Effect of 4-vinylcyclohexene on micronucleus formation in the bone marrow of rats and mice.

This study was conducted to evaluate the potential of 4-vinylcyclohexene (VCH) to induce micronuclei in the bone marrow of mice and rats. Male and female Crl:CD BR (Sprague-Dawley) rats and B6C3F1/CrBR mice were exposed to VCH 6 hr/day for 2 days or for 13 weeks. In the 2-day study, mice were exposed by inhalation to 0, 250, 500, or 1000 ppm, and rats were exposed to 0, 500, 1000, or 2000 ppm. In the 13-week study, mice were exposed to 0, 50, 250, or 1000 ppm, and rats were exposed to 0, 250, 1000, or 1500 ppm. In each study, a separate group of mice was exposed to 1000 ppm 1,3-butadiene (BD) so that a comparison could be made between the two compounds. Likewise, cyclophosphamide was also included for rats as a positive control. Bone marrow was collected from VCH-exposed animals approximately 24 h and 48 h after the final exposure. There were no statistically significant increases in micronucleatedpolychromatic erythrocytes (MN-PCEs) among VCH-treated mice and rats at any dose level or sampling interval at either 2-days or 13-weeks. Also, no statistically significant differences in the polychromatic erythrocytes (PCE) to normochromatic erythrocytes (NCE) ratios were observed in any of the VCH-treated mice and rats compared to air-exposed animals. As expected, both the butadiene-treated mice and the cyclophosphamide-treated rats showed significantly more MN-PCEs than the control animals.

Evaluation of thresholds for benomyl- and carbendazim-induced aneuploidy in cultured human lymphocytes using fluorescence in situ hybridization.

Threshold mechanisms of activity for mutagenic agents have been debated for some time, especially for those substances which induce aneuploidy by inhibiting mitotic spindle function. No observed effect levels (NOELs) or "practical thresholds" have been demonstrated for several aneugens both in vitro and in vivo generally by either counting chromosomes in metaphase preparations or by observing micronuclei. Recently, fluorescence in situ hybridization (FISH) has proven to be a sensitive and useful technique for the assessment of aneuploidy at low concentrations. Using binucleate human lymphocytes coupled with FISH, we have been able to characterize a threshold mechanism of action for two spindle inhibitors, benomyl and its active metabolite, carbendazim. Test chemicals were added 24 h following culture initiation. After a further 20 h, cytochalasin B was added, and cells were harvested 28 h later (72 h post initiation). The distribution of chromosomes between the nuclei of binucleate cells was evaluated by fluorescence microscopy for the simultaneous detection of centromeres labeled with FITC (green) or Cy-3 (red). Six human chromosomes were investigated in pairs (1 and 8, 11 and 18, and X and 17). Abnormalities were classified as chromosome loss (including centromeric positive micronuclei), chromosome gain, non-disjunction, or polyploidy. Dose-response data were generated over a range of closely spaced concentrations at 100 ng/ml intervals. The threshold, defined as the lowest "effect" concentration using statistical methods, was determined for each chromosome. Non-disjunction proved to be the most sensitive endpoint for the detection of aneuploidy occurring at higher frequencies and lower concentrations. Results for the six chromosomes demonstrated similar dose-response data which included a series of concentrations with no statistically significant increase above background, followed by a second range of higher concentrations with a statistically significant, concentration-dependent increase. Nearly equimolar threshold concentrations were determined for benomyl- and carbendazim-induced non-disjunction.

Chronic toxicity, oncogenicity, and mutagenicity studies with chlorotetrafluoroethane (HCFC-124).

