Interaction of cultured mammalian cells with WR-2721 and its thiol, WR-1065: implications for mechanisms of radioprotection.

An isothermal microcalorimeter was used to measure changes in heat flow when radioprotective drugs were added to cultured mammalian cells. The heat produced when WR-2721 was added continued for at least 90 min. WR-2721 was dephosphorylated by the cells to thiol (WR-1065) which oxidizes to disulphide. In the microcalorimeter, thiols give an immediate burst of heat due to this oxidation. A biological oxygen monitor revealed that WR-1065 and cysteamine rapidly consumed all the oxygen in culture medium. (10 mM WR-1065 deoxygenated medium in 2 min.) Rapid consumption of oxygen by radioprotective thiols indicates that they will not co-exist with oxygen for long in cells. This has two important implications with respect to mechanisms of radioprotection: (1) oxygen in tissues will be consumed rapidly and could result in local hypoxia; and, (2) at modest doses of protective agents the thiol will be consumed in oxic cells and hence very little will be available for reactions such as hydrogen donation. Our results indicate that anoxia is probably the principal mechanism of protection by aminothiols in mammals and aerated cells. This has major implications for clinical applications of radioprotectors and these are discussed.

A kinetic analysis of spontaneous rho- mutations in yeast.

Spontaneous mutation to the petite state at the level of the individual cell was studied in a haploid strain of yeast by the technique of pedigree analysis. Results indicated that (1) the mutability of rho+ cells within a population in log phase is variable; (2) rho+ mitotic buds are, on the average, about 50% more mutable than the rho+ cells from which they arose; (3) the mutability of a rho+ cell tends to decrease as it produces consecutive buds: (4) the probability that a mother cell will become rho- at or immediately subsequent to cell division is, on the average, one third the probability that its bud will be rho-; (5) most, if not all spontaneous rho- mutant cells contain mitochondrial DNA as judged from suppressiveness measurements. The data indicate that the spontaneous production of a mutant cell is a multi-step process. Neither a replicative advantage of defective mitochondrial DNA nor the existence of a "master" mitochondrial genome provides a satisfactory explanation of the process. Either selective dispensation of defective mitochondria to the bud at cytokinesis or normal retention by the mother cell of factors influencing the amplification or rate of induction of defective mitochondrial DNA could be involved.

Evidence of preferential pairing of chromosomes at meiosis in aneuploid yeast.

Meiotic pairing in homothallic S. cerevisiae was studied by tetrad analysis, using strains that were trisomic or tetrasomic for chromosomes. I. The disomic segregants of these strains produce tetrasomic spore colonies that can be distinguished by their phenotype. Results indicated the existence of preferential pairing and nonrandom assortment of chromosomes at meisosis I. The frequency of crossing over is apparently normal in at least some regions when nonpreferred pairing occurs.

Lethal sectoring and the delayed induction of aneuploidy in yeast.

Persistent lethal sectoring in a homothallic strain of yeast has been ascribed to tetrasomy for chromosome I. Such aneuploids can appear many generations after irradiation. The data thus indicate that an induced predisposition towards aneuploidy can be prolonged through successive post-irradiation cell divisions. Sporadic cell death in tetrasomics for chromosome I was found to result from a metabolic imbalance and not from a genetic instability conseqent to aneuploidy. This imbalance may be due to a dosage effect involving cistrons for ribosomal RNA since many of these are known to be located on chromosome I. Tetrasomy is not the only cause of persistent lethal sectoring; the phenomenon has been initiated through genetic recombination involving normal diploids. It has also been concluded that, in trisomics, equational division of the supernumerary chromosome sometimes occurs at the first meiotic division.