tRNA-ethylnitrosourea reaction: incorporation of the carbonyl function of ethylnitrosourea into the "pyrimidine-nucleotide-like" fraction.
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Transformation of cultured epithelial cells by ethylnitrosourea. Altered expression of type I procollagen chains.
Cultured epithelial rodent cells were transformed in vitro using ethylnitrosourea as a carcinogen either alone or in combination with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). The frequency of transformation in the absence of TPA was 5 X 10(-4) at 10 micrograms/ml ethylnitrosourea. Growth of ethylnitrosourea-treated cells in TPA-substituted medium increased the transformation frequency 8-fold. Colonies of transformed cells were isolated from soft agar and analyzed for the production of pericellular matrix glycoproteins. The ethylnitrosourea-transformed cells retained pericellular matrix structures, typical of the nontransformed control cells. Parent cells produced into their culture media fibronectin and procollagen types I and III as their major pericellular glycoproteins. The ethylnitrosourea-transformed cells synthesized and secreted altered procollagen polypeptides. The procollagen of ethylnitrosourea-transformed cells apparently consisted mainly of homotrimeric pro alpha 1 molecules, with smaller amounts of basement membrane procollagen-like chains. Fibronectin synthesis or secretion was not affected by ethylnitrosourea-induced transformation, but the production of fibronectin was enhanced in the transformed cultures treated with TPA. Also, the deposition of procollagen and fibronectin into the pericellular matrix was not affected by ethylnitrosourea-transformation. Very similar changes had previously been observed in murine sarcoma virus-transformed cells. The change of procollagen type I thus appears to be a correlate of malignant transformation of cultured epithelial cells. The results indicate that ethylnitrosourea can induce malignant transformation of epithelial cells in culture and modify production and deposition of pericullular glycoproteins.
Animals↗
The antimalarials quinacrine and chloroquine potentiate the transplacental carcinogenic effect of ethylnitrosourea on ependymal cells.
Quinacrine and chloroquine, two widely used antimalarials, bind strongly to deoxyribonucleic acid, thus preventing mutagenesis. We studied a possible chemoprotective effect of these substances on carcinogenesis of the nervous system induced in Wistar rats by transplacental administration of ethylnitrosourea. One experimental group consisted of rats born from mothers treated with quinacrine prior to prenatal exposure to ethylnitrosourea; a second group consisted of rats chronically treated with chloroquine after prenatal exposure to ethylnitrosourea. When compared with controls, no significant differences were observed in tumor incidence. However, early tumor growth was observed in both rats treated with quinacrine (P < 0.0004) and rats treated with chloroquine (P < 0.02). These differences were due mostly to rapid development of ependymomas of the spinal cord. Our results suggest that quinacrine and chloroquine do not prevent the structural alterations induced in DNA by ethylnitrosourea, which lead, in the long term, to a high incidence of neoplasms in the nervous system. Moreover, the antimalarials studied seem to promote the carcinogenic effects of ethylnitrosourea on ependymal cells.
Animals↗
The effect of the interval between dose applications on the observed specific-locus mutation rate in the mouse following fractionated treatments of spermatogonia with ethylnitrosourea.
Our earlier analyses have suggested an apparent threshold dose-response for ethylnitrosourea-induced specific-locus mutations in treated spermatogonia of the mouse to be due to a saturable repair process. In the current study a series of fractionated-treatment experiments was carried out in which male (102 x C3H)F1 mice were exposed to 4 x 10, 2 x 40. 4 x 20 or 4 x 40 mg ethylnitrosourea per kg body weight with 24 h between applications; 4 x 40 mg ethylnitrosourea per kg body weight with 72 h between dose applications; and 2 x 40, 4 x 20 and 4 x 40 mg ethylnitrosourea per kg body weight with 168 h between dose applications. For all experiments with 24-h intervals between dose applications, there was no effect due to dose fractionation on the observed mutation rates, indicating the time interval between dose applications to be shorter than the recovery time of the repair processes acting on ethylnitrosourea-induced DNA adducts. In contrast, a fractionation interval of 168 h was associated with a significant reduction in the observed mutation rate due to recovery of the repair process. However, although reduced, the observed mutation rates for fractionation intervals of 168 h were higher than the spontaneous specific-locus mutation rate. These observations contradict the expectation for a true threshold dose response. We interpret this discrepancy to be due to the differences in the predictions of a mathematical abstraction of experimental data and the complexities of the biological system being studied. Biologically plausible explanations of the discrepancy are presented.
