Isopropyl Methanesulphonate Research Articles

Preliminary results of ethylnitrosourea, isopropyl methanesulphonate and methyl methanesulphonate activity in the testis and epididymal spermatozoa of Muta™Mice

Male Muta™Mice were given a single intraperitoneal dose of either 1/15 M phosphate buffer (pH 6) as the vehicle control, MMS 40 mg/kg, ENU 150 mg/kg or iPMS 200 mg/kg, at a dose volume of 20 ml/kg. Animals from each group were killed 3 or 63 days after dosing, the DNA extracted from whole testes and epididymal spermatozoa and analysed for mutation frequency. In the testes, no increase in mutation frequency was observed, at either timepoint, for the animals treated with either MMS or iPMS. A slight increase in the mutation frequency, above vehicle control values, was seen in the ENU-treated animals with a 3 day expression time. A 4-fold increase was observed in the ENU-treated animals exposed for 63 days. In the epididymal spermatozoa, all of the test chemicals induced increases in mutation frequency, at both timepoints, with the exception of a negative result for MMS after 3 days. ENU induced a 2.5 and iPMS a induced a 4-fold increase above the control mutation frequency after 3 days. For all treatments, the later sampling time of 63 days gave an approximate 2-fold increase above the results of the 3-day timepoint. These increases amounted to a 2, 4.5 and 11-fold increase above control for MMS, ENU and iPMS, respectively. The Muta™Mouse positive selection system appears to be sensitive to both the premeiotic germ cell mutagen, ENU and postmeiotic germ cell mutagens, MMS and iPMS.

Mutation assays in male germ cells from transgenic mice: overview of study and conclusions

Three confirmed mouse germ cell mutagens, ethyl nitrosourea (ENU), isopropyl methanesulphonate (iPMS) and methyl methanesulphonate (MMS), have been evaluated for their activity as mutagens to the germ cell DNA of two strains of transgenic mice (lac I, Big Blue™ and LacZ, Muta™Mouse). Both testicular DNA and epididymal sperm DNA were evaluated. A range of sampling times was studied, from 3 days post-dosing to 100 days post-dosing. ENU and iPMS were mutagenic to both testicular DNA and epididymal sperm DNA. Mutant frequencies were higher for both chemicals in DNA recovered from testicular tissue than in epididymal sperm DNA. Likewise, mutant frequencies were higher for both DNA samples at the later sampling times. MMS was not mutagenic under any condition of test. A good level of qualitative agreement in test results was seen for the two assays and for the same assays conducted in different laboratories. The level of quantitative agreement was not as high, but was, nonetheless, generally good. Recommendations for the future conduct of transgenic rodent germ cell mutation assays are made. The test data are discussed within the context of the larger question of how such assays should be integrated into the chemical hazard assessment process.

The activity of ENU, iPMS and MMS in male mouse germ cells using the Muta™Mouse positive selection transgenic mutation assay

The alkylating agents ethyl nitrosourea (ENU), isopropyl methanesulphonate (iPMS) and methyl methanesulphonate (MMS) are potent male rodent germ cell mutagens. The mutagenic activity of these compounds in male mouse germ cells has been evaluated using the Muta™Mouse positive selection transgenic mutation assay. Both ENU (150 mg/kg) and iPMS (100 mg/kg) gave increased mutant frequencies in testicular DNA recovered 50 days after dosing. During the course of the mutation assays on iPMS its activity as a dominant lethal mutagen was confirmed by mating the treated animals with virgin (non-transgenic) females on day 10 post-dosing. Ova analysis on animals exposed to iPMS confirmed earlier reports that the dose level used caused sterility in mice 40 days after dosing. This sterility was shown to be due to aspermia in the treated mice at day 50 post-dosing. These collected findings indicate that at day 50 post-dosing with iPMS, mutations in testicular DNA can be observed in sterile animals. MMS (100 mg/kg) was not mutagenic to either testicular DNA or epididymal sperm DNA, 10 or 50 days, respectively, after dosing.

