Mutagenicity studies with nitrofurans. I. Mutagenicity of nitrofurylacrylic acid for mammals

Cytogenetic analysis of mouse bone-marrow cells, the dominant lethal test in mice and the cytogenetic analysis of human peripheral lymphocytes in vitro were used to study the mutagenicity of 3-(5-nitro-2-furyl)acrylic acid (5-NFA) for mammals. The bone-marrow cytogenetic analysis was performed in female mice exposed to 5-NFA administered intraperitoneally in single doses of 15--120 mg/kg and in 5 repeated doses of 15 and 30 mg/kg, intragastrically in single doses of 30--240 mg/kg and 5 repeated doses of 30 and 60 mg/kg, and perorally for 12 weeks to 5-NFA concentration of 10, 100 and 1000 mg 5-FNA/1 in drinking water. The bone-marrow analysis was performed in this case after 12 days, 3, 4, 6, 8, 10 and 12 weeks exposure. No increase in chromosome damage attributable to dosing with 5-NFA occurred in any of these experiments. Experiments in which mice were exposed to 5-NFA in drinking water for 12 weeks and then treated with a single i.p. dose of 2 mg of the mutagen TEPA [trix-(1-aziridinyl)phosphine oxide] per kg revealed that, at a concentration of 1000 mg 5-NFA/1, the clastogenic activity of TEPA was reduced to that in untreated animals. The dominant lethal test was performed in male mice exposed to 5-NFA applied intraperitoneally in single doses of 40--120 mg/kg and in 5 repeated doses of 10--30 mg/kg, intragastrically in 5 repeated doses of 20--60 mg/kg, and perorally for 4 weeks in drinking water containing 5-NFA at concentrations of 10, 100, 316 and 1000 mg/l. No significant differences were detected between the exposed and control groups of animals. Experiments in which male mice were exposed to 5-NFA in drinking water and treated after the 4-week exposure to 5-NFA with 1 mg TEPA/kg revealed that a concentration of 1000 mg 5-NFA/1 reduced TEPA-induced dominant lethality to within control values. A reduction in male fertility was observed after the single or repeated 5-NFA doses, but no changes when 5-NFA was applied in drinking water. The cytogenetic analysis of human peripheral lymphocytes exposed in vitro for the last 24 h of culture to concentrations of 1--100 micrograms 5-NFA/Ml did not show any compound-related chromosomal changes. The results of dominant-lethal and bone-marrow cytogenetic studies in mice after consumption of drinking water containing 1000 mg of 5-NFA/1 for 12 weeks and dosed subsequently with TEPA suggests that 5-NFA has some antimutagenic activity. Because none of the studies reported revealed any compound-related genetic activity, the results suggest that 5-NFA is not a chromosome-breaking agent in mammals.     

The genotoxic and cytotoxic effects of nicotine in the mouse bone marrow

The objective of the present study was to investigate the potential of nicotine to induce micronucleated polychromatic erythrocytes (MNPCE) in bone marrow of male and female mice. Cyclophosphamide at 40mg/kg was used as positive control clastogen. Single doses of 4, 8 or 16mg/kg nicotine were given via oral intubation and bone marrow was sampled at 18, 24, 30, 36 and 48h after treatment. Cyclophosphamide yielded the expected positive results. Despite the evident signs of acute toxicity shown by the animals, mainly at the 8 and 16mg/kg doses of nicotine, and the reduction in the % PCE, the results show that the MNPCE frequency in male and female mice was not affected by treatment with any of the selected doses of nicotine, in either of the sampling times 18 or 24h. However, at 30 and 36h after treatment, the MNPCE showed significant increases in both genders after doses of 8 and 16mg/kg. A sex-dependent response was recorded, with males having more MNPCE than females after treatment with 8 or 16mg/kg nicotine and sampling at 30h. However, at 36h more MNPCE were induced in females than in males, suggesting different degrees of dose interaction in the sexes under the conditions of the assay. The response was directly correlated with bone-marrow toxicity, as greater bone-marrow suppression was noted in females than in males when 36h samples were examined. By 48h recovery was observed even though the cytotoxicity was high. These findings suggest that nicotine at high doses and after prolonged time intervals is genotoxic and cytotoxic for mouse bone marrow.     