The chronic toxicity, oncogenicity, and mutagenicity of chlorotetrafluoroethane (HCFC-124) were evaluated. In the chronic toxicity/oncogenicity study, male and female rats were exposed to 0, 2000, 10,000, or 50,000 ppm HCFC-124 for 6 hr/day, 5 days/week, for 2 years. Body weights were obtained weekly during the first three months of the study and every other week for the remainder of the study. Food consumption was determined weekly. Clinical signs of toxicity were monitored throughout the study. An ophthalmological examination was performed on all animals prior to study start, and all surviving rats were examined at approximately 3, 12, and 24 months after study start. Clinical pathology was evaluated at 3, 6, 12, 18, and 24 months. An interim termination was conducted at 12 months. All surviving rats were necropsied at 24 months. A complete set of tissues was collected for microscopic examination, and selected tissues were weighed. There were no compound-related, adverse effects on body weight, food consumption, survival, clinical signs of toxicity, ophthalmoscopically observable ocular lesions, serum hormone concentrations, or clinical pathology parameters at any exposure concentration in either male or female rats. Compared to controls, urine fluoride was increased in males and females at all exposure concentrations, and plasma fluoride was increased in females at all exposure concentrations. Excretion of fluoride represents conversion of the parent molecule, and as such is not considered to be an adverse effect. There were no toxicologically significant, compound-related organ weight changes or gross or microscopic findings in male or female rats at any of the exposure concentrations tested. HCFC-124 was not toxic or carcinogenic in rats of either sex after inhalation exposure at concentrations of up to 50,000 ppm in this two-year chronic toxicity/oncogenicity study. After exposure to HCFC-124 for six hours per day, five days per week, for 24 months, the no-observed-adverse-effect level for male and female rats was 50,000 ppm. HCFC-124 was not mutagenic in Salmonella typhimurium strains TA1535, TA97, TA98, and TA100 with and without activation when evaluated at concentrations up to 60% HCFC-124 for 48 hours. No evidence of clastogenic activity was observed in cultured human lymphocytes at atmospheric concentrations up to 100% HCFC-124 for 3 hours, with and without metabolic activation. In vivo, no micronuclei were induced in mouse bone marrow cells following exposure of mice to concentrations of 99,000 ppm HCFC-124 6 hours/day for 2 days.

Fluorescence in situ hybridisation with chromosome-specific centromeric probes: a sensitive method to detect aneuploidy.

Cytochalasin B-blocked binucleate human lymphocytes from female donors have been used to measure micronucleus induction and other aneuploidy events after treatment with colchicine, vinblastine or carbendazim. For the aneuploidy events, centromeric probes for 6 selected chromosomes (1, 8, X, 11, 17, 18) were used to measure chromosome loss, addition and non-disjunction in the interphase nuclei of these binucleate cells. The chromosomes were probed in pairs using Cy-3 (red) and FITC (green) labels for the 2 different centromeric regions. For colchicine, the total non-disjunction frequencies for chromosomes 1 and 8 were similar to the total micronucleus frequencies, but were detected as significant at lower concentrations. For vinblastine (chromosomes 1 and 8) and carbendazim (all 6 chromosomes) the frequencies of non-disjunction far exceeded (7 and > 2-fold, respectively) the peak frequencies of micronucleus induction. Although most chromosomes exhibited similar sensitivity in all the aneuploidy events measured, there was an indication that chromosome X was more than susceptible to non-disjunction than the other chromosomes. We believe that measurement of non-disjunction in binucleate human lymphocytes using chromosome specific centromeric probes offers a sensitive method for detection of aneuploidy and is particularly appropriate for the establishment of thresholds.

Maternal factors related to parenting practices, developmental expectations, and perceptions of child behavior problems.

Parenting practices of a representative sample of 1,056 urban mothers with very young children were studied via the Parent Behavior Checklist (Fox, 1994) and the Behavior Screening Questionnaire (Richman & Graham, 1971). Potential determinants of parenting practices were also addressed, including maternal age, marital status, education level, number of children living at home, and family socioeconomic status. Less positive parenting practices concerning nuturing and discipline were found among mothers who were younger, had more than one child living at home, were single, had a lower income level, and had lower educational attainment. These mothers also tended to perceive their children as demonstrating more difficult behavior problems. However, the negative influence of some determinants of parenting practices, such as low income, was found to be moderated by the presence of other determinants, such as more education. The present results provide evidence that multiple determinants influence parenting practices among parents of young children.

Evaluation of benomyl and carbendazim in the in vivo aneuploidy/micronucleus assay in BDF1 mouse bone marrow.