Animals↗
The frequency of dominant cataract and recessive specific-locus mutations in mice derived from 80 or 160 mg ethylnitrosourea per kg body weight treated spermatogonia.
A systematic comparison of the frequency of dominant cataract and recessive specific-locus mutations in mice has been extended to include results for 80 and 160 mg ethylnitrosourea per kg body weight spermatogonial treatment. The frequency of confirmed dominant cataract mutations in the historical control, 80 and 160 mg/kg ethylnitrosourea treatment groups was 1/22594, 8/5090 and 14/6435, respectively. The frequency of recessive specific-locus mutations in the same dose groups was, respectively, 19/227805, 20/13274 and 35/8658. These present results confirm previous results, which indicate that ethylnitrosourea is effective in inducing both recessive specific-locus and dominant cataract mutations although the per locus mutation rate to recessive alleles was observed to be approximately 6 times greater than the per locus mutation rate to dominant alleles. The exclusion of certain classes of lens opacity variant phenotypes, previously demonstrated not to be due to a dominant mutation, from the group of suspected dominant cataract mutations subjected to a genetic confirmation test has greatly improved the efficiency of the test. A total of 23 dominant cataract mutations were confirmed from a group of 67 phenotypic variants. Of the 23 confirmed dominant cataract mutations, 8 were shown to have reduced transmission to the following generation of offspring expressing the mutant phenotype. These results are also consistent with previous results for ethylnitrosourea or radiation treatment in which it was shown that approximately one-third of the recovered mutations have reduced penetrance. One group of dominant cataract mutations, with phenotypic effects on the polar, sub-capsular or corneal regions, is overly represented in the group of recovered mutations with a reduced transmission of offspring expressing the mutant phenotype. Two hypotheses are suggested for this observation, both dependent on the fact that the regions affected indicate that the mutations are expressed later in the development of the eye. Either all carrier individuals have not expressed the phenotype at the time of examination and classification, or later acting mutations are more subject to environmental interactions resulting in more variable expression. Finally, it is argued that a dominant cataract mutation test represents a most practicable protocol to screen for induced dominant mutations in germ cells of the mouse. The imposition of the criterion that suspected variants be subjected to a genetic confirmation test has at least two advantages beside the fact that results represent unambiguous mutational events.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
The effect of dose fractionation on the frequency of ethylnitrosourea-induced dominant cataract and recessive specific locus mutations in germ cells of the mouse.
A combined dominant cataract-recessive specific locus mutation experiment for fractionated exposure to ethylnitrosourea (2 X 80 mg/kg, 24-h fractionation interval) was designed to determine if lower doses of ethylnitrosourea are more effective in inducing dominant cataract mutations as suggested by previous results. This observation was not confirmed by the present experiment. The extensive, statistically more reliable specific locus results indicate an additive effect of fractionated ethylnitrosourea treatment. A saturable repair system for ethylnitrosourea-induced DNA damage has been previously documented (Karran et al., 1979; Sega et al., 1986; Van Zeeland et al., 1985). Two parameters inherent to a saturable system, the minimal time required for the saturated system to recover and the minimal dose to saturate the system are important, and results of experiments employing a fractionation exposure protocol must be interpreted relative to these two parameters. Longer fractionation intervals or smaller doses result in a reduced mutagenic effect. Due to the inherently lower experimental variability of the specific locus mutation assay as compared to the dominant cataract assay, the specific locus assay is the test of choice to determine factors affecting the mammalian germ cell mutation rate. The dominant cataract test requires a larger investment of experimental resources to achieve a comparable degree of accuracy. The dominant cataract mutation test is important in assessing the mutation rate to dominant alleles in germ cells of mammals. Due to the immediate expression of the mutant phenotype in newly occurring dominant mutations, a dominant mutation assay screens a genetically relevant endpoint in an assessment of the mutagenic hazard for man in mouse experiments. A multi-endpoint design screening specific locus, dominant cataract, and biochemical mutational endpoints (Ehling et al., 1985) allows a systematic comparison of mutagenic results for different classes of mutations in the same animals.
Abnormalities, Drug-Induced↗
A dose-response analysis of ethylnitrosourea-induced recessive specific-locus mutations in treated spermatogonia of the mouse.
