Protection of hydroquinone-induced apoptosis by downregulation of Fau is mediated by NQO1.

The Fau gene (Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV)-associated ubiquitously expressed gene) was identified as a potential tumor suppressor gene using a forward genetics approach. Downregulation of Fau by overexpression of its reverse sequence has been shown to inhibit apoptosis induced by DNA-damaging agents. To address a potential role of Fau in benzene toxicity, we investigated the apoptotic effects of hydroquinone (HQ), a major benzene metabolite, in W7.2 mouse thymoma cells transfected with either a plasmid construct expressing the antisense sequence of Fau (rfau) or the empty vector (pcDNA3.1) as a control. HQ induced apoptosis via increased production of reactive oxygen species and DNA damage, measured using dihydroethidine (HE) staining and alkaline Comet assay, respectively, in W7.2 pcDNA3.1 cells. In contrast, when Fau was downregulated by the antisense sequence in W7.2 rfau cells, HQ treatment did not cause DNA damage and oxidative stress and these cells were markedly more resistant to HQ-induced apoptosis. Further investigation revealed that there was an upregulation of NAD(P)H: quinone oxidoreductase 1 (NQO1), a detoxification enzyme for benzene-derived quinones, in W7.2 rfau cells. Compromising cellular NQO1 by use of a specific mechanism-based inhibitor (MAC 220) and NQO1 siRNA resensitized W7.2 rfau cells to HQ-induced apoptosis. Silencing of Fau in W7.2 wild-type cells resulted in increased levels of NQO1, confirming that downregulation of Fau results in NQO1 upregulation which protects against HQ-induced apoptosis.

Light scattering and morphology of cataract formation in transgenic mice containing the HIV-1 protease linked to the lens alpha A-crystallin promoter.

Two constructs of transgenic mice, TG61 and TG72, containing the HIV-1 protease linked to the lens alpha A-crystallin promoter develop cataract. The TG61 construct develop cataract in utero, while the TG72 construct exhibit frank opacities on the 24th day (homozygotes) and 26th day (hemizygotes) post natum. Polarized light scattering measurements were performed on cortical and nuclear sections of TG72 lenses from day 19 to day 26 as a function of scattering angle. The parallel components of the scattered light intensity increase gradually during opacification, the perpendicular components show very low values from day 19 to day 22 after which they increase exponentially. Analysis of the scattered light intensities yielded parameters describing the size of the protein aggregates, the size of the domains exhibiting optical anisotropy/birefringence, the difference in refractive index between (a) the protein aggregates and their surroundings and (b) the birefringent units and their surroundings. The last parameter accounts for the major portion of the increase in lens turbidity. The TG72 construct shows normal lens development on day 16. By day 21 the posterior cortex shows some disintegration, while the lens is still clear. By day 26 the lens nucleus migrates toward the posterior pole and there is a major alteration in the cortical fibers. Scanning electron microscopic studies reveal normal fiber cell organizations in control animals. In the TG72 construct the fiber cells are well organized at 16 days after birth but already develop some slight separation in the posterior cortical part of the lens. By post-natal day 21, the nucleus and the anterior cortex still exhibit well aligned fiber cell organization, but the posterior cortex shows disalignment. By day 26 in the TG72 construct, all areas of the lens show complete disintegration of the fiber cells and amorphous masses are present throughout. The light scattering parameters describing changes on the nanometer scale can be correlated with the changes in lens morphology during cataractogenesis that occur on the micrometer scale. In comparison, the light and scanning electron microscopic examinations of the postnatal TG61 construct show that the lens is severely disrupted and contains completely disintegrated fiber cell remnants at an early age.

Light scattering parameters of rat lenses with calcium-induced cataracts.

Light scattering intensities of rat lenses obtained in the I,, and I+ modes were analysed using the random density and orientation fluctuation theory. Rat lenses incubated in calcium rich media had the same density fluctuation parameters as rat lenses incubated in control (low-calcium) media. However, the correlation length of the orientation fluctuations decreased during cataract formation by 100 to 200 nm while the amplitude of the fluctuations increased. The correlation length, or the size of the optically anisotropic domains, is related to the size of the cytoskeleton, especially vimentin. Vimentin has been shown to degrade when calcium activates calpain. This has been observed in SDS-gel electrophoretic experiments in rat lenses in calcium rich media. The amplitude factor of orientation fluctuations, that is, the mean squared deviation from the average refractive index, increased between two- and seven-fold during cataractogenesis. These results indicate that calcium cataract formation at the beginning (first 72 hr incubation) has little to do with aggregation or syneresis but it is largely the result of changes in the intrinsic birefringence of the lens due to vimentin degradation.

The effect of freezing on human cortical cataracts.

To ascertain a direct relationship between the light scattering intensity and the particle scattering the light, thin (6-20 micrometers) sections were cut from frozen human cataracts. Freezing for 15 min (-10 C) and then thawing had no effect on the clarity or light scattering properties of nuclear cataracts. Cortical cataracts treated in the same manner appeared to be much clearer than they were prior to freeze-thawing. Light scattering measurements were obtained as a function of scattering angle, both in the I parallel and I perpendicular mode; a He-Ne laser was used. Results showed a reduction in the light scattering intensity in the I parallel and I perpendicular modes with repeated cyclical freezing and thawing and that this decrease was most pronounced at higher scattering angles. Morphologic data showed that much of the anatomical heterogeneity present in the human cortical and rat lens osmotic cataract disappeared upon freeze-thawing. However, even though the anatomical heterogeneity was less, there was persistence of serious cellular anatomical disorganization in grossly transparent parts of the cortex. This suggests that transparency is not dependent upon the presence of intact fiber cells. The importance of these findings to studies of light scattering, cataract classification and analysis of cryoextracted human lenses is discussed.

Effect of change in concentration upon lens turbidity as predicted by the random fluctuation theory.

Theoretical calculations were performed to predict how the light scattering intensity would change with changes in concentration in the gel state. The theory of light scattering was applied to a random distribution of hard spheres. The spherical particles with constant diameter were embedded in a medium having a different refractive index. The light-scattering intensities obtained as a function of concentration showed that in dilute solutions the scattered light intensity increases with concentration. However, in concentrated solution greater than 0.1 or 0.2 volume fraction, the light-scattering intensity decreases with increase in concentration.

Studies on human cataracts. II. Correlation between the clinical description and the light-scattering parameters of human cataracts.

Eight lenses with nuclear cataracts have been classified. The light-scattering properties of the eight lenses were obtained with thin sections cut perpendicularly to the posterior-anterior axis. Analysis of the light scattering envelop in the I and I+ models yielded eight structural parameters that describe the density and orientation fluctuations. The variation of these parameters within each less corresponded well to the clinical description obtained from the stereoscopic photos.

Studies on human cataracts. III. Structural elements in nuclear cataracts and their contribution to the turbidity.

Gross correlation coefficient between light-scattering intensity and the optical parameters obtained for 48 sections of eight lenses with nuclear cataracts were evaluated. On the basis of these and other data in the literature, the structural elements, within the lens fiber are identified. These give rise to the optical parameters. It is proposed that three processes contribute to nuclear cataractogenesis: (1) syneretic process, (2) increase in the concentration but not in the size of protein aggregates, and (3) association (entanglement) between aggregates and optically anisotropic cytoskeleton or membrane components that leads to a decrease in structural bire-fringence.