Clastogenic effect of thiophosphamide in spermatogonia of inbred strains of mice

Sensitivity of spermatogonia of 11 mouse inbred strains to induction of chromosome damages by thiophosphamide (thioTEPA) was studied. Metaphase chromosome preparations were made 24 h after treatment with thioTEPA (at 2.25 mg/kg, i/p). With respect to frequency of cells with chromosome damages, strains were ranked as follows: A/Sn (17.5 + 4.4%) greater than 101/H greater than TPS greater than WR = C57BL/6 = AKR = NZB greater than CBA/Lac greater than C3H/Sn greater than MRL greater than BALB/c (5.0 + 2.2%). This distribution does not coincide with that for sensitivity of bone marrow cells, though the data support, in general, the estimations obtained earlier for strains' mutability. Comparison of the data presented with those from literature demonstrates that the sensitivity to clastogenic effect of thioTEPA (and other alkylating agents) correlates neither with spontaneous level of SCE, nor with unscheduled DNA synthesis, nor with radiosensitivity of inbred mice. The frequency of induced chromosome aberrations in spermatogonia is relatively low and spermatogonia cannot substitute bone marrow cells as a test system when assaying chemical mutagens.     

Modification of the genetic effect of N-nitrosoethylurea in inbred mice by para-aminobenzoic acid

The ability of para-aminobenzoic acid--vitamin (PABA) to influence the sensitivity of mice to alkylating mutagens was studied. PABA had no influence on the cytogenetic effect of thio-TEPA. It was determined that PABA altered the effect of N-ethyl nitrosourea (ENU). The direction of modification depends on animal genotype: pre-treatment with PABA decreases the frequency of chromosome aberrations in bone marrow cells of CBA/LacY and C57BL/JY mice, but significantly increases it in 101/HY mice. The PABA influence on the frequency of gene mutations induced by ENU in melanocytes of mice and revealed by "spot-test" was not determined.     

Modeling the periodically-changing predictable response to mutagen exposure in CBA/LacY mice in a successive inbred generations]

A hypothesis on the genetic determination of periodic fluctuations of the sensitivity to the mutagen thioTEPA in successive inbred generations of mice has been earlier put forward. This study was the initial stage of testing this hypothesis. The mouse strain CBA/LacY was divided into two substrains, which differed in the rate of generation change. As a result, two colonies of isogenic mice differing by 10-12 generations with respect to the inbred age were obtained. Both the rate and range of variations in the mutagen sensitivity (four generations per period of the cycle and 20-40% of cells with chromosome aberrations after the standard dose of 2.5 mg/kg of thioTEPA, respectively) in 19 generations of the "fast" substrain agreed with earlier data. The response of the "slow" substrain corresponded to the expected response of the "fast" substrain after the given number of generations. In the mice of generations F142 and F146 that lived simultaneously and differed in thioTEPA sensitivity, the effects of the carcinogen benzo[a]pyrene (BaP) were significantly different. The levels of these effects corresponded to the levels of the responses to thioTEPA. The data obtained agree with the hypothesis tested.     

The cytogenetic effect of natural modifiers of mutagenesis in a human lymphocyte culture: the modification of the mutagenic effect by recombinant interferon in vitro in the lymphocytes of bronchial asthma patients and of healthy donors]

The significant protective effect of recombinant interferon in the cultures of lymphocytes of healthy donors and patients with bronchial asthma has been revealed. The cytogenetic damage were stimulated by alkylating agents thioTEPA and photrin during their administration at the stages Gi-S of the cell cycle. No differences were revealed in the action of mutagens and protector in the patients and healthy persons.     

Role of the genetic-physiological interrelationships of mother-progeny in establishing the viability and fertility of mammals. II. The weight of the embryos of BALB, CBA and DBA strain mice developing from transplanted blastocysts

The weights of embryos and their placentas obtained by blastocysts of CBA/LacY and DBA/2 mouse strains transfer in recipient females of BALB/c strain and also by blastocysts of BALB/c mouse strain transfer in recipient females of CBA/LacY strain were analysed on the 16th gestation day. It is shown that placental weights are determined by genotypic peculiarities of embryos, although comparative placental weights of different strain embryos could also depend on females fertility. The weight of allogenic embryos (developed from transferred blastocysts) significantly excessed the weight of singenic embryos, and most significant differences were noted in females with small number of embryos. The possibilities to solve some applied and fundamental problems of mammalian reproduction given by the method of interstrain and interbreed early stage embryos transfer are discussed.     