Naloxone prolongs the survival time of mice treated with neuroblastoma

The effect of naloxone on tumor growth and survival time was studied in mice with neuroblastoma tumors. Daily s.c. injections of either 5, 10, 15, or 20 mg/kg naloxone were initiated either 2 weeks prior to tumor cell inoculations (pre-treated groups) or one week after tumor transplantation (post-treated groups). The S20Y cell line, cloned from A/Jax murine C1300 neuroblastoma, was utilized and each animal was inoculated with 10⁶ cells. All mice in the saline- tumor and naloxone post-treated groups, developed tumors within 3 weeks after tumor cell inoculation. In the naloxone pre-treated groups, 4 of 12 mice exposed to 20 mg/kg, 2 of 12 mice exposed to 15 mg/kg, and 1 of 12 mice exposed to 10 mg/kg, did not develop tumors within the 91-day post-inoculation period. Three animals in the 20 mg/kg naloxone pre-treated group developed tumors between 43 and 63 days after tumor cell inoculation. Tumor dimensions were often reduced in naloxone-treated animals but a dose-response relationship was not found in regard to the magnitude of alterations in tumor size. At the time of death, tumor sizes of control and naloxone-exposed mice were similar. In general, tumor-bearing mice receiving naloxone lived longer than saline-tumor controls, with animals receiving higher drug dosages surviving for the longest time. In contrast to a mean survival time of 27 days for controls, naloxone pre-treated groups had increases in survival times of 25–61%, whereas naloxone post- treated groups exhibited increases of 20–40%. The median day of death for all mice exposed to naloxone was prolonged by 21–75%, occuring 6–21 days after the 28-day median for saline-tumor controls. These results suggest that naloxone, a non-addictive compound, is an effective agent in modulating neoplasia.     

采用SCGE和SCE分析法研究姬松茸菌体多糖(Ab-Mp)对DNA损伤的抑制作用

热水浸提法提取姬松茸菌丝体多糖,每天以10mg/(kg bw)和20mg/(kg bw)两种剂量对小鼠进行灌胃,连续15d.腹腔注射环磷酰胺(CP),运用单细胞凝胶电泳(SCGE)法和姐妹染色体交换(SCE)分析法,研究Ab-Mp对染色体损伤的保护作用.结果表明,Ab-Mp降低了CP损伤后彗星细胞的比例,缩短了彗星尾长;降低了CP损伤的姐妹染色单体交换率.Ab-Mp对环磷酰胺诱发的DNA损伤具有拮抗作用.     

Cytogenetic effects of malathion insecticide on somatic and germ cells of mice

Male mice dermally exposed to single or multiple treatment (5 days/2 weeks) showed that the ability of malathion to induce chromosome aberrations in somatic (bone marrow) and germ cells (primary spermatocytes) was related to the type of treatment and dose used. Statistically significant increases of chromosome aberrations in bone marrow cells occurred after single treatment (500 and 2000 mg/kg body wt) when chromatid gaps were included and after multiple treatment (250 and 500 mg/kg) when they were excluded. No dose-response relationships were observed for either treatment. In germ cells, malathion induced a significant increase of univalents in both types of treatment but structural chromosome aberrations were induced only by multiple treatment. Malathion induced a significant decrease of the mitotic indices in the bone marrow.     

Relationship between experimental results in mammals and man: II. Cytogenetic analysis of bone-marrow cells after treatment of cytembena and cyclophosphamide-cytembena combination