Fate of unfertilized ova in male rodent dominant lethal assays: Extension of the studies by Kratochvilova

Kratochvilova has described a technique whereby ova can be recovered from mated mice and their stage of division determined. This is of value to determine if reduced total implantations in a male dominant lethal (DL) germ cell mutation assay are due to pre-implantation loss of embryos, a presumed mutagenic event, or to chemically induced male infertility. Kratochvilova was not specific about the fate of unfertilized ova, but it was implied that they undergo a process of fragmentation that might be confused with the regular cleavage of fertilized ova. It became important for us to draw a firm distinction between ova fragmentation and regular ova cleavage in the rat. We therfore repeated the ova analyses of female mice mated with males exposed to iso-propyl methanesulphonate (iPMS), as described by Kratochvilova. Following that calibration study the technique was extended to the rat via ova cleavage analysis in mated female rats, coupled to a study of the normal decay of ova in virgin rats. Unfertilized ova are shown to undergo irregular fragmentations that can be clearly distinguished from normal cell division. It is concluded that the individual or combined incidences of single celled ova and fragmented ova (dependent on the cleavage stage of the concurrent control embryos) can provide a measure of male infertility as it relates to reduced implantations in DL assays. This ability to regard two morphological classifications of unfertilized ova as providing evidence for male infertility will simplify the conduct of ova analyses in both the mouse and the rat.

Activity of iPMS and nPMS in mouse bone marrow micronucleus assays: comparison with mouse dominant lethal assay data

isoPropyl methanesulphonate (iPMS) and its n-propyl analogue (nPMS) are shown to be active in mouse bone marrow micronucleus assays using male CBA, male and female ( C3H El × 102 El )F1 and male and female Muta ™Mouse mice. iPMS was significantly more active than nPMS. No significant strain or gender differences were observed. These findings reflect the differences reported earlier for these two chemicals in mouse dominant lethal mutation assays. The earlier described dominant lethal assay data are represented schematically and discussed.

Relationships between ablation of distinct haematopoietic cell subsets and the development of donor bone marrow engraftment following recipient pretreatment with different alkylating drugs

A number of different alkylating chemotherapeutic agents--busulphan, dimethylbusulphan (DMB), isopropylmethane sulphonate (IMS), melphalan, cyclophosphamide (CY) and bischloroethylnitrosourea (BCNU)--were investigated for their cytotoxic effects on different haemopoietic cell populations in host mice and for their ability to induce short- and long-term engraftment of transplanted bone marrow. At 24 h after drug treatment the femoral content of transient and permanent repopulating stem cell subsets was assessed, respectively, from the frequency of early- (day 5-15) and late- (day 25-35) developing cobblestone area-forming cells (CAFCs), growing in vitro in long-term bone marrow cultures (LTBMCs). At this time a fixed complement of 10(7) congenically marked donor bone marrow cells (B6-Gpi-1a-->B6-Gpi-1b) was infused in the drug-treated mice and erythroid engraftment was followed over 36 weeks. Diverse effects on early- and late-developing CAFC frequencies were found for the different drugs; these were generally related to the pattern of donor bone marrow engraftment in treated recipients. Melphalan was more toxic to early-developing than to late-developing CAFC subsets, and the transplant only offered an early wave of blood chimerism followed by return of host cells. CY and BCNU had minimal to moderate effects on CAFC content and engraftment with no apparent preference for any particular haemopoietic cell subset. IMS also had a relatively low toxic effect on host marrow CAFC frequencies but appeared exceptional in its ability to allow for more donor-type engraftment. The dimethane sulphonate compounds busulphan and DMB were especially potent at depleting late CAFC subsets and ensured high and lasting levels of donor bone marrow engraftment. These studies support the value of CAFC measurements for predicting the fate and growth of transplanted bone marrow cells in recipients pretreated with a variety of cytotoxic agents.