Benomyl and its active metabolite carbendazim were investigated in BDF1 mouse bone marrow to establish whether micronuclei induced by these fungicides are caused by clastogenic or aneugenic events. Micronuclei were evaluated for kinetochores using immunofluorescent antikinetochore antibodies. Kinetochore positive (K+) micronuclei are likely to arise from chromosome loss since they presumably contain intact kinetochores and are indicative of aneuploidy. Conversely, kinetochore negative (K-) micronuclei are mostly likely to contain acentric chromosome fragments arising primarily from clastogenic damage. Benomyl and carbendazim were administered as single oral doses of 0.3, 8.6 or 17.2 mmol/kg (for benomyl, equivalent to 100, 2500 or 5000 mg/kg; for carbendazim, equivalent to 66, 1646 or 3293 mg/kg). Both compounds were positive in the micronucleus test at doses of 8.6 and 17.2 mmol/kg, and an average of 82% (benomyl) and 87% (carbendazim) of the total micronucleated polychromatic erythrocytes were K+. No effects were seen with either fungicide at 0.3 mmol/kg. These results are analogous to findings with known aneugens such as vincristine but are in contrast to results with classical clastogens such as cyclophosphamide. Thus, benomyl and carbendazim induce micronuclei in mouse bone marrow cells primarily through an aneugenic mechanism.

Assessing the risk of heritable gene mutation in mammals: drosophila sex-linked recessive lethal test and tests measuring DNA damage and repair in mammalian germ cells.

The former U.S. EPA OPPT tiered test scheme for heritable gene mutations included the Drosophila sex-linked recessive lethal (SLRL) test in which positive results triggered the mouse specific locus (MSL) test. However, review of available literature indicated that the evaluation of mutations in the germ cells of this insect is not a good predictor of the risk of heritable gene mutations in mammals. The database contained 29 compounds for which there were conclusive MSL test results in either spermatogonial and/or postspermatogonial cells. Results in the SLRL test were available for 27 of those compounds. Of the 24 SLRL-positive chemicals, only 13 (54%) induced heritable mutations in mice; the three SLRL-negative compounds were nonmutagenic in mouse germ cells. The overall concordance between the two tests was 59%. In contrast, results of unscheduled DNA synthesis (UDS: 18 chemicals) and alkaline elution (AE: 14 chemicals) assays in rodent testicular cells following in vivo exposure correlated well with results in the MSL test (83% and 86%, respectively). MSL test results in spermatogonia and postspermatogonia were also compared separately to the SLRL, UDS, and AE assays. The concordances for the two cell types in the SLRL relative to the MSL test were 36% and 79%, respectively, indicating that the SLRL test is extremely poor in predicting heritable gene mutations in mammalian spermatogonia. Concordances for UDS and AE assays relative to MSL test results in spermatogonia (53% and 54%, respectively) and postspermatogonia (91% and 100%, respectively) were greater. Based on these analyses, the U.S. EPA OPPT has revised its tiered test scheme using assays for interaction with gonadal DNA (e.g., UDS and AE) in place of the SLRL test.

Tissue-specific genotoxic effects of acrylamide and acrylonitrile.

Acrylamide (AA) has been reported to induce dominant lethal mutations in male rat germ cells and tumors in a variety of organs, including the scrotum, thyroid and mammary glands, but not the liver of rats. The structurally similar vinyl monomer acrylonitrile (ACN) does not induce dominant lethal mutations but does induce tumors of the brain, Zymbal gland, forestomach and mammary gland, but not the liver of rats. Several in vitro and/or in vivo unscheduled DNA synthesis (UDS) assays were employed to examine the potential tissue-specific genotoxic activity of these compounds. Neither AA nor ACN induced DNA repair in either the in vitro or in vivo hepatocyte DNA repair assays. Glycidamide (GA), a mutagenic metabolite of AA, induced DNA repair in the in vitro hepatocyte DNA repair assay. Cyanoethylene oxide (CEO), a mutagenic metabolite of ACN, did not yield a DNA repair response in the in vitro hepatocyte DNA repair assay, but was highly toxic and could not be tested at doses equivalent to GA. AA, but not ACN, produced a DNA repair response in the in vivo spermatocyte DNA repair assay. AA produced a slight response in the in vitro human mammary epithelial cell (HMEC) DNA repair assay in normal cells derived from discarded surgical samples from five different women. GA produced a strong UDS response in all cases in the same assay. CEO, but not its parent compound ACN, produced a response in the HMEC DNA repair assay. These results show a highly tissue-specific pattern of genotoxic activity for AA and ACN that correlates, to the extent that it has been examined, with the tissue-specific pattern of carcinogenic and dominant lethal activity. The induction of DNA repair by GA and CEO confirms the genotoxic potential of these metabolites. While the observation of genotoxic activity of AA in the HMEC DNA repair assay suggests that mammary cells might be a target for carcinogenic activity of this compound in humans, other factors such as pharmacokinetics and epidemiology must be evaluated to establish that effect.