Differences in spermatogenesis in cryptorchid testes among various strains of mice

The purpose of the study reported here was to define strain differences in spermatogenesis in cryptorchid testes in mice. Mice of strains A/J, BALB/c, CBA/N, C3H/He, C57BL/6 (B6), ddY and ICR were found to be sensitive to heat stress attributable to experimentally induced cryptorchidism. In contrast, mice of strains AKR/N (AKR), MRL/MpJ-+/+ (M+) and MRL/MpJ-lpr/lpr (lpr) were resistant to heat stress. Relative increases of apoptotic cells were detected in the sensitive group, but not in the resistant group. A decrease of proliferating cell nuclear antigen-immunoreactive cells after experimentally induced cryptorchidism was observed only in the sensitive group. These results suggested that heat stress-resistant germ cells were present in MRL and AKR strains, possibly originating from the genetic background.     

Effect of cigarette-smoke condensate and norharman on the induction of SCEs by direct and indirect mutagens in CHO cells

Cigarette-smoke condensate and norharman were investigated either alone or in combination with a number of direct or indirect mutagens for the induction of sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells. Cigarette-smoke condensate and norharman induced SCEs in these cells, only in the presence of a metabolic activation system. The number of SCEs induced by the direct-acting mutagens mitomycin C and N-methyl-N'-nitro-N-nitrosoguanidine was decreased in the presence of cigarette-smoke condensate or norharman. However, cigarette-smoke condensate and norharman showed synergistic effects in combination with the indirect mutagens 2-acetylaminofluorene, 2-aminofluorene, N-hydroxy-acetylaminofluorene, 2-aminoanthracene and benzo[a]pyrene. No synergism was observed when CHO cells were treated simultaneously with cigarette-smoke condensate or norharman and the indirect mutagen cyclophosphamide.     

Detection of differences in liver protein composition between recombinant strains of mice with different ThioTEPA sensitivities

The sensitivity of recombinant mouse strains subjected to 23-27 generations of inbreeding to the clastogenic effect of thioTEPA (triethylene thiophosphoramide) was reestimated, and their characteristics were confirmed. Six 1XC3 recombinant strains were obtained from crossing strains 101/H x C3H/Sn, which differed from one another with respect to the sensitivity to thioTEPA. The protein composition of the liver tissue was studied in the recombinant strains by means of two-dimensional electrophoresis. Interstrain differences with respect to five liver proteins were found, which were correlated with the differences in the response to the mutagen.     

Hypersensitivity to mitomycin C cell-killing in Roberts syndrome fibroblasts with, but not without, the heterochromatin abnormality

Roberts syndrome (RS) is a rare genetic disorder, characterized clinically by severe pre- and post-natal growth retardation and symmetric limb reduction deformities. Some patients with RS have a distinctive abnormality of the constitutive heterochromatin (the RS effect) which has been described as a premature separation of the paracentromeric and nucleolar organizing regions of the chromosomes and the distal portion of the long arm of the Y chromosome (German, 1979). These patients [denoted RS(+)] are clinically indistinguishable from the RS(-) patients who lack the cytogenetic marker for Roberts syndrome. Recently, a mutant in Drosophila has been described which has both heterochromatin undercondensation and hypersensitivity to mutagen treatment (Gatti et al., 1983). The authors suggested that the uncondensed heterochromatin may be more accessible to damage by mutagens. Thus, the present study was an investigation of the mutagen sensitivity in Roberts syndrome, to determine whether there is a similar relationship between abnormal heterochromatin structure and mutagen sensitivity. Plating efficiency experiments were performed with RS(+) fibroblasts, RS(-) fibroblasts, RS heterozygous fibroblasts and a large assortment of appropriate control cells. The RS fibroblasts with the heterochromatin abnormality were consistently more sensitive (based on D10 values) to mitomycin C treatment than were any of the other cell strains tested, including RS(-) cells. These results support the hypothesis that mitomycin C sensitivity and abnormal heterochromatin structure in Roberts syndrome are related.     