Cytogenetic analysis and the micronucleus test of bone-marrow cells was used to study the possible extrapolation of results from experimental animals to man.Cytembena was given i.p. in doses of 5, 10, 20, 40 and 80 mg/kg body wt. to Wistar rats in doses of 20, 40 and 80 mg/kg body wt. to ICR mice an dto Chinese hamsters. Five patients with various types of malignancy, so far medically untreated, received 20 mg Cytembena/kg body wt i.v.A combination of Cytembena and cylophosphamide was applied i.p. in single equal doses 1 : 1 of 5, 10, 20, and 40 mg/kg body wt to ICR mice, Chinese hamsters and Wistar rats. Patients were given i.v. 20 mg Cytembena and 20 mg cyclophosphamide/kg body wt.Bone-marrow cells were examined 24 h after the administration.The frequency of abnormal metaphases and chromosomal breaks after Cytembena treatment was low; nonetheless, the indicated dose-effect relationship was found in all the rodents used. The frequency of chromosomal breaks was 2–3 times higher in rodents in comparison with man, after treatment with a dose of 20 mg Cytembena/kg body wt.Highest frequencies of induced aberrations were found in mice. The rodents appeared to be 3–4 times more sensitive to the induction of chromosomal breaks and abnormal metaphases than man, after a dose of 20 mg Cytembena and 20 mg cyclophosphamide/kg body wt.     

6-Mercaptopurine,an inducer of cytogenetic and dominant-lethal effects in premeiotic and early meiotic germ cells of male mice

Dominant-lethal effects of 6-mercaptopurine on male mice were studied using eight doses, ranging from 150 to 482 mg/kg. Effects of the 150-mg/kg dose were studied over the entire spermatogenic cycle, and those of the higher doses for matings made between days 28.5 and 41.5 after treatment. It was found that, with low doses, there was only one period in which clearcut increases in induced dominant-lethal mutations were detected, namely in matings that occurred 32.5 to 35.5 days after treatment. With higher doses, effects could be detected beyond that period through day 39.5. Spermatozoa utilized for matings during the period of greatest response were presumably derived from germ cells that were in late differentiating spermatogonial and early meiotic spermatocyte stages at the time of treatment. These results are similar to those of Ray and Hyneck. To date, 6-mercaptopurine is unique in inducing dominant lethality only at these particular stages.A study of chromatid aberration induction in the treated males themselves was carried out for 150 and 250 mg/kg doses of 6-mercaptopurine over the period of 9 to 16 days after treatment. A considerable increase in isochromatid and chromatid deletions was observed in diakinesis-metaphase-I spermatocytes on days 14 and 15 after treatment. For reasons discussed, the cells sampled at this time may be assumed to have been in early meiosis (preleptotene), with some in late differentiating spermatogonial stages, at the time of treatment. The rough agreement in sensitive cell type for dominant lethality and chromatid aberration induction suggests that chromatid deletions are the cause of dominant lethality in this study. Conservative estimates of the frequency of dominant lethality expected from the chromatid aberration frequencies tend to substantiate this suggestion.     

cis-Dichlorodiammine platinum(II) induced aberrations in mouse bone-marrow chromosomes

The clastogenic effect of cis-dichlorodiammine platinum(II) (cis-platin) on mouse bone-marrow chromosomes has been studied. Cis-platin was injected at 3 different doses. Cells were fixed at different time intervals after treatment. Different types of aberrations together with the percent of mitotic index and frequency of abnormal metaphases were studied. The aberrations observed were primarily chromatid breaks, although isochromatid breaks, interchanges, and multiple breaks were also observed. A dose- and time-dependent effect was observed for both inhibition of mitotic index and frequency of abnormal metaphases. Trypsin-Giemsa staining of bone-marrow metaphase chromosomes from normal mice was compared with the bands of metaphase chromosomes obtained after Giemsa staining of chromosomes from platinum-treated mice and they were observed to be identical. Bands were present up to 120 h and aberrations were also induced in such plates.     

Induction of chromosome aberrations in lymphocytes of mice after subchronic exposure to benzene

The induction of chromosome aberrations in lymphocytes of mice after subchronic exposure to benzene was investigated. 4 groups of 5 Swiss (ICR) male mice were given orally a solution of benzene every day for 14 days except days 5 and 10. The daily doses were 0, 36.6, 73.2 and 146.4 mg/kg. Mice were sacrificed on day 15, lymphocytes were obtained by perfusion of the spleen and the cells were cultured in RPMI 1640 medium. After 48 h of culture, cells were harvested for cytogenetic analysis. A significant dose-dependent increase in the frequency of cells with chromatid aberrations were found (p less than 0.001). A significant increase in polyploid cells were also observed (p less than or equal to 0.05). This study represents the first report on the induction of chromosome aberrations and polyploid cells in lymphocytes of mice after subchronic exposure to benzene. Such dual activity of benzene suggests that benzene may be responsible for more human health problems than currently estimated.     

