Direct chemical measurement of DNA synthesis and net rates of differentiation of rat lens epithelial cells in vivo: Applied to the selenium cataract

Abstract

This report describes a direct chemical method for rapidly estimating DNA synthesis and net rates of epithelial cell differentiation in the ocular lens in vivo. DNA synthesis in the lens of control and selenium-treated rats (12- or 13 days of age) was estimated by chemically isolating and measuring trichloroacetic acid (TCA)-insoluble 3H from the lens following injection of [3H]thymidine. Labeled substrate for DNA synthesis peaked in the lens at 1 hr after injection, decreased markedly by the third hour and was essentially gone by hour 12. Synthesis of labeled DNA in the lens was largely complete by about 3 hr. The [3H]DNA content of the whole lens, measured as TCA-insoluble 3H, remained constant for at least 4 months. The distribution of labeled epithelial cells between the epithelial-cell layer and fiber-cell mass was followed for up to 1 month after injection by measuring the ratio of [3H]DNA in the capsule (epithelial-cell layer) to lens body. Between days 2-3 and day 14 after injection, the ratio of [3H]DNA in the epithelial-cell layer to lens fiber cells decreased linearly in a semilogarithmic plot of the ratio vs. time; i.e. the rate of change of the ratio followed first-order kinetics. Thus, the rate constant (k) for the rate of change in the ratio of [3H]DNA in the capsule layer to lens body can provide an estimate of the percentage of the labeled epithelial cells which leave the capsule per day through differentiation into fiber cells. An apparent rate constant of 0.27 day-1 was estimated from the mean of five experiments; i.e. 27% of labeled epithelial cells were differentiating into cortical fiber cells per day. Therefore, about 70% of the germinative epithelial cells would be replaced every 4 days in these rats. This value is in good agreement with results of studies using autoradiographic technics. The selenium cataract is reported to involve rapid damage to lens epithelial cells. Incorporation of [3H]thymidine into DNA was decreased by at least 60% in the lens of selenium-treated rats. Selenium did not decrease the availability of substrate in the lens for DNA synthesis. The cause of the decreased incorporation of [3H]thymidine into the selenium-exposed lenses remains to be determined.