Mothers and fathers of young children: comparison of parenting styles.

This study compared the developmental expectations and parenting behaviors of 52 mothers and fathers with children between the ages of 1 and 4 years, using the Parenting Inventory: Young Children. While both mothers and fathers were nurturing parents, mothers obtained significantly higher nurturing scores. Possible reasons for this difference were discussed. Developmental expectations and discipline strategies did not differ between mothers and fathers.

Differences in the quality of semen in outdoor workers during summer and winter.

BACKGROUND AND METHODS: In warm climates throughout the world, there is a deficit of births during the spring season. To determine whether this deficit might reflect a deleterious effect of heat on the male reproductive capacity during the previous summer, we obtained semen specimens in summer and winter from normal men who worked outdoors in the vicinity of San Antonio, Texas, and we performed automated semen analyses with an image-analysis system. RESULTS: Pairwise comparisons among 131 men without azoospermia who contributed specimens in both summer and winter revealed significant reductions during the summer in sperm concentration, total sperm count per ejaculate, and concentration of motile sperm. The mean decreases in these values after adjustment for potential confounding characteristics were 32 percent (95 percent confidence limits, 28 and 44 percent), 24 percent (95 percent confidence limits, 18 and 43 percent), and 28 percent (95 percent confidence limits, 24 and 44 percent), respectively (P less than 0.0001). The lower a subject's average sperm concentration and motile-sperm concentration, the greater the reduction. We found no correlation, however, between the number of hours per day spent working during summer in settings without air conditioning and either the summer sperm concentration or the difference in concentration between summer and winter. Among the children of the men in the study whose wives were living near San Antonio during the year before they gave birth, a disproportionately low number were born during the spring. CONCLUSIONS: Semen quality deteriorates during the summer. This phenomenon may account at least in part for the reduction in the birth rate during the spring in regions with warm climates.

Computer-assisted semen analysis: results vary across technicians who prepare videotapes.

Automated semen analyses revealed differences of 21% to 30% in concentration-related parameters and 5% to 11% in motion-related parameters between means of groups of replicate specimens. Disparities among videotapes produced by two laboratory technicians accounted for the divergence in concentration-related parameters. This resulted partially from differences between the two technicians in propensity to dilute concentrated specimens. The causes of the greater portion of disparities between videotaping technicians, however, have not been identified. Differences in motion-related parameters could not be ascribed to technicians, but the basis for these differences is also unknown. The results suggest that values obtained from the CellSoft image analysis system may not be comparable between technicians or laboratories, despite use of identical computer parameter settings. Until effective quality control measures have been implemented, such comparisons must be made with caution.

Spontaneous formation of foci of morphologically transformed cells in populations of C3H 10T1/2 (clone 8) cells.

Spontaneous formation of morphologically altered foci of types II and III (neoplastic transformation) was examined in populations of C3H 10T1/2 (10T1/2) cells. Initial surviving cell densities ranged from 3 to 3 x 10(5) cells/100-mm dish and the final cell density was approximately 2 x 10(6) cells/dish, yielding widely differing numbers of population doublings but similar numbers of cell births from the time of cell plating to the attainment of confluence. Spontaneous formation of foci was independent of the initial surviving cell densities (and, therefore, of the number of population doublings) but was related to the number of cell divisions (cell births) between the time the cell population was plated and when suppression of proliferation of wild-type cells occurred in confluent cultures. In 418 pooled asynchronously proliferating cultures in 100-mm dishes the 95% confidence limits for the fraction of dishes containing foci was 0.041-0.089 for type II foci and 0.008-0.036 for type III foci; for cell populations in 2041 pooled cultures in 100-mm dishes, the proliferation of which was synchronized by release from confluence-induced arrest of proliferation, the 95% confidence limits for the fraction of dishes containing foci were 0.150-0.166 for type II foci and 0.017-0.032 for type III foci. Using the Poisson method, the 95% confidence limits for rates of spontaneous transformation in asynchronously proliferating populations of 10T1/2 cells were 1.4-3.2 x 10(-8)/cell/division for type II foci and 0.28 to 1.3 x 10(-8)/cell/division for type III foci; in populations in which proliferation was initially synchronized by release from confluence-induced arrest, spontaneous transformation rates were 5.6-6.3 x 10(-8)/cell/division for type II foci and 0.59-1.1 x 10(-8)/cell/division for type III foci. Spontaneous transformation occurred in populations of wild-type 10T1/2 cells at the rates and with the characteristics expected of the mutation of a single gene locus.

