In vitro toxicology of heavy metals using mammalian cells: an overview of collaborative research data.

The uptake and distribution of selected heavy metals were followed and related to cytotoxicity using various parameters of proliferation and viability of cultured cells. The effects of short-term lead exposure on DNA synthesis were reversible, indicating that lead does not significantly influence genetic cellular function. In contrast, nickel effects persisted, indicating that DNA is one of the main nickel targets. Heavy metals affected all cycle phases, but those related to preparation and commencement of DNA synthesis were the most susceptible. Tolerance appeared in chronic exposure to lead and cadmium. Lead combined with X-rays had additive effect, while manganese acted synergistically and appeared to inhibit the DNA repair processes. Zinc and manganese showed a protective effect against the toxic effects of cadmium. Similar antagonistic interaction was seen for nickel v. manganese cytotoxicity. This model system makes it possible to compare heavy metal effects at the cellular level and to identify cellular targets and metabolic processes.

Cell survival after the combined action of manganese (MnCl2) and X-rays in synchronized Chinese hamster cells.

The interactions between the effects of manganese chloride and X-rays were studied in synchronized populations of V79 Chinese hamster fibroblasts. The cells were selected by shaking off asynchronous cultures for detachment of mitotic cells which were plated in petri dishes and exposed to various treatments. Irradiation was carried out with a Philips RT-100 X-ray unit. A final concentration of 0.25 mM MnCl2 was used. The main parameter was the colony forming ability of the surviving cell fraction. When MnCl2 was administered over 1 h, its toxicity was low regardless of the phase of the cell cycle. Administered separately, 2 Gy irradiation produced only a slight decrease in survival, less marked in the S phase. However, the two agents together induced a synergistic inhibition of the surviving fraction in the S phase when the metal was given immediately after irradiation. If manganese was administered 3 h after irradiation the two inhibitory effects apparently remained only additive. It seems that MnCl2 can impair some repair processes starting immediately after irradiation.

Cyclic nucleotides affect growth and differentiation of cultured rat embryonic shields.

The modified organ culture of rat embryonic shields provides favorable conditions during 2 weeks for the differentiation of main tissue types. Since the terminal differentiation in explants is inferior to that obtained in the homografts of the same shields under the kidney capsule, we tried to improve the culture medium by adding some known regulatory molecules: db-cAMP, db-cGMP, ATP, AMP, and butyric acid. These agents were added to the liquid medium in the concentration of 1 mM. In the first part of the study the explants were fixed and weighed after 8 or 14 days in vitro culture, and histological sections were examined. When the explants were treated with db-cAMP during the second week of culture, the skeletal muscle appeared more frequently in the treated series than in controls, and the weight of the treated explants was sometimes increased when compared with the control series. The db-cGMP had no effect on differentiation, but stimulated the growth of the explants when applied during the first week of culture. On the contrary, the db-cAMP when added during the first week, severely impeded the growth of explants. Other agents seem to be ineffective. In the second part, the content of cAMP and cGMP was measured in normal explants. The radioimmunoassay showed the same content of cAMP and cGMP during the entire culture period. In the third part of our study the incorporation of tritiated uridine and tritiated thymidine was measured during the second week of culture after the addition of db-cAMP. During the first days of treatment with db-cAMP the uptake of tritiated uridine and thymidine was inhibited, whereas on the seventh day the uptake was similar to that of the control. We can conclude that both cyclic nucleotides have a visible effect on growth whereas only cAMP has a positive impact on the differentiation of myotubes in cultured rat embryonic shields.

Toxicity of nickel for mammalian cells in culture.