Marrow repopulation in mice treated with busulphan or isopropyl methane sulphonate and bone marrow

By using karyotypic analysis of female mice treated with busulphan or isopropyl methane sulphonate (IMS), and injected with male bone marrow the donor contribution to both total marrow cellularity and spleen colony forming cells (CFU-S) was assessed for up to 6 months after transplant. In the mice treated with busulphan the marrow cells yielded metaphases of which between 40% and 83% were of donor type. Between 60% and 97% of metaphases in spleen colonies formed in irradiated mice were of donor type during the 24-week study period. In contrast, mice prepared for the transplant with IMS showed no cells of donor type at any time after transplant, neither did they possess CFU-S of donor type. We were therefore led to conclude that the donor cells made no contribution to longterm engraftment in mice prepared with IMS, whilst in those prepared with busulphan they were the predominantly active haemopoietic cells. These results are consistent with a model of haemopoiesis in which the most primitive cells reside in a 'niche' where they are resistant to the effects of IMS but susceptible to the action of busulphan. Busulphan may vacate some niches to allow engraftment by transplanted marrow, whilst IMS yields no unoccupied niches for grafted cells to occupy, and cannot therefore lead to a stable chimaerism.

Marrow repopulation in mice treated with busulphan or isopropyl methane sulphonate and bone marrow

Marrow repopulation in mice treated with busulphan or isopropyl methane sulphonate and bone marrow

Cell migration velocities in the crypts of the small intestine after cytotoxic insult are not dependent on mitotic activity.

The role of mitotic activity in the normal process of intestinal epithelial cell migration was investigated. The movement of [3H]TdR-labelled cells in the crypt-villus column was used to study migration both in the crypts and on the villi. Radiation alone or in conjunction with other cytotoxic agents (hydroxyurea, cyclophosphamide and isopropyl-methane sulphonate) was used to eliminate cell division activity and to decrease crypt cellularity. This was done in order to determine the role of 'mitotic pressure' in driving cell migration. It has been clearly demonstrated in this study that cell migration, both within the crypts and on the villi, can take place in the complete absence of mitotic activity and after a drastic decrease in crypt cellularity. These results add to the continually mounting evidence against the idea that the 'pressure' generated by mitoses within the crypt or indeed in other epithelial regions is responsible for propelling epithelial cells. The data also demonstrate that the migration mechanisms are resistant to cytotoxic exposure.

Clonogenic response of cells of murine intestinal crypts to 12 cytotoxic drugs.

The in situ response of clonogenic cells in murine intestinal crypts to graded doses of 12 clinically used cytotoxic drugs has been measured by the crypt microcolony assay. For the alkylating agents mechlorethamine hydrochloride and BCNU, survival curves down to three logs of crypt depletion could be obtained. However, with isopropyl methane sulphonate only one log of depletion was measurable, and for a fourth alkylating agent, cyclophosphamide, no crypt killing occurred before other toxicities resulted in death of the animal in less than 3-4 days. This is the major constraint on the use of the microcolony assay with cytotoxic drugs. Of four antibiotics, survival curves were obtainable for adriamycin, bleomycin and mitomycin C, but not for actinomycin D. 5-Fluorouracil gave only a limited degree of crypt kill. Of three S-phase-specific drugs, hydroxyurea and vincristine resulted in little or no crypt depletion, but there was dose-dependent crypt depletion after methotrexate. The complexities of applying cellular-kinetic classifications of cytotoxic drugs to in situ endpoints are discussed.

Ablation of murine jejunal crypts by alkylating agents

The gut microcolony assay has been used to measure damage to intestinal crypts by single and split doses of 3 alkylating agents: mechlorethamine hydrochloride (HN2), bis-chloroethyl-nitrosourea (BCNU) and isopropyl methane sulphonate (IMS). The single-dose survival curves for whole crypts were distinguished by extrapolation numbers (3.0, 176 and 1.5 respectively) that were lower than most previously published values for assay by irradiation. Significant sparing of crypts occurred when doses of HN2 or BCNU, but not IMS, were given in 2 equal fractions separated by more than 2 h. Deduced D0 values for those cells from which crypts regenerate were 1.9 mg/kg HN2, 19 mg/kg BCNU and 487 mg/kg IMS.