Induction of foci of morphologically transformed cells in synchronized populations of 10T1/2 cells by N-methyl-N'-nitro-N-nitrosoguanidine and the effect of spontaneous transformation on calculated transformation frequency.

Exposure of synchronized C3H10T1/2 (clone 8) cell populations of various sizes to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) at a concentration of 2 micrograms/ml for 30 min at 24 h after release from confluence-induced arrest of proliferation produced neoplastic transformation (formation of foci of morphologically altered cells) by a random but episodic process in a small fraction of the cells at risk soon after treatment. The fraction of dishes that contained type II or type III foci increased as the number of cells at risk increased. In contrast, the development of spontaneous foci is a stochastic process that depends on the number of new cells that form during population growth and is independent of the number of cells that are plated (J. W. Grisham et al., Cancer Res., 48: 5969-5976,1988). When there were small numbers of cells at risk, spontaneous formation of foci was a source of considerable error in evaluating MNNG-induced transformation frequency. In surviving cell populations of less than 1000-3000 cells/100-mm dish, the frequency of induction of foci by MNNG could not be distinguished statistically from the frequency with which foci were expected to form spontaneously. When the fraction of MNNG-treated dishes that contained foci was adjusted for the fraction of pooled control dishes that contained foci, the number of foci induced by a uniform dose of MNNG was found to vary with the number of surviving cells. However, the MNNG-induced transformation frequencies calculated by the Poisson method were independent of the size of the population of cells at risk, provided the population of cells at risk was of sufficient size to allow spontaneous and induced transformation to be distinguished statistically. The results of this study show that the frequency of MNNG-induced transformation can be quantitated in cultures of 10T1/2 cells that contain varying but sufficient numbers of cells at risk when spontaneous transformation is considered. Furthermore, these observations suggest that MNNG-induced transformation of 10T1/2 cells occurs with the frequency and characteristics of a mutation-like change involving a single gene.

Activity of germ-cell mutagens and nonmutagens in the rat spermatocyte UDS assay.

The ability of 13 chemicals of known germ-cell mutagenicity to induce unscheduled DNA synthesis (UDS) in rat spermatocytes was examined. At selected times following i.p. injection of test compounds, spermatocytes were isolated from Fischer 344 rats by enzymatic digestion of the seminiferous tubules and cultured for 24 h in the presence of [3H]thymidine. 7 compounds, methyl methanesulfonate, triethylenemelamine, cyclophosphamide, methylnitrosourea, ethylnitrosourea, procarbazine, and dibromochloropropane produced positive UDS responses in spermatocytes. These chemicals are also positive for specific locus mutations, heritable translocations, or dominant lethal mutations when administered to male rodents. Mitomycin C, which produces DNA interstrand crosslinks and induces heritable mutations and translocations in male germ cells, failed to stimulate UDS in rat spermatocytes. Germ-cell nonmutagens N-methyl-N'-nitro-N-nitrosoguanidine, dimethylnitrosamine, 4-nitroquinoline 1-oxide, and ethylene dibromide were negative in the rat spermatocyte UDS assay. Correlation of these results with those of other assays for heritable mutations in germ cells indicates that the in vivo/in vitro spermatocyte DNA repair assay is useful in predicting the mutagenic potential of chemicals in male germ cells.

Use of seminiferous tubule segments to study stage specificity of unscheduled DNA synthesis in rat spermatogenic cells.