The effects of nickel chloride were studied in two human cell lines, HeLa and diploid embryonic fibroblasts, as well as in V79 Chinese hamster cells and in L-A mouse fibroblasts. NiCl2 produces a dose-dependent depression of proliferation and mitotic rate. Effects on viability are accompanied by an increasing release of the intracellular enzyme lactic dehydrogenase. Lactic acid production is stimulated. The plating efficiency is reduced, as are DNA and protein synthesis and, to a lesser degree, RNA synthesis. Comparing these results with those of previous studies of the cytotoxicity of other heavy metals in the same test systems, similar effects are observed though with different intensities and slight differences between the cell lines employed. As regards lethal effects (LC50) the following rank order of cytotoxicity can be established: Ni2+ approximately equal to Pb2+ less than Mn2+ less than Hg2+ less than Cd2+; as regards growth inhibition the same rank order is observed as in the case of the LC50 in HeLa and human fibroblasts, but in L-A cells Ni2+ is more inhibitive than the other metal ions listed above with the exception of Cd2+. With respect to colony formation NiCl2 is less effective than PbCl2, MnCl2, and CdCl2. NiCl2 effects in serum-free medium are much faster and more severe than in medium containing serum or serum albumin indicating that serum constituents, notably albumin, bind the metal effectively and inhibit cellular uptake; this confirms reports of other authors on the serum binding and slow uptake of NiCl2. Synchronized cells are most sensitive in the G1 and early S phases of the cell cycle. Together with the finding that thymidine incorporation is affected to a considerable degree this contributes an explanation of the known genotoxic effects of nickel.

The rate of DNA synthesis in HeLa cells during combined long-term and acute exposures to lead.

Asynchronous populations of HeLa cells growing as monolayers were incubated in a nutritive medium supplemented with a low concentration of 10(-5) M lead chloride for several months. At various time intervals the [3H]thymidine uptake was measured in the DNA of the cells by a scintillation counting technique. During the first 2 weeks of incubation, the rate of DNA synthesis was slightly reduced. After 3 weeks the synthesis returned to normal and later it was a little enhanced. The pretreated cells tolerated acute intoxication by a high lead concentration of 2.5 x 10(-4) M without a change in the rate of DNA synthesis. The cells also proliferated 1 year in a culture medium supplemented with a progressively increasing lead concentration. However, the survival of cells at the highest lead concentration lasted only a few days. After return to a metal-free medium the cells again became very sensitive to lead as shown by the reduced rate of DNA synthesis. The transitory character of adaptation indicates that there was no direct genetic involvement.

[Cytotoxicity of manganese for mammalian cells in vitro--comparisons with lead, mercury and cadmium].

The effects of manganese chloride were studied in two human cell lines HeLa and human embryonic diploid fibroblasts in V79 Chinese hamster lung cells, and L-A mouse fibroblasts. Manganese produces a dose-dependent depression of proliferation along with a decrease of the mitotic rate. Effects on viability are accompanied by increased release of the intracellular enzyme lactic dehydrogenase. Lactic acid is stimulated indicating enhanced glycolytic activity. The colony forming ability and the rate of DNA synthesis are inhibited in a dose- and time-related fashion. Comparing these results with those of previous studies of the cytotoxicity of lead, mercury, and cadmium in the same test systems, similar effects are observed, though with different intensities and slight differences between the cell lines. As regards depression of viability, the following rank order of toxicity can be established: Pb2+ < Mn2+ < Hg2+ < Cd2+; as regards reduction of proliferation lead is less and cadmium more effective than manganese, while mercury effects vary depending on the cells tested. Concerning colony formation and DNA synthesis the toxicity of manganese is again higher than that of lead and manganese, especially the influence on DNA synthesis, show immediate recovery after cessation of exposure indicates that the genetic material is not directly involved and that the effects on proliferation colony formation, and DNA synthesis are the consequences of several elaborate processes at the cellular level.

[Lead and cadmium toxicity in established mammalian cell lines]. / Toxicité du plomb et du cadmium pour des lignées continues de cellules de Mammifères.

Lead and cadmium toxicity was evaluated in three mammalian cell lines (tumour : HeLa, transformed : XC and normal : NRK) by means of the modifications of the 3H-TdR incorporation rate in the nucleus of the treated cells. The three cell lines showed different degrees of sensitivity. Sensitivity depended on the line, metal, its concentration and duration of incubation. Cadmium was found to be at least five times more toxic than lead except at low concentration. The normal cell line was more sensitive to cadmium and less sensitive to lead than other lines.

Reversible inhibition of DNA, RNA and protein synthesis in human cells by lead chloride.

An acute intoxication by lead chloride (conc. 2.5 - 10(-4)M) produces a temporary reduction of macromolecular syntheses in HeLa cells growing asynchronously. The reduction is similar for DNA, RNA and proteins and differs only in the intensity. After a one-day intoxication, if the cells are put back in a fresh medium, the syntheses return to normal within 10 h. The histochemical sulphide-silver method shows that lead is present in the cells during the inhibition.