Flow cytometric analysis of protein thiol groups in relation to the cell cycle and the intracellular content of glutathione in rat hepatocytes

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Abstract
The relationship between protein thiols (PSH) and cell proliferation was examined in ethanol‐fixed rat hepatocytes. A new protocol was developed for simultaneous measurement of protein thiol vs. DNA content by flow cytometry. The fluorescent dye o‐phthalaldehyde (OPT) was used for flow cytometric measurements of protein thiol groups. The influence of nonprotein thiols was examined by monitoring the cell cycle of cells in which the glutathione content (GSH) was modified by treatment with buthionine sulphoximine (BSO). Three rat liver cell lines (IAR 20, IAR 6.1, IAR 6.1RT7) were used: these cell lines possess different growth characteristics and degrees of tumorigenicity, which made it possible toanalyse changes in PSH during normal and deranged cell proliferation. The effects on the cell cycle of the changes in PSH due to the depletion of GSH were measured by 5‐bromo‐2'‐deoxyuridine (BrdUrd) incorporation and flow cytometry. The data obtained can be summarised as follows: (a) OPT fluorescence increases with increasing DNA content in all rat liver cell lines examined; (b) the greatest variation in PSH content occurs in G1. There is a smaller variation in G2 + M, and PSH levels are relatively invariant throughout S‐phase; (c) a higher content of PSH is found in the tumorigenic cell lines; (d) the amount and distribution of PSH is not affected by BSO treatment; (e) kinetic studies indicate that BSO treatment has no effect on the ability of the IAR rat liver cell lines to progress through the cycle.