In vivo genotoxicity of nitrates and nitrites in germ cells of male mice. I. Evidence for gonadal exposure and lack of heritable effects

Effects of nitrate (doses of 600 and 1200 mg/kg/day during 14 days) and sodium nitrite (60 and 120 mg/kg/day during 14 days) on germ cells of male mice were investigated. The mode of application was stomach intubation. The germ cell stages analysed were spermatids (for the heritable effects) and differentiating and stem-cell spermatogonia (for direct effects).A lack of heritable translocations, sperm abnormalities, as well as morphological changes, such as changes in eyes, coat colour, testes and body weight, was demonstrated in F₁ males originating from treated P males. Significant effects in treated males were found with respect to: (1) sex-chromosomal univalency in the diakinesis-methaphase I stage after the treatment of stem spermatogonia (both doses of sodium nitrate and the higher dose of sodium nitrite), (2) sperm-head abnormalities after treatment of differentiating spermatogonia (the higher dose of sodium nitrate and both doses of sodium nitrite), and (3) fertility after treatment of spermatids (the higher dose of sodium nitrite). Nonmutagenic effects and possible carcinogenic potential of the tested doses are discussed.     

Unscheduled DNA synthesis induced by the antitumor drug vincristine in germ cells of male mice

The cytotoxic and genotoxic effects of vincristine (VCR) on germ cells of male mice were investigated. Several parameters (the scale and the time course of unscheduled and scheduled DNA synthesis in spermatocytes and spermatids and the number of sperm present in caudal epididymides) were analyzed. Our results show that intraperitoneal administration of a single dose of VCR resulted in: (1) damage to DNA in spermatocytes and spermatids; (2) a reduction in the rate of germ-cell development; and (3) killing of the non-proliferating spermatid cells. Damage of DNA in germ cells indicates that VCR may have potential genetic hazards to patients who receive it in antitumor therapy.     

Unscheduled DNA synthesis induced in mouse spermatids after combined treatment with methyl methanesulfonate and X-rays.

Unscheduled DNA synthesis (UDS), which is considered to be DNA repair, has been studied in early- to mid-spermatid stages of the mouse after combined treatments with X-rays and methyl methanesulfonate (MMS). UDS in spermatids was detected by giving testicular injections of [methyl-3H]thymidine ([3H]dThd) and making use of the fact that no scheduled DNA synthesis occurs in the germ cells after the last S period in primary spermatocytes. X-rays and MMS are each able to induce UDS in mouse spermatids. However, there was a statistically significant reduction in the amount of UDS observed when X-ray exposures of from 200 to 600 R were given 4 h before an i.p. injection of 75 mg/kg of MMS and concurrent testicular injections of [3H]dThd. This reduction in UDS is more than can be explained by the completion of repair of X-ray-induced DNA lesions. We suggest that the reduction in UDS is the result of an X-ray-produced impairment of a least a part of the repair mechanism involved in correcting MMS-induced DNA lesions. When the time interval between a 600-R X-ray exposure and MMS treatment was between 3 and 20 h (latest time interval s;udied) there was a statistically significant reduction of UDS in the spermatids. No significant decrease in UDS response occurred when the time interval between radiation exposure and MMS treatment was less than approximately 3 h.     

Unscheduled DNA synthesis in isolated hepatic nuclei after treatment of rats with methylnitrosourea in vivo

Administration of the carcinogenic methylating agent, methylnitrosourea, to rats caused a significant increase in endogenous DNA synthesis assayed subsequently in isolated hepatic nuclei $\text{in}\text{vitro}$. DNA synthesis was related directly to the dose of carcinogen and inversely to the interval between treatment and isolation of nuclei. This synthesis appears to represent the continuation $\text{in}\text{vitro}$ of unscheduled, reparative DNA synthesis initiated in damaged cells $\text{in}\text{vivo}$.     

Induction of shape abnormality and unscheduled DNA synthesis by arecoline in the germ cells of mice

The ability of arecoline, an alkaloid of betel nut, to induce abnormality in the shape of sperm heads and unscheduled DNA synthesis (UDS) in the early spermatid stages of Swiss albino mice was studied. Treatment of mice with arecoline at the dose levels of 20, 40 and 80 mg/kg elicited dose-related increase in the number of abnormal sperm heads, as well as the unscheduled incorporation of [3H]thymidine into the DNA of early spermatids. Such increase in the production of abnormally shaped sperms and UDS response of the early spermatids following arecoline treatment expressed its genotoxic potential in the mouse germ cells.     