Reproductive capacity and dominant lethal mutations in female guinea-pigs and Djungarian hamsters following X-rays or chemical mutagens

The reproductive capacity and induction of dominant lethal mutationns in adult female guinea-pigs and Djungarian hamster were tested follwing treatement with 400 rad X-rays, 1.6 mg/kg triethylenemelamine (TEM) or 75 mg/kg isopropylmethanesulphonate (IPMS). A fairly high of dominant lethals were observed in female guinea-pigs mated at the first oestrus after irradiation (23.4 ± 6.4%) with a lower yield at 3 months (9.6 ± 8.2%). Neither of the chemical caused any significant induction of dominant lethals at either mating time. In the reproductive capacity experiments, the mean litter size of irradiated female guinea-pigs was reduced for about 12 months and this was especially marked in the first 6 months following treatment. Neither of the chemicals caused any significant differences in early litter sizes but there was a noticeable reduction in the litter sizes of TEM-treated females in the 18–24 month interval. With Djungarian Hamsters a marked effect of X-rays on reproductive capacity was apparent. After 400 rad a smaller proportion of irradiated females littered im the first 25-day interval than after the other treatment, and no irradiated females produced more than one litter. Neither of the chemicals caused such a drastic reduction in fertility but TEM-treated females produced fewer litters and became sterile at an earlier age than control or IPMS-treated females. With IPMS, the number of litters produced was similar to the controls. Both chemical caused a significant reduction in litter-size but further work is needed to establish whether this was due to induction of dominant lethals. No translocations were observed in the sons of treated female guinea-pigs or hamsters, but the numbers of animals studied were too small for any conclusions to be drawn.

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.

Alkylation of cytosine and 5-hydroxymethyl-cytosine by methyl methanesulphonate and N-methyl- N-nitrosourea: Its relevance to mutagenesis

The reaction of cytosine and 5-hydroxymethyl-cytosine (OHMeCyt) with a variety of monofunctional alkylating agents has been investigated to evaluate further the possible role of cytosine alkylation in mutagenesis and the possibility that the immunity of T-even phages to mutation by methyl methanesulphonate (MMS) was due to the unreactivity of OHMeCyt towards this agent. Both cytosine and OHMeCyt reacted equally well with the methylating agents MMS and N-methyl- N-nitrosourea (MNU) affording 6% and less than 1% respectively of the 3-substituted derivative. No product was isolated following subjection of the bases to reaction with ethyl methanesulphonate (EMS), N-ethyl- N-nitrosourea (ENU) or iso-propyl methanesulphonate (iPMS).

Haemopoietic stem cells are organised for use on the basis of their generation-age.

THE spleen-colony assay of Till and McCulloch1, which is a clonal assay2, measures the content of haemopoietic cells in blood-forming tissues. Stem cells (colony forming units in spleen, CPUS) form red cells, granulocytes, platelets and themselves (self-renewal) in these colonies3,4. Worton et al.5 reported that stem cells separated by velocity sedimentation differed in their capacity for self-renewal. Schofield and Lajtha6 reported that CPUS from mice treated with iso-propyl methane sulphonate have a low self-renewal capacity. We have studied the functional capacity of myeloid stem cells from normal mice (NBM) and mice treated with hydroxyurea. CPUS from mice treated with five injections of hydroxyurea (5 HUBM, G.S.H. and N. M. Blackett, unpublished) formed 3 times more CPUS and 2.5 times more colony forming units in culture per original colony (CFUC, a committed precursor of granulopoiesis7) than those from NBM. When these grafted CPUS were retransplanted they still formed three times more CFUC and CPUS per secondary colony than those from NBM (Tables 1 and 2).