Arsenic trioxide inhibition of the thiophosphamide induction of mutations in mouse germ and somatic cells

After single i. p. injection of arsenic trioxide, at the dosage range of 1/4 to 1/40 LD50 into hybrid mice (CBA X C57B1/6J)F1, no induction of dominant lethals in male germ cells was observed. However, it led to an increase in the number of micronuclei in the erythrocytes of bone marrow. Treatment with the effective dose of thioTEPA, causing an increase in the number of dominant lethals in male germ cells and in the number of micronuclei in the erythrocytes of bone marrow, followed by injection of arsenic trioxide, resulted in inhibition of the mutagenic activity of thioTEPA. This inhibition increased proportionally with the dose of arsenic trioxide.     

Modeling of Periodically Changing Predictable Response to Mutagens in Successive Inbred Generations of CBA/LacY Mice

A hypothesis on the genetic determination of periodic fluctuations of the sensitivity to the mutagen thioTEPA in successive inbred generations of mice has been earlier put forward. This study was the initial stage of testing this hypothesis. The mouse strain CBA/LacY was divided into two substrains, which differed in the rate of generation change. As a result, two colonies of isogenic mice differing by 10–12 generations with respect to the inbred age will be obtained. Both the rate and range of variations in the mutagen sensitivity (four generations per period of the cycle and 20–40% of cells with chromosome aberrations after the standard dose of 2.5 mg/kg of thioTEPA, respectively) in 19 generations of the fast substrain agreed with earlier data. The response of the slow substrain corresponded to the expected response of the fast substrain after the given number of generations. In the mice of generations F₁₄₂and F₁₄₆that lived simultaneously and differed in thioTEPA sensitivity, the effects of the carcinogen benzo[a]pyrene (BaP) were significantly different. The levels of these effects corresponded to the levels of the responses to thioTEPA. The data obtained agree with the hypothesis tested.     

Genotype- and age-associated in vivo cytogenetic alterations following mutagenic exposures in mice

We studied mice from eight genetic strains at two ages (young, 10 weeks; and old, more than 80 weeks) for cytogenetic alterations (sister chromatid exchange (SCE), micronuclei, and metaphase indices) following challenges by two known mutagens: N-nitrosoethyl urea (ENU, 17 mg/kg) and cyclophosphamide (CP, 4.5 mg/kg) on bone marrow cells in vivo. The data were used to evaluate the effect of age, genotype, and differential aging patterns of genotypes in relative susceptibility to chromosomal breakage and instability in otherwise normal individuals. The older animals had a higher frequency of micronuclei, reduced metaphase indices, and lower SCE/cell as compared with their younger counterparts. Treatment with both mutagens significantly increased micronuclei and SCEs/cell in almost all strains at both ages but had little effect on the frequency of cells in metaphase. Among individual differences for SCEs/cell at most treatment combinations were not significant. In general, the induced SCEs (treatment-control) are significantly higher in older animals, variable among strains, and relatively higher as a result of CP than the ENU treatment. When the age effect was evaluated as the difference of SCE/cell in old and SCE/cell in young animals of each genotype-treatment combination, an age-dependent pattern was evident. In the presence of a mutagen the pattern in aging response was highly variable and strain (genotype) dependent. This variability may be viewed as subtle inherent genetic predisposition of sensitivity to mutagens that could be evaluated only using sensitive measures (e.g., SCE and not micronuclei) following more than one mutagenic challenges. These subtle differences could become pronounced when these parameters are evaluated at different ages on the same genotype.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytogenetic effects of 900 MHz (GSM) microwaves on human lymphocytes

The cytogenetic effects of 900 MHz radiofrequency fields were investigated with the chromosome aberration and sister chromatid exchange frequency methods. Three different modes of exposure (continuous, pseudo-random and dummy burst) were studied for different power outputs (0, 2, 8, 15, 25, 50 W). The specific absorption rates varied between 0 and 10 W/kg. We investigated the possible effects of the 900 MHz radiation alone as well as of combined exposure to the chemical or physical mutagens mitomycin C and X-rays. Overall, no indication was found of a mutagenic, and/or co-mutagenic/synergistic effect of this kind of nonionizing radiation.     

