins into optically dense high molecular weight aggregates that cause local shifts in refractive index and thus light-scattering. ²⁴ In cataractous lenses, 70% of protein thiols are present as mixed disulfides, whereas in normal lenses only 10% of protein thiols are found as mixed disulfides. ²⁵  

To counteract oxidative stress, the lens is equipped with a well-developed oxidation defense system, comprising reducing nonenzymatic molecules (e.g., glutathione, vitamin C, vitamin E, and carotenoids) and enzymatic antioxidant systems, including superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and the glutathione-dependent thioltransferase (TTase). ^{7,15,26 –29} We have found that vitamin C supplementation, per se, has no protective effect on in vivo UVR-induced light-scattering in the lens, ³⁰ whereas vitamin E supplementation, per se, protects against UVR-induced light-scattering. ^5,15,16  

TTase, also called glutaredoxin (Grx or sometimes GRx or GLRX), is a subfamily of the thiol-disulfide oxidoreductase family ²⁹ and contributes to oxidation defense by specifically reducing PSSG to free sulfhydryl groups by dethiolation. ³¹ The isoform Grx1 is a cytosolic heat-stable protein (11.8 kDa) that is found ubiquitously in eukaryotic and prokaryotic cells. ³² The first evidence of presence of Grx1 in the lens was made in 1996. ³³ It was demonstrated that Grx1 activity showed strong resistance to oxidative conditions. ^34,35 Furthermore, the Grx1 gene expression doubles in lens epithelial cells exposed to oxidative stress. ^36,37 Löfgren et al. ³⁸ showed that lens epithelial cells from Grx1 ^−/− mice are more vulnerable to oxidative stress. Moreover, they demonstrated that when Grx1 ^−/− cells are restored with Grx1, the antioxidant activity returns to normal. It was demonstrated in the lens, that under persistent oxidative stress, Grx1 expression increases and that this increase is important for maintenance of thiol groups in the reduced state. ³¹  

The absolute sensitivity to a toxic agent is commonly estimated as the minimal dose required to induce effect, the threshold dose. The threshold dose can be estimated in a small sample experiment as the dose for which 16% of exposed individuals express more light-scattering than is expected in 97.7% of a nonexposed group of animals, the maximum tolerable dose (MTD_2.3:16). ³⁹ The MTD_2.3:16 for cataract formation after in vivo exposure of the C57BL/6 mouse to UVR-300 nm was found to be 2.9 kJ/m². ⁴⁰ We recently demonstrated that the absence of Grx1 sensitizes the lens to oxidative damage from in vivo exposure to UVR-300 nm. ⁴¹  

The protection provided by sunscreens is commonly measured as the threshold dose of UVR for erythema with the sunscreen, H_Th:Sunscreen, compared with the threshold dose for erythema without the sunscreen, H_{Th:No sunscreen}, protection factor (PF) (equation 1).  

 

 

The interpretation of the PF is that for the same irradiance of UVR, the use of the sunscreen allows a PF longer exposure time before erythema develops, compared with exposure without the sunscreen. In analogy, the in vivo PF for a gene (e.g., Grx1 with regard to a toxic response, such as UVR-induced cataract) can be quantitatively estimated as the ratio of the threshold dose with the gene compared with the threshold dose without the gene. 

The purpose of the present study was to measure the PF for the Grx1 gene (PF_Grx1) for in vivo UVR-300-nm–induced cataract. 

Materials and Methods

Experimental Animal

Six-week-old C57BL/6, Grx1 double-knockout mice (Grx1 ^−/−) and the same strain with intact Grx1 gene (Grx1 ^+/+) were the experimental animals. The Grx1-knockout mouse was characterized by depletion of exons 1 and 2 of the mouse Grx1 gene ⁴² and was bred on a 129SV and C57BL/6 hybrid background. All animals were kept and treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The use of animals for this experiment was approved by the Northern Stockholm Animal Experiments Ethics Committee, protocol N241/08. All animals were genotyped at the animal care facility, St. Erik Eye Hospital. 

Exposure to Ultraviolet Radiation

UVR Source.

The radiation from a high-pressure mercury arc lamp (HBO 350W; Osram Sylvania, Mississauga, ONT, Canada) was collimated and passed through a water filter and a double monochromator. This process resulted in UVR-B in the 300 nm wavelength region (full width at half maximum = 10 nm), ⁴³ or UVR-300 nm. 

UVR irradiance at the corneal plane was measured with a thermopile (model 7101; Oriel, Stratford, CT) calibrated to a traceable source from the American National Standard Institute. UVR irradiance was checked before and after each UVR exposure. 

UVR Exposure.

Ten minutes before UVR exposure, the animal was anesthetized with a mixture of 40 mg/kg ketamine and 5 mg/kg xylazine injected subcutaneously. Five minutes after the injection, tropicamide was instilled into both eyes to induce mydriasis. Before exposure, all animals were checked with a slit lamp to exclude preexisting cataract but none was found. The left eye in each mouse was exposed in vivo to UVR. The exposure time was 15 minutes based on previous studies. ^44,45 The contralateral, nonexposed eye was lubricated and shielded during exposure and kept as a control. After exposure, lubricating eye ointment was instilled into both eyes. 

