Robert B. Cumming

Induction of dominant lethal mutations by alkylating agents in male mice

Abstract Methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) induced a high frequency of dominant lethal mutations in mouse spermatozoa of vas and epididymis, testicular sperm, and late spermatids, and a low frequency in early spermatids. In an equimolar dosage, MMS is about 4 times more effective than EMS in inducing dominant lethal mutations in these cell stages. Neither N -methyl- N ′-nitro- N -nitrosoguanidine (MNNG) nor 2-methoxy-6-chloro-9-[3-ethyl-2-chloroethyl)amino propylamino]acridine dihydrochloride (ICR-170) induced dominant lethal mutations in spermatozoa of vas and epididymis, testicular sperm, or spermatids. These results are in good agreement with the expected effect of the chemicals based on work with microorganisms.

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..

Dosimetry studies on the ethylation of mouse sperm DNA after in vivo exposure to [3H]ethyl methanesulfonate ☆

Abstract Methods for determining the chemical dose of ethyl methanesulfonate (EMS) to the DNA of mouse spermatozoa in the vasa deferentia and epididymides have been developed. These include procedures for the removal of contaminating protamine, which, like DNA, possesses nucleophilic sites that can be ethylated by EMS. At least 99% of all sperm protamine (at a 95% confidence level), as well as any other cellular contaminants, is removed during purification of the DNA. The purified DNA recovered from spermatozoa gives no indication of a preferential recovery of either (G+C)-rich or (A+T)-rich regions of the mouse genome: the [ 14 C]dT/[ 3 H]dC ratios for whole sperm and sperm DNA were the same for each animal tested. The spermatozoa of males used in the dosimetry studies were labeled with [ 14 C]thymidine, and then the animals were given various [ 3 H]EMS doses intraperitoneally. A constant exposure time of 4 h was used. The ratios of 3 H and 14 C activities in whole sperm and purified sperm DNA were used to measure the percentage of the total sperm ethylation occurring in the DNA. The maximum percentage found was about 18% in the dose range of 100–400 mg/kg. Values for the ethylations per nucleotide ( E / N ) ranged from ∼ 10 −7 at 3.3 mg/kg up to ∼ 10 −4 at 400 mg/kg, and the data indicated that E / N increased with the 1.5 power of the dose. E / N was also measured in testicular DNA, and the values obtained were close to those found for spermatozoan DNA. The results of such chemical dosimetry studies will be far-reaching in the interpretation of molecular events responsible for genetic alterations. As an example, dominant lethal studies by others, using EMS in the dose range considered in the present paper, have shown little or no effect until two or more days after injection of the mutagen into male mice. Since many sperm DNA ethylations are found after a 4-h exposure to EMS it appears that most of these DNA ethylations are not genetically important, at least in the production of dominant lethals, and that perhaps genetic damage occurs only at rarely ethylated DNA sites.

Modification of the acute toxicity of mutagenic and carcinogenic chemicals in the mouse by prefeeding with antioxidants

Abstract Butylated hydroxytoluene (BHT) fed to male mice for 4 wk gave significant protection against mortality caused by ethyl methanesulphonate (EMS), n -propyl or isopropyl methanesulphonate, ethylene dibromide, diethylnitrosamine and cyclophosphamide. It did not protect male mice against X-rays, methyl methanesulphonate (MMS), N -methyl- N ′-nitro- N -nitrosoguanidine or dipropylnitrosamine, but female mice were protected from the lethal effects of MMS. Other agents, such as butylated hydroxyanisole, 1,2-dihydro-6-ethoxy-2,2,4-trimethyl quinoline and sodium phenobarbitone also gave protection against EMS toxicity. The protection, when it occurred, may have been due to the induction of drug-metabolizing enzymes.

Fate and metabolism of some mutagenic alkylating agents in the mouse. I. Ethyl methanesulfonate and methyl methanesulfonate at sublethal dose in hybrid males.

Abstract Ethyl methanesulfonate (EMS) or methyl methanesulfonate (MMS) with a [ 14 C]alkyl label were injected intraperitoneally into (101 × C 3 H)F 1 hybrid male mice. Radiactivity levels were measured in various tissues and tissue fractions at eight time periods ranging from 15 min to 24 h after injection. Activity levels were also measured in the blood and urine, and respiration pattern analysis was carried out on exhaled 14 CO 2 . Distribution of these compounds is rapid, and approximately the injected dose w/w reaches the testis in an active form. However, EMS is rapidly hydrolyzed in vivo , and MMS is 4–6 times more effective in alkylating biological molecules. The amount of genetic damage done in the mouse (chromosome breakage) is correlated with in vivo alkylation of macromolecules. It is suggested that the great difference in the spectrum of genetic damage observed in mammals relative to that reported for other organisms is due to differences in the repair system.

Unscheduled DNA synthesis in spermatogenic cells of mice treated in vivo with the indirect alkylating agents cyclophosphamide and mitomen.

Abstract Cyclophosphamide (CPA) and mitomen (DMO) are chemical mutagens that require metabolic activation to produce their biological effect. We have used an in vivo UDS assay in various meiotic and postmeiotic germ-cell stages of male mice to study DNA repair after treatment with these chemicals. EMS, a compound requiring no metabolic activation, was also used for comparative purposes. CPA and DMO induced UDS in meiotic through early-to-midspermatid stages, but no UDS was detected in late spermatids and mature sperm. While EMS produced a maximum UDS response in the germ cells immediately after treatment, CPA and DMO did not produce a maximum response until ∼0.5 to 1 h after injection. This delay is attributed to the time required for CPA and DMO to be enzymatically vonverted active alkylating metabolites. Unlike the results found with EMS, mutation frequencies (dominant lethals, translocations, specific-locus mutations) following CPA treatment are not noticeably reduced in germ-cell stages in which UDS occurred. In the case of DMO, mutations are induced only in mature spermatozoa, and these germ-cell stages represent only a fraction of those in which no UDS is detected. The results with CPA and DMO thus still leave unclear the relationship between DNA repair and the differential spermatogenic response of mice to genetic damage.