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

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.     

Unscheduled DNA synthesis after treatment with 20-methylcholanthrene and N-nitrosodimethylamine in primary culture of human gallbladder epithelial cells

Unscheduled DNA synthesis (UDS) induced by treatment with 20-methylcholanthrene (MCA) and N-nitrosodimethylamine (DMN) was measured in cultured human gallbladder epithelial cells. MCA induced UDS very efficiently, while DMN was far less effective than MCA. Addition of rat S9 mixture did not affect the amount of UDS by the chemicals. Differences between the present results in human cells and our previous findings in bovine cells could be due to species and tissue specificity.     

DNA metabolism during maturation of male germ cells. II. Amplification structures in the nuclei of rat germ and somatic cells

Amplification structures have been found in preparations of histone-depleted somatic (liver) and sex (spermatogonia, spermatocytes 1) rat cells. Multi-forked chromosomal (2-4 replicative forks originating from a single strand of DNA) and extrachromosomal circular amplification structures have been detected in the nuclei of sex cells. All the circular molecules of DNA detected belong, according to size, to the class of small nuclear polydispersed circular DNAs. Chromosomal amplification structures (eye-in-eye or several replicative forks originating from one DNA strand) have been only detected in the nuclei of somatic cells.     

Unscheduled DNA synthesis in xeroderman pigmentosum cells after microinjection of yeast photoreactivity enzyme

Photoreactivating enzyme (PRE) from yeast causes a light-dependent reduction of UV-induced unscheduled DNA synthesis (UDS) when injected into the cytoplasm of repair-proficieint human fibroblasts (Zwetsloot et al., 1985). This result indicates that the exogenous PRE monomerizers UV-induced dimers in these cells competing with the endogenous excision repair. In this paper we present the results of the injection of yeast PRE on (residual) UDS in fibroblasts from different excision-deficient XP-strains representing complementation groups A, C, D, E, F, H and I (all displaying more than 10% of the UDS of wild-type cells) and in fibroblasts from two excision-proficient XP-variant strains.In fibroblasts belonging to complementation groups C, F and I and in fibroblasts from the XP-variant strains UDS was significantly reduced, indicating that pyrimidine dimers in these cells are accessible to and can be monomerized by the injected yeast PRE. The UDS reduction in the XP-variant strains is comparable with the effect in wild-type cells. In cells from complementation groups C, F and I the reduction is less than in wild-type and XP-variant cells. Fibroblasts belonging to groups A, D, E and H did not show any reduction in UDS level after PRE injection and illumination with photoreactivating light. These result give evidence that the genetic repair defect in some XP-strains is probably due to an altered accessibility of the UV-damaged sites.     

gamma-Tubulin overexpression in Sertoli cells in vivo. II: Retention of spermatids,residual bodies,and germ cell apoptosis

The degree of germ cell dependence on Sertoli cell-mediated activities has been a subject of considerable attention. Sertoli cell secretory pathways have been extensively studied both in an effort to understand their normal physiologic roles and as targets for pharmacologic and toxicant activity. To determine the degree to which normal spermatogenesis depends on key functions of the Sertoli cell microtubule network, adenoviral vectors that overexpress the microtubule nucleating protein, gamma-tubulin, were delivered to Sertoli cells in vivo. gamma-Tubulin overexpression disrupts the Sertoli cell microtubule network (as described in the companion article); leads to gross disorganization of the seminiferous epithelium, inducing retention of spermatids and residual bodies; and causes germ cell apoptosis. These data are consistent with earlier studies in which toxicants and pharmacologic agents were used to disrupt microtubule networks. These data confirm that Sertoli cell microtubule networks play an important role in maintaining the organization of the seminiferous epithelium and that in the absence of an intact Sertoli cell microtubule network, germ cell viability is impaired.     