Genotoxic effect of 2,4-dichlorophenoxy acetic acid and its metabolite 2,4-dichlorophenol in mouse.

The cytogenetic effect of 2,4-dichlorophenoxy acetic acid (2,4-D) and its metabolite 2,4-dichlorophenol (2,4-DCP) was studied in bone-marrow, germ cells and sperm head abnormalities in the treated mice. Swiss mice were treated orally by gavage with 2,4-D at 1.7, 3.3 and 33 mg kg(-1)BW (1/200, 1/100 and 1/10 of LD(50)). 2,4-DCP was intraperitoneally (i.p.) injected at 36, 72 and 180 mg kg(-1)BW (1/10, 1/5, 1/2 of LD(50)). A significant increase in the percentage of chromosome aberrations in bone-marrow and spermatocyte cells was observed after oral administration of 2,4-D at 3.3 mg kg(-1)BW for three and five consecutive days. This percentage increased and reached 10.8+/-0.87 (P<0.01) in bone-marrow and 9.8+/-0.45 (P<0.01) in spermatocyte cells after oral administration of 2,4-D at 33 mg kg(-1)BW for 24 h. This percentage was, however, lower than that induced in bone-marrow and spermatocyte cells by mitomycin C (positive control). 2,4-D induced a dose-dependent increase in the percentage of sperm head abnormalities. The genotoxic effect of 2,4-DCP is weaker than that of 2,4-D, as indicated by the lower percentage of the induced chromosome aberrations (in bone-marrow and spermatocyte cells) and sperm head abnormalities. Only the highest tested concentration of 2,4-DCP (180 mg kg(-1)BW, 1/2 LD(50)) induced a significant percentage of chromosome aberrations and sperm head abnormalities after i.p. injection. The obtained results indicate that 2,4-D is genotoxic in mice in vivo under the conditions tested. Hence, more care should be given to the application of 2,4-D on edible crops since repeated uses may underlie a health hazard.     

Clastogenic effects of hydroquinone: induction of chromosomal aberrations in mouse germ cells

The clastogenic activity of hydroquinone (HQ) in germ cells of male mice was evaluated by analysis of chromosomal aberrations in primary spermatocytes and differentiating spermatogonia. In the first experiment with treated spermatocytes the most sensitive stage of meiotic prophase to aberration induction by HQ was determined. Testicular material was sampled for microscopic analysis of cells in diakinesis-metaphase I at 1, 5, 9, 11, and 12 days after treatment with 80 mg/kg of HQ, corresponding to treated diplotene, pachytene, zygotene, leptotene and preleptotene. The frequencies of cells with structural chromosome aberrations peaked at 12 days after treatment (p less than 0.01). This indicates that the preleptotene when DNA synthesis occurred was the most sensitive stage of meiotic prophase. In the second experiment the dose response was determined 12 days post treatment by applying 2 additional doses of 40 mg/kg and 120 mg/kg. The clastogenic effects induced by 40 and 80 mg/kg were significantly different from the controls (p less than or equal to 0.01) and higher than the results obtained with 120 mg/kg of HQ. A humped dose-effect relationship was observed. In a third experiment the same doses were used to analyse chromosomal aberrations in dividing spermatogonia of mice 24 h after treatment with HQ. All the administered doses gave results statistically different from the control values (p less than or equal to 0.01) and the data were fitted to a linear equation. HQ was found to be clastogenic in male mouse germ cells. It is concluded that the clastogenic effect in male germ cells is of the same order of magnitude as in mouse bone marrow cells.     

Mutagenic evaluation of tromaril (N-beta-phenylethylanthranilic acid), a novel anti-inflammatory drug, using mammalian test systems

The mutagenic effect of tromaril, a new anti-inflammatory drug, was assessed in Swiss male mice employing bone-marrow micronucleus induction, abnormal sperm formation, and the induction of meiotic chromosome anomalies in spermatocytes as the test parameters. Administration of tromaril induced significantly higher percentages of MN and abnormal sperm at 600 and 900 mg/kg body wt., whereas chromosomal anomalies in spermatocytes were significantly higher at all the 3 doses of 300, 600 and 900 mg/kg body wt. at time intervals of 7, 15 and 30 days as compared to parallel controls.     

