Mouse embryos at the preimplantation stage were used in vitro for assessing the risk exerted by arsenic on early stages of embryonal development. We studied the effects of sodium arsenite on morphological development, proliferation, and micronucleus formation. A concentration of 100 microM sodium arsenite immediately killed the embryos, and 1 microM almost completely inhibited formation of blastocysts. Micronuclei were induced starting from 0.7 microM arsenite. Concentrations below 0.1 microM (0.1 microM corresponds to 7.5 ppm arsenic) did not show significant effects with respect to any of the end-points examined. Arsenic did not influence radiation risk under various experimental conditions; all the effects after combining moderately toxic doses of sodium arsenite and X-rays corresponded to the effect obtained by the addition of the single effects.

The influence of a combination of caffeine (0.1 mM, 1 mM, or 2 mM) and X-rays (0.24 Gy, 0.94 Gy, or 1.88 Gy) on preimplantation mouse embryos in vitro was studied under different conditions. The agents were applied either singly or in combination. The embryos were irradiated in the G2-phase of the two-cell stage (28 h p. c. or 32 h p. c.) either 1 h after or immediately before application of caffeine. Caffeine was present during the whole incubation period (until 144 h p. c.). The effects on the microscopic visible development (formation of blastocysts 96 h p. c., hatching of blastocysts 144 h p. c.) and on the cell numbers of embryos at different times (48 h p. c., 56 h p. c., 96 h p. c., 144 h p. c.) were determined. We found conditions under which caffeine markedly enhanced radiation risk, i.e., under which the combination effect exceeded the sum of the single effects. This is true, in particular, for the embryonal development, for which the risk may almost be doubled, whereas the enhancement of risk is not so great for the proliferation of cells. In some cases the combination results lie even outside the envelope of additivity in the range of supra-additivity. The amount of caffeine necessary for such marked effects, however, is so high (at least 1 mM caffeine for rather long times), that it is almost impossible to reach them in vivo by consumption of caffeine-containing beverages.

Mercuric chloride (3 microM or 10 microM) increased several effects of ionizing radiation (1 Gy) on preimplantation mouse embryos in vitro. Blastocyst formation, hatching of blastocysts, and the number of cells per embryo were affected by this increase in radiation risk. The formation of micronuclei, however, was not influenced either in experiments using mercury alone or in combination experiments with X-rays. Not only was the sum of the single effects exceeded in some of the combination experiments, but the dose-pairs, which were necessary for obtaining a certain effect, clearly fell to the left of the envelope of additivity. That is, the enhancement of effects cannot be explained merely by the shape of the dose-response curves, but there is an interaction between mercury and ionizing radiation.