The induction of chromosomal structural changes in male chickens by the alkylating agents: triethylene melamine and ethyl methanesulfonate

E. coli chromosomal DNA wastreated with various Pt co-ordiantion compounds and then used as donor DNA in E. coli transformation. Genetic analysis of transformants obtained with Pt-treated DNA showed effects of cis-diamminedichloroplatinum(II) (cis-Pt(II)) and cis-dimethyl-1,3-diaminopropane CL₄ (cis-Pt(IV) (DMDAP) on the processing of DNA. With trans-diamminedichloroplatinum(II) (trans-Pt(II)) appllied in similar concentrations no effects were found.The effects of cis-Pt(II) and cis-Pt(IV) (DMDAP) on the genetic processing were different. The effects of cis-Pt(II) could be explained by assuming intra-strand crosslinks as an important lesion.     

An evaluation of the micronuclei test using triethylenemelamine,trimethylphosphate, hycanthone and niridazole

To determine the feasibility of the micronuclie procedure for cytogenetic studies, a comparatively weak chromosome breaking agent, trimethylphosphate (TMP) and the potent alkylating agent, triethylenemelamine (TEM) were evaluated. The procedure followed was that of Matter and Schmid with the following modifications: (a) direct flushing of bone marrow with 0.2 ml calf fetal serum, (b) air drying slides for a period of only 1 h, and (c) the use of pH 6.0 phosphate buffer to dilute both the Wright and Giemsa stains.With this technique a dose-response curve was generated for both TMP and TEM, using mice as the experimental animal. With TMP, a doubling over background was found when a concentration of 0.5 g/kg per day for five days was administered. To establish a statistically significant doubling dose over the control, a minimum of five animals must be used woth 2000 polychromatic cells being analyzed per animal.Of the two antischistosomal agents tested, hycanthone yielded an increase of 20-fold in the number of mircronuclei over control at 40 mg/kg administered i.p. for five days, while with niridazole no increase in micronuclei at several concentrations tested both by single and multiple injection was found.The results obtained with these compounds compare favorably woth what has been reported for the standard in vivo metaphase analysis.     

Expression of TEM-induced damage to postmeiotic stages of spermatogenesis of the mouse during early embryogenesis. I. Investigations with in vitro embryo culture.

After treatment of postmeiotic stages of spermatogenesis of the mouse with TEM, dose and stage of spermatogenesis-dependent disturbances of the early embryonic development can be observed both in vivo and after in vitro culture of the embryos. The observations in both systems can be correlated. The embryo-culture system thereby enables analysis of the expression of mutagen-induced damage more accurately than the in vivo dominant lethal test. With the doses used (0.2 and 0.4 mg/kg) TEM-treatment of the fathers did not affect the rate of fertilized and cleaving eggs during the first three weeks post-treatment but severely disturbed the further development of the embryos at all stages up to implantation, exhibiting a maximum effect on morulae.     

Induction of translocations by cyclophosphamide in different germ cell stages of male mice: cytological characterization and transmission.

Cytological and fertility tests were performed in F1 male mice derived from different germ-cell stages of male parents treated with cyclophosphamide (350 mg/kg body weight). The objectives of the present experiment were: (I) to determine the sensitivity of the male germ-cell stages to the induction of translocations by the compound, and (2) to characterize translocation configurations in F1 and F2 males, in order to obtain information about the pattern of chromosome breakage induced and its transmission to subsequent generations. Of 508 F1 males studied, 39 were partially sterile. The group of males conceived 8-21 days after treatment contained by far the highest proportion of partially sterile animals (30%). It was also the only group in which totally sterile animals (11%) were found. Of 25 semisterile males from this group, 24 gave evidence of translocations when spermatocytes were scored at diakinesis. The translocation frequencies in F1 derived from treated spermatozoa and spermatocytes were 14 and 1%, respectively. No translocations were detected cytologically in 6 semisterile males derived from treated spermatogonial stages. These results indicate that spermatid stages are especially sensitive to the mutagenic action of cyclophosphamide. In 21 of the 31 semisterile translocation males (68%), the majority of the spermatocytes contained 18 bivalents plus a ring-of-four configuration, indicating that both breakpoints were relatively centrally located; and in several of these males, the frequency of cells with rings was close to 100%. In another 9 F1 males (29%) the predominant multivalent configuration was a chain-of-four, indicating one of the breakpoints to be relatively more terminally located; and in one male (3%), the majority of cells had two unequal bivalents, indicating both breakpoints to be fairly close to the ends of the chromosomes involved. Determination of centromere positions by the use of C-banding showed that chain-of-four configurations in any one male were predominantly of a given type..     