Inactivation and mutation of coliphage T4 by aliphatic nitrosamides and methanesulphonates: In vitro recovery of infectivity of T4 inactivated by isopropyl methanesulphonate

The inactivation and mutation (to r phenotype) of extracellular coliphage T4 wild-type by the monofunctional alkylating agents N-methyl- and N-ethyl- N-nitrosourea and isopropyl methanesulphonate were investigated. The rate and extent of change in phage infectivity observed during the post-treatment period were found to correlate with what is known of the mechanisms by which these agents react in vitro. Loss of phage infectivity was found to occur during the period following treatment with agents, but that resulting from treatment with isopropyl methanesulphonate was preceded, in the first 24 to 48 h, by a recovery of infectivity. This suggested that changes in phage infectivity occuring after treatment with monofunctional alkylating agents are resultant of various processes which diversely promote loss and recovery of infectivity. The mutagenicity of N-methyl- N-nitrosourea was similar to that of its ethyl homologue at a level of phage survival of 4 × 10 −3, but less than that of isopropyl methanesulphonate. At a level of survival of 3 × 10 −2 ethyl methanesulphonate was a mutagenic as its isopropyl homologue, but methyl methanesulphonate was only slightly if at all mutagenic. These results could not be correlated with the compounds' reaction mechanisms. The efficiency of isopropyl methanesulphonate (compared with its toxicity to phage) was found to decrease as the severity of the dose was increased.

Prophage inductive efficiency of alkylating agents and radiations.

The prophage inducing efficiency in E. coli K-12 (lambda) of a number of agents--alkylating agents and radiations--has been compared at a high survival of bacteria. The inducing effectiveness (per alkylation in DNA) and efficiency (compared with mutation frequency in E. coli Sd-4) was found to decrease in the order 2-hydroxyethylating agents (2-hydroxyethyl methanesulphonate and ethylene oxide) greater than isopropyl methanesulphonate approximately equal to methyl methanesulphonate greater than ethylating agents (diethyl sulphate, ethyl methanesulphonate). The low inducing activity of the ethylating agents could not be explained with respect to their reactivity towards targets of differing nucleophilicity, nor could the effectiveness in mutagenicity or the ability to break the chromosomes in the presence of metal ions be invoked. The high inducing efficiency of hydroxyalkylating agents may be related to their ability to break DNA strands.

Effect of isopropyl methane sulphonate (IMS) on haemopoietic colony-forming cells.

Summary. A dose of isopropyl methane sulphonate (IMS) has been selected which depletes the population of CFU in the femur to 30–50%. During the subsequent 5–7 days the surviving population declines to between 0.5% and 2% of controls. It is shown that the IMS‐treated host is a satisfactory environment for growth of CFU in grafts from either normal or IMS‐treated donors although the grafts themselves behave differently from each other. The colonies produced from marrow from IMS‐treated donors are virtually devoid of CFU, whereas 70% of those from control donors contain CFU. It is proposed that IMS, at the dose selected, kills primary, self‐maintaining CFC but does not kill the derived non‐self‐maintaining cells which, nevertheless, are also capable of spleen colony formation. It is suggested that the chemical provides a useful tool for the study of the haemopoietic stem cell complex.

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

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.

Differential response of mutagenic effects to storage of barley seds treated with propyl methanesulphonate and isopropyl methanesulphonate

Barley seeds were treated with propyl methanesulphonate (PMS) or isopropyl methanesulphonate (iPMS) and strored at 25°. Both storage of PMS- and iPMS-treated seeds at 30% water content resulted in recovery from the induced mutagenic effects while at 5% seed water content no significant changes in the frequency of chromosome aberrations or in the frequency of M 2 chlorophyll mutants were detected. Marked differences between the storage effects of PMS and iPMS were observed at 15 and 20% water content. During storage of PMS-treated seeds, the mutagenic effects of PMS increases in contrast to a slight recovery from these effects after iPMS treatment. The differences during storage are explained by the different stabilities of the two compounds.