On the mutagenicity of methadone hydrochloride. Induced dominant lethal mutation and spermatocyte chromosomal aberrations in treated males

The mutagenicity of methadone hydrochloride was tested in male mice using the dominant lethal mutation technique and the spermatocyte test of treated mice. Male mice of C3H inbred strain received one of the following doses, 1, 2, 4 or 6 mg/kg body weight once a day for 3 consecutive days. Another group of mice served as control and received saline instead. Treated males were then mated to virgin females at 3-day intervals for a period of 45 days. Pregnant females were dissected at mid-term and the corpora lutea and intrauterine contents were recorded. The spermatocytes of treated males were examined 45-50 d after treatments with methadone and abnormal pairing configurations were scored. The methadone treatment was found to increase the rate of preimplantation deaths consistently in all post-meiotic stages with all doses used. In addition, the higher doses, 4 and 6 mg, affected spermatogonia stages. Quantitatively, the dose-response relationship cannot be demonstrated though the spectrum of effect increased with higher doses as more spermatogenesis stages became more sensitive to the treatment. In many cases the frequency of live implants showed a positive correlation with preimplantation deaths in contrast with the frequency of early deaths which showed only sporadic variation. The mutation indices based on total embryonic death indicate that methadone hydrochloride affected several stages of germ-cell maturation namely, spermatozoa (M.I. 14-35), late spermatids (M.I. 15-48), early spermatids (M.I. 14-50), late spermatocytes (M.I. 15-43) and spermatogonial stages (M.I. 12-63). Chromosome analysis at diakinesis-metaphase 1 revealed significant increase in the frequency of sex chromosome and autosome univalents with different doses of methadone. The smallest dose applied was quite effective and the data represent direct dose-response relationship. Of the multivalent configuration, the most frequent type was chain quadrivalents. The frequencies of total translocations per cell were estimated as 0.1, 0.16 and 0.2 for the 4 applied doses illustrating a dose-response relationship for the doses: 1, 2 and 4 mg, whereas with the higher dose, 6 mg, an abrupt decrease was apparent (0.05). This study calls for concern regarding the possible genetic hazards this drug may impose upon human populations.     

The biological activity of hydrogen peroxide. I. Induction of chromosome-type aberrations susceptible to inhibition by scavengers of hydroxyl radicals in human embryonic fibroblasts

The cytogenetic effect of hydrogen peroxide (H2O2) was investigated in human embryonic fibroblasts. Chromosome-type aberrations were found together with chromatid-type aberrations in metaphase cells harvested 24 h after a single 10-min treatment with 10(-5)-10(-3) M H2O2 in 0.9% NaCl solution. The chromosome-type aberrations were observed to be predominantly dicentrics and deletions. Both types of aberration showed a dose-response relationship to the dose of H2O2 over the range of 10(-5)-1.5 X 10(-4) M H2O2. The intercellular distribution of dicentrics showed a Poisson distribution. Centric and acentric rings and abnormal monocentrics were a minor fraction of the chromosome-type aberrations. The chromatid-type aberrations observed, such as breaks, exchanges and gaps, showed no dose-response relationship. The frequency of isochromatid breaks was higher than that of chromatid breaks and approximately 70% of the isochromatid breaks were found in the centromeric or pericentromeric region. The intercellular distribution of chromatid exchanges showed an over-dispersed distribution. The generation of aberrations by H2O2 was effectively suppressed by catalase and several scavengers of hydroxyl radicals (.OH) such as ethanol, dimethyl sulfoxide (DMSO) and mannitol. This result suggest that .OH plays an essential role in the generation of the chromosome aberrations by H2O2.     