An autoradiographic study of unscheduled DNA synthesis in the germ cells of male mice treated with X-rays and methyl methanesulfonate.

Unscheduled DNA synthesis (UDS) in the germ cells of male mice after in vivo treatment with X-rays or methyl methanesulfonate (MMS) was assayed by use of a quantitative autoradiographic procedure. MMS induced UDS in meiotic through type III elongating spermatid stages, whereas X-rays induced UDS in meiotic through round spermatid stages. No UDS was detected in the most mature spermatid stages present in the testis with either MMS or X-rays. Taking into account differences in DNA content of the various germ-cell stages studied, we concluded that X-rays induced a maximum UDS response in spermatocytes at diakinesis--metaphase I. The level of UDS induced by MMS was about the same in all the stages capable of repair. Chromosome damage and UDS were measured simultaneously in the same spermatocytes at diakinesis 90 min after X-irradiation or MMS treatment. The level of UDS in most of the X-irradiated cells paralleled the extent of chromosome damage induced. A statistical analysis of these results revealed a positive correlation. As expected, MMS induced no chromosome aberrations above control levels. Therefore no correlation was determined between UDS and chromosome damage in this case. The distribution of UDS over the chromosomes treated at diakinesis with MMS or X-rays was studied. It was found that UDS occurred in clusters in the irradiated cells, whereas it was uniformly distributed in the MMS-treated cells.

Effects of butylated hydroxytoluene alone or with diethylnitrosamine in mice

Abstract Mice (18 months old) given butylated hydroxytoluene (BHT) at a dietary level of 0·75% for 16 months had 63·6% of lung tumours compared with 24·0% in controls. Mice given dietary BHT as well as diethylnitrosamine (DENA) in the drinking-water (average total intake 330 mg DENA/kg) had more lung tumours per mouse (4·0) than did the controls or those given DENA alone or BHT alone (1·4–2·2). The incidence of reticulum-cell sarcomas in mice killed at 12 months of age in the group treated with BHT plus DENA exceeded control values (52·6 and 22·7%, respectively), while the incidence in groups receiving DENA alone or BHT alone was not significantly different from that in controls. At 18 months, the incidence of reticulum-cell sarcoma in both groups given BHT was significantly less than in the groups not receiving BHT. At 18 months of age, a higher percentage of squamous-cell carcinomas was seen in the forestomach of mice given BHT plus DENA than in mice receiving DENA alone. The production of cysts within the liver parenchyma after DENA treatment also appeared to be potentiated by BHT treatment.

Specific-locus mutation rates in the mouse following inhalation of ethylene oxide, and application of the results to estimation of human genetic risk

Male (101 X C3H)F1 mice were exposed in an inhalation chamber to ethylene oxide (EtO) in air at a concentration of (generally) 255 ppm. After accumulating total exposures of 101 000 or 150 000 ppm.h in 16-23 weeks, the males were mated to T-stock females for a standard specific-locus mutation-rate study in which 71387 offspring were observed. The spermatogonial stem-cell mutation rate at each exposure level, as well as the combined result, does not differ significantly from the historical control frequency. At the lower and higher exposure levels, the results rule out (at the 5% significance level) an induced frequency that is, respectively, 0.97 and 6.33 times the spontaneous rate; the combined results rule out a multiple of 1.64. The relationship between mouse spermatogonial stem-cell mutation rates and EtO-induced testis ethylations was compared with the relationship between Drosophila post-stem-cell mutation rates and sperm ethylations (Lee, 1980). The comparison does not rule out equal mutability per ethylation; but it cannot prove parallelism. An assessment of the mouse-Drosophila relationship will require a more efficient alkylator than EtO and the use of comparable germ-cell stages. More meaningful conclusions may be drawn by utilizing the data for direct estimation of human risk by expressing the induced mutation frequency that is ruled out (at the 5% significance level) as a multiple of control rate and extrapolating to human exposure levels. The probable absence of major stem-cell killing (and thus, possibly, cell selection) by EtO indicates that such extrapolation probably does not produce an underestimate. For a human exposure concentration of 0.1 ppm on working days during the reproductive lifespan, the mouse experimental results rule out (at the 5% significance level) an induced spermatogonial stem-cell gene mutation rate greater than 8% of the spontaneous rate; for 1.0 ppm, they rule out an induced rate roughly equal to the spontaneous rate. The induced rate for any one poststem-cell stage would have to be about 3 orders of magnitude higher than that for stem cells to constitute an equivalent risk.

Hyperplasia of hepatic bile ducts in mice following long-term administration of butylated hydroxytoluene

Abstract Six of 18 male BALB/c mice regularly givena diet containing 0·75% butylated hydroxytoluene (BHT) and killed at 12 months of age showed a marked hyperplasia of the hepatic bile ducts, with an associated subacute cholangitis. None of the 64 untreated control mice nor 19 BHT-treated control mice that additionally received diethylnitrosamine in their drinking-water, developed proliferation of the bile-duct epithelium. While the cause, pathogenesis and fate of the observed lesion is uncertain, further investigation is needed to determine the possible hazard arising from long-term human consumption of BHT.