A dose-response analysis was carried out with 2 independent data sets available for ethylnitrosourea-induced specific-locus mutations in spermatogonia of the mouse. It was assumed that the occurrence of mutation is binomially distributed and maximum-likelihood procedures were employed to determine the appropriateness of 4 alternative models, Linear, Linear-Quadratic, Power, and Threshold, in describing the dependence of the binomial parameter on dose. For both data sets, the Threshold model yielded a far superior fit and the threshold dose was estimated to be between 34 and 39 mg/kg. These results are supported by the relatively inefficient response of ethylnitrosourea at lower doses in inducing DNA adducts. Relevant specific-locus mutation results in the mouse for low-dose fractionated treatment as well as the recovery of mutation mosaics indicate the threshold model to be an oversimplification. Rather than a threshold dose below which 100% of the induced DNA adducts are repaired, we propose that some DNA adducts which may eventually be fixed as a mutation persist through a number of repair-competent cell divisions and do not interfere with normal cell function nor do they induce a repair response before being eventually fixed as a mutation. We interpret the thresholded response for ethylnitrosourea-induced specific-locus mutations to be due to a saturable repair process which at lower doses results in ethylnitrosourea being less efficient in inducing mutation. Once this repair process is saturated, a clear dose-related increase in the mutation rate is observed.
Animals↗
Specific-locus test shows ethylnitrosourea to be the most potent mutagen in the mouse.
Use of the specific-locus test to measure the frequency of transmitted gene mutations induced in mouse spermatogonia has shown ethylnitrosourea to be by far the most potent mutagen yet discovered in the mouse. The dose used, 250 mg/kg, gave a mutation rate 5 times as high as had been obtained with 600 R, the most effective acute dose of x-rays. Compared to procarbazine, heretofore the most mutagenic chemical known in the mouse, ethylnitrosourea proved to be 15 times more mutagenic than the peak effect obtained with the most effective dose of procarbazine. Because of its high mutagenicity, ethylnitrosourea can serve as a model compound in exploring the effect of such factors as dose response, dose fractionation, sex, and cell stage on the mutagenic action of a chemical. Ethylnitrosourea is clearly the mutagen of choice for the production of any kind of desired new gene mutations in the mouse.
Alleles↗
Macromolecular synthetic activity in mice regenerating liver after ethylnitrosourea injection.
The present report is a continuation of our previous studies on the biochemical mechanisms of carcinogenesis; studying the nature of interactions taking place between Ethylnitrosourea and DNA, RNA and protein of various stages of their synthetic activity. As a model system we chose partially hepatectomized mice live 36 hrs after surgery. Synthetic macromolecule activity in the remaining liver segment was determined by means of 3H-thymidine, 3H-uridine and 3H-leucine. We observed complete depression of DNA synthetic activity (immediately after Ethylnitrosourea administration it remained depressed almost through out the whole period of our observations) while protein synthetic activity was highly elevated. Qualitative changes of soluble proteins which were analyzed by isoelectric fractionation on 5% polyacrylamide after previous 3H- and 14C-leucine incorporation, could not be detected. Our biochemical data are correlated with histological studies and with the tumour incidence following the Ethylnitrosourea treatment of partially hepatectomized mice in the course of long-term experiments. The results provide guideline for further analysis, which should be modified according to the information concerning Ethylnitrosourea carcinogenesis induced 36 hours after partial hepatectmoy.
Animals↗
In vivo mutagenesis in the cynomolgus monkey: time course of HPRT mutant frequency at long time points following ethylnitrosourea exposure.
We have monitored mutant frequency at the HPRT locus in peripheral blood lymphocytes of cynomolgus monkeys using a clonal assay in which mutants are selected by resistance to 6-thioguanine. Among untreated animals, the mean spontaneous mutant frequency was 2.9 +/- 2.9 x 10(-6) (standard deviation, based on 131 determinations in 33 animals), in good agreement with HPRT mutant frequencies in other species. In four animals treated with a single intraperitoneal dose of 77 mg/kg ethylnitrosourea, mutant frequency increased with time, peaking 70 to 100 days after treatment. Mutant frequency in two of the four animals was monitored at intervals for 6 years, and a second identical treatment was given about 830 days after the first. Mutant frequency again peaked in these two animals 70 days after the second dose and decreased following peak values, declining to a plateau that was higher than the predose mutant frequency in both animals. This pattern was repeated following the second ethylnitrosourea treatment. Fractionating the dose of ethylnitrosourea into five equal daily injections had no effect on mutant frequency in two animals when compared to a single dose.
Animals↗
Study of postnatal effects of chemopreventive agents on offspring of ethylnitrosourea-induced transplacental carcinogenesis in rats. I. Influence of retinol acetate, alpha-tocopherol acetate, thiamine chloride, sodium selenite, and alpha-difluoromethylornithine.