H-gene (histocompatibility) mutations induced by triethylenemelamine in the mouse

Based on a simple dermal-graft procedure, the H-test for histo-compatibility mutations screens the mouse “H-system” of not less than 29 and perhaps considerably more than 100 loci that are scattered throughout the genome. Graft-tests with normally heterozygous or hemizygous loci permit scoring mutations as gains, losses, and gains+losses. Tests with normally homozygous loci screen only for gains, but further analysis can detect accompanying losses. Assayed by the H-test, triethylenemelamine (TEM) increased the spontaneous mutation rate per generation by approximately four- to five-fold in the case of BALB/c spermatogonia and F₁ hybrid oocytes (BALB/c females × C57BL/6 males), but had a much smaller effect, if any, on the rate of C57BL/6 spermatogonia. Single intraperitoneal doses of 2.4–4.0 mg/kg were given.     

Dominant lethal effects of triflupromazine in hybrid C3D2F 1 -J mice

In three separate experiments, triflupromazine (TFP), a phenothiazine tranquilizer, was active in a dominant lethal test using hybrid male mice. The effect for a single intraperitoneal injection was dose-related and the major effect was observed during the spermatocyte and spermatogonial stages of spermatogenesis.     

The induction of chromosome aberrations in mouse dictyate oocytes by X-rays and chemical mutagens.

Oocytes were collected from female mice and matured in vitro to Metaphase I during the first or third week after treatment with a dose of 400 rad X-rays, 1.6 mg/kg triethylenemelamine (TEM) or 75 mg/kg isopropylmethanesulphonate (IPMS). In week 1 the mean number of oocytes per female was similar for all treatments but in week 3 irradiated females yielded fewer oocytes than the chemically treated or control females. In week 1 the proportion of oocytes maturing was smaller in irradiated females than in the other groups but in week 3 was similar in all groups.Structural chromosome aberrations, scored in the Metaphase I oocytes, were of the chromatid or isochromatid type and involved gaps, breaks, fragments, intrachanges and interchanges. Aberration frequency did not increase with time after either of the chemical mutagens but after irradiation was higher in the third week than in the first week. The aberration yield from IPMS-treated females was similar at both sampling times, while a lower yield was recorded in week 3 following TEM treatment than in week 1.     

Alkylating esters X. The reaction of some aziridine alkylating agents with methionine and S-methyl cysteine

Two biologically active aziridine ring-containing compounds, N,N-ethylene urethane (I) and N,N-ethylene urea (II), have been shown to react with methionine in dilute phosphate buffer (pH 7.4) at 37°C. Degradative procedures indicate that the aziridine ring effectively alkylates the thio ether group of methionine and other thio ether-containing amino acids to produce sulphonium salts (V). By using [³⁵S]methionine, the sulphonium salts have been shown to be quite stable under physiological conditions ($\text{t}{}_{\mathrm{<mtext>built1</mtext><mtext>2</mtext>}}$ 7–9 days) hydrolysing to convert the methionine residue to homoserine.It is proposed that similar alkylations of methionyl residues in vivo by aziridine-alkylating agents may explain the complex, and as yet unknown, metabolic fate of the aziridine ring and could also be a factor contributing to the diverse effects that these agents have on living cells.     

Cytogenetic effects of hycanthone in the rat

Hycanthone methanesulfonate (HCT), a new drug used for the treatment of schistosomiasis, was examined for its ability to produce chromosomal abnormalities in rat bone marrow cells. Rats were given intraperitoneal injections of HCT at 40, 80, or 100 mg/kg and were killed at 6, 24, or 48 h. All levels produced a significant increase cells with abnormalities at all three time periods. There was a significant linear relationship between arithmetic dose and the proportion of cells affected. Based on two of the three experiments peformed, assuming the same slope holds in the vicinity of 0, the upper 95% confidence limits on the expected risk at level of 0.5] mg/kg would be a 0.055% increase in affected cells above control values.