Formation of micronucleated erythrocytes in mouse bone-marrow under conditions of hypothermia is not associated with stimulation of erythropoiesis

Conditions of marked and long-lasting hypothermia have been shown to increase the formation of micronucleated polychromatic erythrocyte (MNPCE) in mouse bone-marrow. Stimulation of erythropoiesis as a consequence of anoxic conditions associated with decreased body temperature has been suggested as a possible mechanism for hypothermia-induced micronucleus formation. We examined whether chemically induced hypothermic conditions that produced increased MNPCE formation were associated with stimulation of erythropoiesis by measuring erythropoietin (EPO) concentrations in blood. Marked and long-lasting hypothermia was induced in male mice by oral administration of the antipsychotic compounds E-5842 (200 mg/kg) or chlorpromazine (100 mg/kg). Maximum decreases from the basal temperature, achieved 8 h after treatment, were 14.8 and 12.8 degrees C, respectively. A statistically significant increase in bone-marrow MNPCE frequency was observed 48 h after administration of E-5842 (p<0.01) or chlorpromazine (p<0.05). Mice made anaemic by retro-orbital bleeding (0.5 ml), which acted as positive control for stimulation of erythropoiesis, showed no relevant variation in mean rectal temperature and a slight non-statistically significant increase in MNPCE frequency after 48 h. Blood samples for determination of EPO levels were obtained 4 (bleed-control animals only), 8, 16 and 24 h after treatment. In spite of the induced hypothermia, no significant variation in EPO blood levels was observed after administration of E-5842 or chlorpromazine. Bleed-induced anaemic mice showed a clear increase in EPO blood levels at all sampled time points, differences from baseline values being statistically significant (p<0.001) at the 8-h samplings and beyond. These results indicate that induction of MNPCE secondary to chemically induced hypothermia is not mediated by stimulation of erythropoiesis.     

Chlornaphazine and chlorambucil induce dominant lethal mutations in male mice

Two antineoplastic agents, chlornaphazine (CN) and chlorambucil (CHL), were tested for the induction of dominant lethal mutations in male mice. Both compounds are nitrogen mustard derivatives and have been shown to be genotoxic in a variety of organisms. CN was administered intraperitoneally to DBA/2J male mice at a dosage of 0, 500, 1000, or 1500 mg/kg body weight (bw). Immediately following treatment, each male was mated at 4-day intervals to two virgin C57BL/6J females. CHL was administered intraperitoneally to C3H/HeJ and DBA/2J males at a dosage of 0, 2.5, or 5.0 mg/kg bw. These males were mated at weekly intervals to two virgin T-stock females. CN and CHL clearly induced dominant lethal mutations. CN induced dominant lethal effects in all post-meiotic germ-cell stages of treated DBA males, with a clear dose-response relationship. The results with CHL-treated DBA males indicated that all post-meiotic germ-cell stages, except late-spermatids, were affected by CHL treatment, while in C3H males, CHL induced dominant lethal effects in all post-meiotic germ-cell stages. A dose-response relationship was also observed with CHL in C3H male mice. In the present experiments, regardless of the agent or the mouse strain used, spermatids appeared to be the germ-cell stage most sensitive to dominant lethal induction.     

Induction of specific-locus and dominant-lethal mutations by cyclophosphamide and combined cyclophosphamide-radiation treatment in male mice

Cyclophosphamide is the most widely used antineoplastic agent. It is also used to condition patients for bone-marrow transplantations. Because of the general interest of this compound we initiated a systematic study of the induction of dominant-lethal and specific-locus mutations in male mice. In addition, we investigated the induction of specific-locus mutations by the combined treatment of cyclophosphamide and ionizing radiation.A dose of 40 mg/kg bw of cyclophosphamide caused dominant-lethal mutations in male mice only in the 1st and 2nd week after treatment. A dose of 120 mg/kg induced dominant-lethal mutations in the mating intervals 1–21 days posttreatment. No dominant lethal mutations were observed after the 3rd week. The same differential spermatogenic response was observed for the induction of specific-locus mutations. Cyclophosphamide induced recessive mutations exclusively in spermatozoa and spermatids. No mutations were recovered from treated spermatocytes and spermatogonia. In contrast to cyclophosphamide, radiation induces specific-locus mutations in all germ-cell stages.The pretreatment with cyclophosphamide 24 h before radiation enhanced the frequency of specific-locus mutations in spermatogonia. The distribution of the observed mutations among the 7 loci and their viability supports the hypothesis that these mutations were induced by radiation rather than by cyclophosphamide. The compound causes an immediate inhibition of DNA and RNA synthesis in spermatogonia. The inhibition very likely interferes with the repair process. The disturbance of the repair process is probably the cause of the synergistic effect for the induction of specific-locus mutations in spermatogonia of mice after pretreatment with cyclophosphamide 24 h before irradiation.     