Generation of functional oocytes and spermatids from fetal primordial germ cells after ectopic transplantation in adult mice

Primordial germ cells (PGCs) are undifferentiated germ cells in developing fetuses. As these cells give rise to definitive oocytes and spermatozoa that contribute to new life in the next generation, their development must be under strict control, regarding genetic and epigenetic aspects. However, we do not know to what extent their development depends on the specific milieu. In this study, we transplanted mouse PGCs collected from male and female gonads at 12.5 days postcoitum, together with gonadal somatic cells, under kidney capsules of adult mice. The transplanted PGC and gonadal somatic cells constructed testis-like and ovary-like tissues, respectively, under the kidney capsules within 4 wk. Normal-appearing round spermatids and fully grown germinal vesicle (GV) oocytes developed within these tissues. Ectopic spermatogenesis continued thereafter, while oogenesis consisted of only a single wave. The injection of these round spermatids directly into mature in vivo-derived oocytes led to the birth at term of normal pups. PGC-derived GV oocytes were isolated, induced to mature in vitro, and injected with normal spermatozoa. The injected oocytes were successfully fertilized and developed into normal pups. Our findings demonstrate the remarkable flexibility of PGC development, which can proceed up to the functional gamete stage under spatially and temporally noninnate conditions. This transplantation system may provide a unique technical basis for induction of the development of early germ cells of exogenous origins, such as those from embryonic stem cells.     

Unscheduled DNA synthesis after beta-irradiation of mouse skin in situ

The skin of ICR mouse was irradiated with beta-rays from 90Sr-90Y with surface doses up to 30 krad. Unscheduled DNA synthesis (UDS) was measured by autoradiography after labeling the skin with radioactive thymidine using the forceps-clamping method. The level of UDS in epithelial cells of the skin was detected as an increasing function of radiation dose. Fibroblastic cells, compared with epithelial cells and hair follicle cells at the same depth of the skin, showed a lower level of UDS, indicating a lower DNA repair activity in fibroblasts. Cancer risk of the skin was discussed.     

Transgenic mice produced by retroviral transduction of male germ line stem cells in vivo

Spermatogonial stem cells are the only stem cells in the postnatal body that can transmit parental genetic information to the offspring, making them an attractive target cell population for animal transgenesis. Although transgenic mice and rats were recently produced by retrovirus transduction of these cells in vitro, with transplantation of the transduced cells into infertile recipients, the difficulty of restoring fertility and preparing recipients using spermatogonial transplantation limits practical application of the technique. In this article, we describe a novel approach for producing transgenic animals by transducing spermatogonial stem cells in vivo using a retrovirus vector. Microinjection of retrovirus into immature seminiferous tubules resulted in the direct transduction of spermatogonial stem cells in situ, and the animals produced transgenic offspring after mating with females. Transgenic mice were produced in C57BL/6, BALB/C, A, and C3H backgrounds, with an average efficiency of 2.8%. The transgene was transmitted stably and expressed in the next generation. The technique overcomes the drawback of the in vitro-transduction approach, and will be useful as a novel method for producing transgenic animals as well as providing a means for analyzing the self-renewal and differentiation processes of spermatogonial stem cells in vivo.     

Mutagenicity studies on paracetamol in human volunteers. II. Unscheduled DNA synthesis and micronucleus test

The possible genotoxic effect of paracetamol (PC) was studied in a group of 11 healthy volunteers. PC was administered in the form of tablets 3 x 1000 mg in the course of 8 h. Blood samples and buccal mucosa cells were taken 0, 24, 72 and 168 h after the first administration of the drug. Each blood sample was used for the termination of the unscheduled DNA synthesis (UDS) in peripheral lymphocytes and ascorbemia in plasma. Buccal mucosa cells were analysed for micronuclei. After PC administration the level of UDS induced by MNNG was decreased to T/C = 4.11 +/- 0.56 after 24 h vs. T/C = 5.02 +/- 0.47 (p less than 0.01) at 0 h. The frequency of micronucleated cells in the buccal mucosa was increased after 72 h to 0.38 +/- 0.07% vs. 0.19 +/- 0.06% (p less than 0.01) before PC administration. If PC was administered simultaneously with ascorbic acid (AA), also in a dose of 3 X 1000 mg, a decreased level of UDS was observed after 24, 72 and 168 h and the increased number of micronuclei was qualitatively the same as the PC alone: 0.38 +/- 0.09% after 72 h vs. 0.20 +/- 0.05% at 0 h AA did not decrease the genotoxic effect of PC, but prolonged the influence of PC on UDS.     

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.     

Protective role of Codiaeum variegatum against genotoxicity induced by carmustine in somatic and germ cells of male mice

Molecular Biology Reports - Carmustine (Cr) is an important chemotherapeutic drug, widely used in the treatment of brain tumors. Herein, the protective role of Codiaeum variegatum leaves ethyl...