We studied the influence of the vitamins retinol acetate, alpha-tocopherol acetate and thiamine chloride; the antioxidant sodium selenite and an inhibitor of polyamine biosynthesis, alpha-difluoromethylornithine, on the offspring of transplacental carcinogenesis by ethylnitrosourea in rats. Ethylnitrosourea was given to pregnant rats as a single i.v. injection, at a dose of 75 mg/kg body wt. or 5.5 mg/kg body wt., on the 21st day after conception. Retinol, tocopherol or thiamine was added to the diet, and selenite and alpha-difluoromethylornithine to drinking water of the offspring throughout their postnatal life at moderate doses. In control groups, ethylnitrosourea induced tumors of brain, spinal cord, peripheral nervous system and kidneys in the offspring. alpha-Difluoromethylornithine exerted a slight inhibitory effect; this agent decreased the total tumor multiplicity and the multiplicity of peripheral nervous system tumors and also prolonged survival time. Retinol, tocopherol, thiamine and selenite did not influence the development of the transplacentally-induced tumors.
Animals↗
The mutagenic activity of ethylnitrosourea at low doses in spermatogonia of the mouse as assessed by the specific-locus test.
Ethylnitrosourea is the most efficient chemical mutagen in spermatogonial stem cells of the mouse and its mutagenic activity has been intensively studied. The pertinent specific-locus mutation test results for a discussion of low dose-effect studies have been summarized and indicate: (1) A threshold dose response best characterizes the relationship between dose and mutation rate. (2) The reduced effectiveness of ethylnitrosourea in the low dose range is likely due to a saturable repair process. (3) The recovery of the saturable repair process as assessed in fractionated dose experiments is long (ca. 168 h). The dynamics of stem cell spermatogonia suggests a long time interval before the cell population passes through at least one cell division and this may be relevant to an interpretation of the fractionation effects. (4) There is a slight but important discrepancy between the predicted and observed mutagenic activity of ethylnitrosourea in the low dose range. This is interpreted to be due to the differences between a mathematical abstraction and the biological realities of the system being studied.
Animals↗
Somatic and germ cell mutagenesis in lambda lacZ transgenic mice treated with acrylamide or ethylnitrosourea.
The transgenic Muta Mouse in vivo mutagenesis assay was employed to determine the activity of acrylamide and ethylnitrosourea in liver and germ cells after 3, 10 and 100 days following treatment. Each cell of the Muta Mouse carries 80 copies of the lambda gt10 phage including the bacterial lacZ gene, which act as the target gene for the mutagenesis assay. Groups of Muta Mice were given a single intraperitoneal injection of 80 or 160 mg/kg ethylnitrosourea or 50 or 100 mg/kg acrylamide. The tissues were prepared 3, 10 or 100 days post treatment. The liver genomic DNA was extracted with the manufacturer's standard protocol, while the genomic germ cell DNA was extracted with 4 different methods due to problems encountered in DNA yields and packaging efficiency. The mutation analysis of the lacZ gene was carried out by the positive selective assay method [Gossen et al. (1989) Proc. Natl. Acad. Sci. USA, 86, 7971-7975; Dean and Myhr (1994) Mutagenesis, 9, 183-185]. There was a slight increase due to treatment of the observed mutation frequencies in the acrylamide liver group for all three assay times. From the day 3 group to the day 100 group a time dependent decrease in all the absolute mutant frequencies was detectable. The ethylnitrosourea liver group showed a time- and dose-dependent increase in the mutant frequencies from day 3 to day 100. No meaningful results were obtained for the germ cell tissue assays due to the low amount of genomic DNA extracted which was not packageable in the lambda lacZ assay. At present for the mutagenesis assay of isolated spermatozoa in our laboratory we would be forced to pool tissues from animals to obtain enough DNA for an assay. Since 'jackpot'-animals may exist [Heddle et al. (1992) Mutation Res., 272, 195-203] the individual animals of such a pooled analysis group must be tested before pooling.
Acrylamide↗
The effect of nerve growth factor and antibodies to nerve growth factor on ethylnitrosourea-induced neoplastic proliferation in rat trigeminal nerves.