DNA repair in spermatogenic cells at various stages of maturity was determined by quantitation of unscheduled DNA synthesis (UDS). Male F-344 rats were exposed (i.p.) to methyl methanesulfonate (MMS, 35 mg/kg); 1 hr later, segments of seminiferous tubules corresponding to spermatogenesis stages II, IV-V, VI, VII, VIII, IX-X, XII, and XIV were isolated with the transillumination pattern of the tubules as a guide. Intact tubule segments were cultured 24 hr in the presence of [3H]thymidine, and UDS was quantitated by autoradiography as net grains/nucleus (NG). In primary spermatocytes from treated rats, NG count increased with increasing maturity from leptotene primary spermatocytes (3.5 NG) up through stage VIII and IX-X pachytene spermatocytes (22 NG), after which NG decreased in stage-XII pachytene and diplotene spermatocytes (to 16 NG and 8 NG, respectively). Round spermatids of steps 2-8 of spermiogenesis all exhibited approximately the same UDS response (8 NG). Elongating spermatids as mature as step 14 underwent UDS after exposure to MMS, but step-15 and later-step spermatids did not. The DNA repair response of pachytene spermatocytes cultured within segments of seminiferous tubule corresponding to stages VIII and IX-X was 4 to 25 times greater, depending on the dose of MMS, than pachytene spermatocytes isolated by enzymatic digestion and cultured in suspension [Bentley and Working, Mutat Res 203:135-142, 1988]. Thus, the use of segments of seminiferous tubule both increased the sensitivity of UDS as an indicator of DNA damage in rat germ cells and enabled the study of UDS in spermatogenic cells at different stages of maturity.

Mutagenic potency at the Na+/K+ ATPase locus correlates with cycle-dependent killing of 10T1/2 cells.

Perturbation of DNA replication by chemical-DNA adducts produced by exposure to mutagenic/carcinogenic chemicals results in mutagenic or cytotoxic damage in the DNA. Demonstration of a correlation between cell cycle dependency of cytotoxicity and point mutation at the Na+/K+ ATPase gene could suggest that the two consequences of chemical exposure are caused by the same damage in the template DNA and that both are mediated through DNA replication-associated mechanisms. N-methyl-N'-nitro-N-nitrosoguanidine, N-ethyl-N'-nitro-N-nitrosoguanidine, 4-nitroquinoline-1-oxide, and benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide demonstrated cell cycle-related patterns of cytotoxicity in 10T1/2 cells, with maximal cell killing produced by exposure in early S phase, and were highly efficient mutagens of the Na+/K+ ATPase gene relative to their cytotoxic potential. In contrast, methyl methanesulfonate and N-acetoxy-N-2-fluorenylacetamide were maximally cytotoxic in cell populations exposed in early G1 phase and were weak mutagens of the Na+/K+ ATPase gene at comparable levels of cytotoxicity. These data suggest that mutagenic/carcinogenic chemicals that are effective at producing mutations by misreplication kill cells by a related mechanism that may be associated with the perturbation of DNA replication.

Comparison of the dominant lethal effects of acrylonitrile and acrylamide in male Fischer 344 rats.

Acrylonitrile (ACN) and acrylamide (AA), structurally similar vinyl monomers, are both animal carcinogens. ACN is weakly mutagenic in bacteria and induces sister-chromatid exchange, unscheduled DNA synthesis and cell transformation in cells in culture. AA induces chromosomal aberrations in bone marrow, blood and germ cells in vivo, and dominant lethal mutations in the germ cells of male mice and rats. In the current study, the ability of AA and ACN to induce dominant lethal mutations in the germ cells of male Fischer 344 rats was compared. Three groups of 50 males were gavaged daily for 5 days with ACN (60 mg/kg in normal saline), AA (30 mg/kg in normal saline) or vehicle only; an additional group of 20 males received a single i.p. injection of 0.2 mg/kg triethylenemelamine (TEM) on the afternoon of day 5. Starting 1 day after exposure, each male was bred to one female per week for 4 weeks (TEM-exposed group) or 10 weeks (ACN, AA and control groups). Mating rates were reduced only during week 1 in the TEM-treated group; pregnancy rates were reduced only during week 2 in the AA-exposed group and week 4 in the TEM-treated group. Females were necropsied 13 days after the end of the appropriate mating week and the amount of pre- and post-implantation loss calculated. ACN treatment of male rats induced no increases in either pre- or post-implantation loss in females in any of the 10 weeks post-exposure examined.(ABSTRACT TRUNCATED AT 250 WORDS)