Quantification of Intensity of Forward Light-Scattering

Each animal was euthanized at 48 hours after the start of the UVR-exposure. The latency period was chosen to record the maximum intensity of light-scattering induced at short delay after in vivo exposure to UVR in the C57BL/6 mouse. ⁴⁶ Then, the eyes were enucleated and the lenses were extracted microsurgically using an operation microscope. Remnants of the ciliary body were removed from the lens equator while keeping the lens in balanced salt solution (BSS; Alcon Sverige, Bromma, Sweden). One of the Grx1 ^−/− lenses was damaged during the dissection and was excluded from data analysis. 

Experimentally induced cataract was quantified as intensity of forward lens light-scattering, measured with a light dissemination meter (LDM). ⁴⁷ The instrument uses the principle of dark-field illumination. In this instrument, light trans-illuminates the mouse lens measured, parallel to the lateral surface of a cone at 45° against the horizontal plane. At this angle, the light cannot enter the objective aperture. If the mouse lens scatters light, a defined fraction of the scattered light reaches the objective and is measured by a photodiode. An opaque lipid emulsion of diazepam (Stesolid Novum; Dumex-Alphapharma, Kobenhaven, Denmark) was used as a scattering standard. Light-scattering was therefore expressed in equivalent diazepam concentration (EDC). The readings expressed in EDC were further log transformed to tEDC to allow normal distribution statistics. ⁴⁷  

Macroscopic Structure of Light-Scattering

Immediately after lens light-scattering measurements, the macroscopic structure of light-scattering was visualized in a dark-field illumination, and images were recorded with digital photography. 

PF Associated with the Grx1 Gene

The PF associated with the Grx1 gene, PF_Grx1, was estimated in analogy to the PF for sunscreens (equation 1) as the ratio between the threshold UVR dose for light-scattering induction in Grx1 ^+/+ animals, MTD_2.3:16(Grx1 ^+/+), and the threshold UVR dose for light-scattering induction in Grx1 ^−/− animals, MTD_2.3:16(Grx1 ^−/−) (equation 2).  

 

 

The maximum tolerable dose 2.3:16 (MTD_2.3:16) was estimated as described elsewhere. ³⁹  

Experimental Design

Altogether, 20 Grx1 ^−/− and 20 Grx1 ^+/+ mice were used. For each genetic strain, the 20 animals were evenly distributed in groups of four animals on one of the dose groups: 0.0, 2.1, 2.9, 3.6, or 4.1 kJ/m² UVR-300 nm. Each animal was unilaterally exposed to UVR, with a dose of UVR depending on group belonging. Each lens was measured three times for forward light-scattering. 

The doses used were chosen to optimize the precision of the estimate of the regression parameters in accordance with equation 3. ³⁹  

 

 

The expected MTD_2.3:16, E(MTD_2.3:16), used was 2.9 kJ/m². ⁴⁰  

Statistical Parameters

The confidence coefficient was set to 0.95, considering the sample size and the expected precision of estimates. 

Results

Macroscopic Appearance

Both the Grx1 ^−/− and the Grx1 ^+/+ animals developed more lens opacity with increasing dose of UVR-300 nm, but the presence of the Grx1 gene appeared to be associated with less lens opacity than if the Grx1 gene was absent (Fig. 1). 

Figure 1.

 

Dark-field illumination macroscopic appearance of lenses from C57BL/6 mice after in vivo exposure to UVR-300 nm. Grx1 ^−/− indicates double knockout for glutaredoxin 1 and Grx1 ^+/+ indicates the same strain with the glutaredoxin gene intact. Scale bar, 1 mm.

Exposed lenses showed anterior subcapsular and cortical cataract. There was no nuclear cataract in any of the groups. 

Light-Scattering Measurement and PF

The difference of forward light-scattering between exposed and contralateral unexposed lenses increased with an increasing dose of UVR in both the Grx1 ^−/− and the Grx1 ^+/+ groups, respectively, but the increase was less expressed in the Grx1 ^+/+ group (Fig. 2). 

Figure 2.

 

Light-scattering difference between exposed and contralateral nonexposed lens at 48 hours after exposure, as a function of in vivo UVR-300 nm dose. Gray spots: lenses from glutaredoxin double-knockout animals (Grx1 ^−/−); dark spots: lenses from the same strain of animals with the glutaredoxin gene intact Grx1 ^+/+. Solid lines: the least-squares fit for differences between lenses originating from Grx1 ^−/− animals (gray curve) and Grx1 ^+/+ animals (black curve).

The threshold dose for least significant light-scattering increase in the lens after exposure to UVR-300 nm, estimated as MTD_2.3:16, was 3.0 kJ/m² in the Grx1 ^−/− mice (CI 0.95 = 2.3–4.0, df = 17) and for Grx1 ^+/+ mice, 3.8 kJ/m² (CI 0.95 = 2.8–6.4; df = 18). 

The PF for the Grx1 gene for protection against light-scattering in the lens after in vivo exposure to UVR-300 nm was 1.3 as calculated according to equation 2. 

