Clastogenic effects of cis-diamminedichloroplatinum. I. Induction of chromosomal aberrations in somatic and germinal cells of mice.

The clastogenicity of cisplatin, cis-diamminedichloroplatinum(II), an extensively used antitumor drug, has been studied employing (101/E1 X C3H/E1)F1 mice, aged 12-14 weeks. Chromosomal aberrations were assessed in mitotic divisions of bone marrow cells and differentiating spermatogonia. The drug was tested at 3 doses, 0.5, 1.0 and 2.5 mg/kg and 1.0, 2.5 and 5.0 mg/kg, respectively, for bone marrow and spermatogonia. Cisplatin had a clastogenic effect which was dose-dependent in both cell types. The frequencies of aberrant cells increased non-linearly in bone marrow and the dose-response relationship could be best described by a linear-quadratic equation. At the highest dose the affected cells carried multiple aberrations. An average of 2.7 aberrations per aberrant cell was observed 12 h after treatment of the mice with 2.5 mg/kg of cisplatin. In differentiating spermatogonia the dose response for aberrant cells could be described by a linear equation. The damage to the individual affected cell was less dramatic than in bone marrow, averaging 1.4 aberrations per damaged cell at the highest dose tested. Gaps were excluded from these considerations but they generally also showed a dose-related increase. A quantitative comparison of the clastogenic response to cisplatin was based on the dose-response relationships using 2 criteria, the doubling dose and the dose of unit increase (DUI). For both comparisons the general conclusion was that bone marrow cells were twice as sensitive as differentiating spermatogonia to the clastogenic action of cisplatin.

Molecular analysis of the TK locus in L5178Y large and small colony mouse lymphoma cell mutants induced by hycanthone methanesulfonate.

Mouse lymphoma cells of the L5178Y TK + /-3.7.2C line were exposed to varying concentrations of the anti-schistosomal drug hycanthone methanesulfonate. The trifluorothymidine (TFT)-resistant cells fell into two classes based on colony size. Southern blot analyses were performed using NcoI-digested DNA from a number of large and small mutant colonies from each treatment group. Two different restriction fragment banding patterns were identified in these analyses, those colonies that contained the 6.4 kb NcoI restriction fragment and those that did not. A total of 471 mutant colonies were analyzed and 84.5% (398) of these colonies did not exhibit the 6.4 kb fragment. There did not appear to be a hycanthone methanesulfonate dose response effect in the number of colonies that did not contain the 6.4 kb fragment among the treated groups. In addition, 82% (154 out of 188) of spontaneous mutants did not contain the 6.4 kb fragment. The results imply that greater than 80% of all spontaneous mutations found in the mouse lymphoma assay regardless of colony size do not contain the 6.4 kb fragment and each may be considered to be a large scale mutation. In addition, greater than 80% of the hycanthone induced mutations in the mouse lymphoma assay do not contain the 6.4 kb fragment and thus may be considered to be a large scale mutation.

Clastogenic effects of cis-diamminedichloroplatinum. II. Induction of chromosomal aberrations in primary spermatocytes and spermatogonial stem cells of mice.

The clastogenic effect of the anticancer drug cis-diamminedichloroplatinum (II) (cisplatin) on meiotic prophase in primary spermatocytes and on spermatogonial stem cells of male (101/E1 x C3H/E1)F1 mice was studied. The intraperitoneal doses of cisplatin tested were 5.0, 7.5 and 10.0 mg/kg. Chromosomal aberrations were examined at diakinesis-metaphase 1 of meiosis 1-13 days after treatment, representing cells treated at diplotene, pachytene, zygotene, leptotene an preleptotene. Reciprocal translocations were evaluated 63-70 days after treatment, representing treated stem-cell spermatogonia. Cisplatin had a toxic effect in zygotene to preleptotene of meiosis, as indicated by the significant reduction in testicular weight. At diplotene, pachytene and zygotene no enhancement of aberrations was found. An increase in aberrant cells was observed during leptotene with preleptotene being the most sensitive stage. The dose-response relationship for aberrant cells was linear on day 13 after treatment. It is concluded that, like mitomycin C (Adler, 1976), cisplatin primarily caused aberrations during the premeiotic phase of DNA synthesis. No significant increase of translocation multivalents was found after treatment of stem-cell spermatogonia.

Clastogenic effects of acrylamide in mouse bone marrow cells.