Alkaline elution of rat testicular DNA: detection of DNA cross-links after in vivo treatment with chemical mutagens

The alkaline elution technique was used to measure DNA damage in the rat testis after intraperitoneal injection of 3 chemicals known to cause heritable mutations in rodents. These 3 chemicals are triethylenemelamine (TEM), mitomycin C, and cyclophosphamide. All three of these chemicals produced DNA damage which was readily detectable by alkaline elution. Both TEM and mitomycin C produced DNA interstrand cross-links, although TEM was a more potent cross-linker on an equimolar basis than mitomycin C. Cyclophosphamide produced both DNA cross-links and DNA strand breaks. Alkaline elution in the absence of proteinase K indicated that some of the strand breaks appeared to be closely associated with protein. These studied indicate that the alkaline elution technique is capable of detecting DNA damage in mammalian germ cells produced by chemical mutagens. This technique may prove useful as a screening tool for identifying chemicals which cause heritable mutations in mammals.     

Isolation, Characterization, and Genetic Analysis of Mutator Genes in Escherichia coli B and K-12

Twenty-one Mut mutants were obtained from Escherichia coli B (B/UV) and K-12 (JC355) after treatment with mutagens. These Mut strains are characterized by rates of mutation to streptomycin resistance and T-phase resistance which are significantly higher than the parental (Mut(+)) rates. Mutator genes in 12 strains have been mapped at three locations on the E. coli chromosome: one close to the leu locus; five close to the purA locus; and six close to cysC. In addition, eight mutator strains derived from E. coli B/UV are still unmapped. Some effort was made to deduce the mode of action of the mutator genes. These isolates have been examined for possible defects in deoxyribonucleic acid repair mechanisms (dark repair of ultraviolet damage, host-cell reactivation, recombination ability, repair of mitomycin C damage). By using transductional analysis, it was found that the ultraviolet sensitivity of NTG119 and its mutator property results from two separate but closely linked mutations. PurA(+) transductants that receive mut from NTG119 or NTG35 are all more sensitive to mitomycin C than is the PurA recipient. Unless transduction selects for sensitivity, a probable interpretation is that defective repair of mitomycin C-induced damage is related to the mode of action of mut in these transductants and the donor. Abnormal purine synthesis may be involved in the mutability of some strains with cotransduction of the mutator properly and purA (100% cotransduction for NTG119). Three mutators are recombination-deficient and may have a defective step in recombination repair. One maps near three rec genes close to cysC.     

Aging and sister chromatid exchange. IV. Reduced frequencies of mutagen-induced sister chromatid exchanges in vivo in mouse bone marrow cells with aging.

Induction of sister chromatid exchanges (SCE's) was examined in bone marrow cells of young and old C57BL/6J mice exposed to three different DNA-damaging agents (cyclophosphamide, mitomycin C, and doxorubicin). At low concentrations of all three mutagens, the levels of induced SCE's were similar in young and old cell populations. However, at higher mutagen concentrations, SCE induction was significantly reduced in old cell populations. Studies of mice aged 5 to 32 months revealed that induced SCE frequencies remain stable during early adulthood (5 to 12 months) and then begin to decline as a function of age. These results indicate that with aging there exists a gradual alteration of cellular response to DNA damage.     

Rapid detection of mutagen induced micronucleated erythrocytes by flow cytometry

Summary The micronucleus test is a cytogenetic method for the screening of mutagens and carcinogens which exhibit clastogenic mechanisms of action. After application of clastogenic agents chromosomal fragments or even whole chromsomes can remain as conspicuous structures (micronuclei) in a small fraction of anucleated polychromatic erythrocytes which can be visually scored using a microscope following staining with May-Grünwald-Giemsa solutions. These time-consuming, painstaking, and tedious manual evaluations are often sources of unreliability and uncertainty. Here, a fluorescence technique is presented which applies DNA and protein fluorochromes to discriminate normal anucleated erythrocytes from micronucleated erythrocytes using a fluorescence microscope. This method is particularly tailored to be applied to flow cytometric instrumentation. Data obtained manually and automatically in flow show a strong linear correlation with high significance (r=0.96) as far as the percentage of micronucleated erythrocytes as an indicator for the mutagenicity of a given drug is concerned. These results have been obtained by means of the established clastogens cyclophosphamide and mitomycin C.     