Sprague-Dawley (CD) rats were injected intravenously with ethylnitrosourea at a dose of 20 mg/kg on day 20 of gestation. This exposure resulted in early neoplastic proliferation or development of a neurinoma of the trigeminal nerve in 58% of the offspring at 90 days of age. Implantation of osmotic microinfusion pumps containing 2.5S nerve growth factor prior to ethylnitrosourea administration significantly reduced the incidence of early neoplastic proliferation. Postnatal implantation of microinfusion pumps containing 2.5S nerve growth factor also resulted in a significant but less pronounced reduction of early neoplastic proliferation. Immunoglobulin G directed against nerve growth factor (anti-nerve growth factor) did not influence the incidence of early neoplastic proliferation when administered via microinfusion pumps implanted on day 15 postnatally. These findings suggest that nerve growth factor has a protective effect on the developing nervous system against ethylnitrosourea-induced carcinogenesis.
Animals↗
Reduction of ethylnitrosourea-induced neoplastic proliferation in rat trigeminal nerves by nerve growth factor.
Ethylnitrosourea administered to pregnant rats on the 21st day of gestation at a dose of 50 mg/kg resulted in early neoplastic proliferation or neurinomas of trigeminal nerves in 90% of the offspring at 90 days of age. Treatment with nerve growth factor either postnatally following ethylnitrosourea administration or transplacentally prior to ethylnitrosourea treatment appeared to result in a reduction of neurinomas at 90 days of age.
Age Factors↗
Prenatal multicarcinogenesis by ethylnitrosourea in mice.
The Role of prenatal age, sex, and the maternal background upon the incidence, multiplicity, and spectrum of tumors induced by ethylnitrosourea has been studied in the offspring of reciprocal hybrids of the same genotype. The first generation (F1) offspring of C57BL/6J X C3HeB/FeJ and the reciprocal hybrids were observed throughout their life-span for tumor development following single i.p. injections of ethylnitrosourea (60 microng/g) given to pregnant mothers at 12, 14, 16, or 18 days of gestation. Animals exposed to ethylnitrosourea survived on the average for only 90 weeks. They developed by that age tumors in lungs, livers, ovaries, nervous system, and forestomach. Control mice killed at 90 weeks were essentially free of tumors. The fetal age at the time of administration of the carcinogen was one of the most significant modulators of tumor development in lung, liver, ovaries, and nervous system. The sex of the animals influenced the rate of development of liver tumors, whereas maternal background affected the multiplicity of lung tumors.
Animals↗
Transplacental induction of neurogenic tumors in BD IX rats by intragastric administration of ethylnitrosourea precursors.
The concurrent daily intragastric administration of ethylurea at two dose levels (50 mg/kg and 100 mg/kg bodyweight) together with one dose level of sodium nitrite (50 mg/kg bodyweight) by a stomach tube to pregnant BD IX rats from day 15 to day 22 of gestation resulted in the induction of neurogenic tumors in all offspring. Since both ENU-precursors alone do not produce neurogenic tumors, these results are evidence of ENU formation from its precursors under the influence of gastric juice. Differences in the survival time and the incidence of tumors at both dose levels were not significant. The amount of ethylnitrosourea synthesized in the animals was very close at both dose levels, and was dependent on the amount of sodium nitrite applied. The experimental results are consistent with the conclusion, that the rat fetuses had been exposed to a total amount of about 60 mg/kg ethylnitrosourea. Neurogenic tumors dominated with 98% incidence over the non-neurogenic. The incidence of neurogenic tumors per rat was high (6.0 for Group I and 6.7 for Group II). Neurogenic tumors were equally distributed among the central and peripheral nervous systems. The neurogenic tumors induced with the precursors of ethylnitrosourea were morphologically similar in all aspects to those induced with the carcinogen itself and could be classified as oligodendroglioma, astrocytoma, mixed glioma, anaplastic glioma, glioependymoma, ependymoma, and neurinoma. Three unusual tumors were found: one early anaplastic "septum ependymoma" in the dorsal column of the spinal cord, and two special mixed tumors of the cranial nerves, i.e. a neurinoma with portions of an oligodendroglioma and a neurinoma with parts of an invasive ependymoma.
Animals↗
Strand breakage in rat brain DNA and its repair induced by ethylnitrosourea in vivo.
The damage and repair of rat brain DNA was studied in vivo after a single carcinogenic dose of ethylnitrosourea. Fragmentation of the brain DNA produced by this carcinogen was demonstrated on alkaline sucrose gradients. By the 24th hrs after treatment with ethylnitrosourea the single-strand damage to DNA was not completely repaired. As the highly differentiated cells of the central nervous system do not proliferate, it is possible that during brain carcinogenesis delayed repair of DNA of primitive cells might be needed for the formation of tumor anlage.
Animals↗