Discussion

The present study was designed to elucidate the PF for the thioltransferase gene (Grx1). 

There have been reports that ketamine/xylazine anesthesia causes a transient increase of light-scattering within 5 hours of induction. ⁴⁸ However, it has also been shown that at extended periods after induction, there is no significant increase of light-scattering. ⁴⁹ In the present study, we observed the lenses at 48 hours after exposure to UVR. Therefore, it is unlikely that observed opacities were associated with the anesthetics. 

The fact that the lenses macroscopically developed more opacities with increasing dose of UVR at close to threshold dose (Fig. 1) is consistent with our previous findings for the C57BL/6 mouse. ⁴⁰ In the present study, only subcapsular and cortical opacities were observed macroscopically both in Grx1 ^−/− and in Grx1 ^+/+ mice lenses (Fig. 1). Our laboratory previously reported that also nuclear cataract may occur in Grx1 ^−/− and in Grx1 ^+/+ mice lenses, with an incidence of around 10%, ^40,46 but in a subsequent study, we found only anterior subcapsular opacities. ⁴¹ A similar low incidence of nuclear cataract was observed in the rat lens exposed to a near-threshold dose of UVR. ⁵⁰ Nuclear cataract was reported as an end stage of short-delay onset lens damage in the rat lens after a 10-times-threshold dose. ⁵¹ It was demonstrated that the in vivo penetration depth for UVR-300 nm in the rat lens is on the order of 0.5 mm. ⁵² Therefore, we believe that the infrequent nuclear cataract observed after a near-threshold dose reflects an occasional more serious biological response to the initial damage at the anterior surface caused by the exposure to UVR-300 nm. 

Our observation that the differences of light-scattering in lenses from Grx1 ^+/+ mice increased less than the differences of light-scattering in lenses from Grx1 ^−/− mice (Fig. 2) strongly support the notion that the Grx1 gene is protective. This conclusion is consistent with previous in vitro findings. ³⁸ Further, the current result supports the previous observation that Grx1 is resistant to oxidative stress. ^34,35  

The confidence intervals for MTD_2.3:16 presently estimated are relatively wide. This is an inherent problem in all threshold dose estimation. Threshold dose estimation relies on an estimate of the dose–response relationship and a criterion for least significant response. If the dose–response relationship is estimated at doses far from the least significant response and for a large enough interval, the change in response per change of dose within the interval studied can be estimated with high precision in a small sample. However, the dose–response is not reliable at low doses evoking close to a least significant response. At doses small enough to cause close to a least-significant response, variability is inherent and a highly precise estimate of threshold dose would require a sample size that is questionable in animal experiments. 

Considering UVR exposures at the same irradiance with and without protection, the PF expresses how much longer UVR exposure the protection allows before the toxic effect of the UVR exposure occurs. In the present study, we found that Grx1 ^+/+ compared with Grx1 ^−/− is associated with a PF of 1.3. Thus, an intact Grx1 gene allows a 1.3-times longer exposure to UVR-300 nm than if the Grx1 gene is absent before significant short-delay onset of light-scattering occurs. This finding is consistent with our previous results that Grx1 ^−/− increases lens susceptibility to UVR-B induced oxidative stress in the mouse. ⁴¹ It should be emphasized that both the current observation and the previous finding hold for short-delay–onset light-scattering, after single exposure to a near-threshold dose in vivo exposure to UVR. Whether Grx1 has the same protective effect on long-term daily subthreshold exposure is beyond the scope of the present study. 

Several extrinsic and intrinsic antioxidant systems have been described for the lens. ^{7,15,26 –29} A problem in determining the significance of each of these systems has been the lack of a method for quantitative comparison of the in vivo importance. The PF as presented herein aids in making that comparison. It may be that, at a low degree of oxidative stress, the relative impact of various antioxidants differs. However, the same strategy could be used for quantitative comparison of antioxidant systems in daily subthreshold exposures if the accumulated effect of daily subthreshold exposure evokes a least-significant toxic response. 

Conclusions

Estimation of MTD_2.3:16 permits comparison of the PFs among different antioxidant enzyme systems and antioxidants. In vivo, the Grx1 gene is associated with a PF of 1.3 for short-delay onset of light-scattering after in vivo exposure to UVR-300 nm. This indicates the important role of Grx1 in the oxidation defense system of the lens. Considering potential Grx1 polymorphism and chronic exposure to oxidant stress from UVR, the creation of drugs that stimulate or replace Grx1 function may be an option to delay cataract development. 

Footnotes

 Supported by the Swedish Radiation Protection Authority, Gun och Bertil Stohnes Stiftelse, Ögonfonden, Konung Gustav V:s och Drottning Viktorias Frimurarstiftelse, Swedish Research Council Project 2007-2859, and an Uppsala University fellowship for PhD students.

Footnotes

 Disclosure: M. Kronschläger, None; K. Galichanin, None; J. Ekström, None; M.F. Lou, None; P.G. Söderberg, None

The authors are grateful to the research group at Gullstrand Laboratory for inspiration and fruitful discussions and to Monica Aronsson for keeping the animals in good shape and spirit.