Quantitative assessment and characterization of visceral nociception and hyperalgesia in mice

Colorectal distension (CRD) is a well-characterized model of visceral nociception, which we adapted to the mouse. CRD reproducibly evoked contractions of the abdominal musculature [visceromotor response (VMR)], which was graded to stimulus intensity. The magnitude of VMR was greater in male C57BL6 and female 129S6 mice than in male 129S6 and B6.129 mice. In 129S6, C57BL6, and B6.129 mice strains, VMR was reduced dose dependently by morphine (1-10 mg/kg) and by the kappa-opioid agonist U-69593 (0.2-2 mg/kg), although U-69593 was significantly less potent in C57BL6 mice. In additional experiments, the VMR was recorded from adult male 129S6 mice before and after intracolonic administration of various irritants. Only 30% ethanol significantly enhanced responses to CRD. The colon hyperalgesia persisted for 14 days and was associated with a significant shift of the morphine dose-response function to the left. We believe this will be a useful model for study of visceral nociception and hyperalgesia, including studies of transgenic mice with mutations relevant to pain.     

The dominant lethal effect of dietary triethylenemelamine.

Triethylenemelamine (TEM) was administered in the diet to adult male mice at doses of 0.1, 0.3, 1, 10 or 50 mg/kg body weight for 45 days or at doses of 0.1 or 0.3 mg/kg b.w. for 10 days. As a comparison, male mice were treated intraperitoneally with 5 daily doses of 0.25 or 0.5 mg TEM/kg b.w. At the end of the treatment period, males were mated sequentially with 2 untreated virgin females each for 2 or 3 weeks. Near mid-pregnancy the number of implantation sites and fetal deaths were determined. TEM, administered in the diet at 10 or 50 mg/kg b.w. for 45 dyas, was lethal to male mice. Surviving males from the 1 mg/kg level failed to impregnate any females during the two matings. TEM, given in the diet at 0.1 or 0.3 mg/kg for 10 or 45 dyas, decreased fertility and increased dominant lethal mutations in a dose and time dependent manner. These results were comparable to those obtained from males treated i.p. with TEM at 0.25 or 0.5 mg/kg b.w.     

Effects of busulfan on murine spermatogenesis: cytotoxicity, sterility, sperm abnormalities, and dominant lethal mutations

The alkylating agent busulfan (Myleran) adversely affects spermatogenesis in mammals. We treated male mice with single doses of busulfan in order to quantitate its cytotoxic action on spermatogonial cells for comparison with effects of other chemotherapeutic agents, to determine its long-term effects on fertility, and to assess its possible mutagenic action. Both stem cell and differentiating spermatogonia were killed and, at doses above 13 mg/kg, stem cell killing was more complete than that of differentiating spermatogonia. Azoospermia at 56 days after treatment, which is a result of stem cell killing, was achieved at doses of over 30 mg/kg; this dose is below the LD50 for animal survival, which was over 40 mg/kg. Busulfan is the only antineoplastic agent studied thus far that produces such extensive damage to stem, as opposed to differentiating, spermatogonia. The duration of sterility following busulfan treatment depended on the level of stem cell killing and varied according to quantitative predictions based on stem cell killing by other cytotoxic agents. The return of fertility after a sterile period did not occur unless testicular sperm count reached 15% of control levels. Dominant lethal mutations, measured for assessment of possible genetic damage, were not increased, suggesting that stem cells surviving treatment did not propagate a significant number of chromosomal aberrations. Sperm head abnormalities remained significantly increased at 44 weeks after busulfan treatment, however, the genetic implications of this observation are not clear. Thus, we conclude that single doses of busulfan can permanently sterilize mice at nonlethal doses and cause long-term morphological damage to sperm produced by surviving stem spermatogonia.