Assessment of the relationship between the antimutagenic action of riboflavin and glutathione and the levels of antioxidant enzymes

In this study the role of antioxidant enzymes on the antimutagenic actions of riboflavin and reduced glutathione against mutagenic potentials of 4-nitroquinoline 1-oxide and mitomycin C have been investigated. For this purpose the activities of catalase and superoxide dismutase enzymes have been determined in Salmonella typhimurium TA102 and TA100 strains preincubated with different combinations of 4-nitroquinoline 1-oxide, mitomycin C, riboflavin and reduced glutathione for thirty minutes. Also in part of the same samples, the mutagenicity has been determined for each combination of chemicals by using Salmonella preincubation test. The correlation between the levels of antioxidant enzymes and mutagenicity and antimutagenicity has been investigated.While riboflavin displayed a weakly antimutagenic effect on 4-nitroquinoline 1-oxide mutagenicity in TA102 and TA100 (0.25, 0.35 inhibition respectively), it did not have any effect on the strong mutagenicity of mitomycin C in both strains. Reduced glutathione, a well known antioxidant, had no antimutagenic effect against the mutagenicity of both compounds in TA102 and TA100 strains. The antioxidant enzymes, catalase and superoxide dismutase, seemed to have no direct effect on the antimutagenic action of riboflavin and mutagenic action of 4-nitroquinoline 1-oxide and mitomycin C because no change in the activities of catalase and superoxide dismutase was detected in relation to antimutagenicity of riboflavin and mutagenicity of 4-nitroquinoline 1-oxide and mitomycin C in both strains. It should be noted that many antimutagens have more than one mechanism of action and their effect depends on the mutagens being tested.     

Isolation and physiological characterization of mitomycin C-sensitive/UV-sensitive mutants in Bacteroides fragilis

Mutants of Bacteroides fragilis sensitive to mitomycin C were isolated after mutagenesis with ethyl methane sulphonate. One mutant (MTC25) was markedly sensitive to mitomycin C but was unaffected as regards UV sensitivity; another mutant (UVS9) was sensitive to UV radiation but was only moderately sensitive to mitomycin C. Caffeine decreased the survival after UV-irradiation of the wild-type, MTC25 and UVS9 strains by the same relative amount. Aerobic liquid holding recovery occurred in each of the three strains. The MTC25 and UVS9 mutants showed reduced host cell phage reactivation. The wild-type, MTC25 and UVS9 strains all showed UV- and H2O2-induced phage reactivation. The physiological characterization of the MTC25 and UVS9 mutants indicates that it is possible to differentiate between mechanisms for the repair of mitomycin C- and UV-induced DNA damage in B. fragilis.     

In vitro screening of carcinogens using DNA of the His- mutant of Salmonella typhimurium

Carcinogens: 7,12-dimethylbenz(a)anthracene (DMBA), 2-acetylaminofluorene (AAF), 3-methylcholanthrene (MC), daunorubicin, thio-TEPA, cyclophosphamide, 1-(2-chloroethyl)2-cyclohexyl-1-nitrosourea (CCNU) and mitomycin C, which mutate Salmonella typhimurium tester strains in the Salmonella/microsome mutagenicity test, strongly stimulate the in vitro synthesis of DNA isolated from His- mutants (TA-1538, TA-1537, TA-1535), and induce in vitro His- DNA strand separation. Four other carcinogens, ethionine, actinomycin D, bleomycin and reserpine, which were not known as mutagens in the Ames test, also strongly stimulate His- DNA synthesis and His- DNA strand separation in local areas. Those substances which are neither carcinogenic nor mutagenic: d-lactose, fluorene, cholesterol, and saccharin, do not stimulate either His- DNA (or His+ DNA) synthesis or DNA in vitro strand separation. Steroid hormones, which are carcinogenic only for steroid target tissues, do not react with bacterials DNA.