Acrylamide, known to induce dominant-lethal mutations (Shelby et al., 1986; Smith et al., 1986) and heritable translocations (Shelby et al., 1987) in rodent germ cells, was hitherto a questionable clastogen in rodent bone marrow (Shiraishi, 1978). Therefore, it was tested for chromosomal aberrations in mouse bone marrow cells, spermatogonia and by the micronucleus test. The intraperitoneally injected doses ranged from 50 to 150 mg/kg. In the chromosomal bone marrow test and the micronucleus assay positive results were obtained with acrylamide, and in the latter test the effect increased linearly with dose. Chromosomal aberrations were not induced in differentiating spermatogonia by the acute acrylamide treatment. Cisplatin was used as a positive control and gave the expected positive response in all 3 tests. The present results demonstrate that acrylamide is no exception among clastogens. It breaks chromosomes not only in mammalian germ cells but also in somatic cells.

Mutagenicity of antiviral substances of nucleobase analogue type in Salmonella typhimurium employing metabolic activation by mouse liver homogenate or cell-free plant extracts.

Five nucleobase analogues with antiviral properties were tested for their mutagenic activity in his mutant strains TA 1535, TA 1537, TA 1538, TA98, and TA 100 of S. typhimurium by means of preincubation tests with and without metabolic activation by cell free fractions from mouse liver (S-9) and maize seedlings (S-14). In one bacterial strain 6-azathymine increased the revertant counts in the absence of metabolic activation systems. In the presence of S-9 mix, the same substance became mutagenic for another tester strain. Metabolic activation by S-14 resulted in weak mutagenicity of 5-azadihydrouracil in high concentrations. 6-Azauracil, 5-azauracil, and 5-azadihydro-1,3-diacetyluracil were without mutagenic activity in all Salmonella-strains used. Cyclophosphamide, like other standard promutagens, was shown to become mutagenic in the presence of S-14 plant fraction. Thus S-14 activation system besides the S-9 liver system can be employed in mutagenicity testing with microbial systems.

Mutagenicity assay with Salmonella typhimurium revealing biotransformation of antiphytoviral substances by cell-free plant extract.

The mutagenic activity of four antiphytoviral substances was tested in reversion mutagenicity assays with a set of histidine auxotrophic strains of Salmonella typhimurium by means of the preincubation method. A possible metabolic activation of the substances by cell free fractions from maize seedlings (S-14-fraction) and for comparison from mouse liver (S-9 mix) was examined. None of the guanidine, phenyl urea and thiadiazole compounds exerted mutagenic activity in the bacterial strains in experiments without metabolic activation. Cyanoguanidine and N-phenyl-N-carboxyphenylurea became mutagenic for Salmonella strain TA98 after metabolic activation by the S-14 plant fraction. Both substances were not mutagenic in the presence of S-9 mix made from mouse liver. The promutagen cyclophosphamide proved highly mutagenic in experiments with S-14 mediated plant metabolic activation. This kind of bacterial mutagenicity assay is valuable in investigations of potential agrochemicals, as the examples have shown.

Yield and activity of Autographa californica multicapsid nucleopolyhedrovirus and Phthorimaea operculella granulosis virus in cloned and uncloned cell lines of P. operculella.

Three selected uncloned Pop 2, Pop 3, Pop 4 and two cloned cell lines Pop cl1A and Pop cl2B were derived from the original cell line established from Phthorimaea operculella (ORS-Pop-93). Three new non-selected cell lines ORS-Pop-94A, ORS-Pop-94B and ORS-Pop-95 were also established from embryos of the same insect. Differences in morphology, growth rate and polypeptide profile were determined between these cell lines. All the cell lines were susceptible to the Autographa californica nucleopolyhedrovirus (AcMNPV). The cloned cell lines produced higher levels of AcMNPV (TCID-50 and PIB) than the parental cells and at the same rate as the Sf9 reference cell line. Substantial amounts of viral DNA were synthesized in the clone Pop cl 2B after infection with the granulosis virus of the potato tuber moth P. operculella (PTMGV) and a complete multiplication was obtained in the ORS-Pop-95 cell line. The comparison between Pop cell lines which support limited or complete replication of certain baculoviruses can offer insights into some of the molecular barriers which restrict the host range of these viruses. These cell lines with variable susceptibility to baculoviruses could also be used for in vitro recombinations, increasing their virus host range to be used for the control of this pest.