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Acrylamide (AA), a mutagen and rodent carcinogen, recently has been detected in fried and baked starchy foods, a finding that has prompted renewed interest in its potential for toxicity in humans. In the present study, we exposed Big Blue rats to the equivalent of approximately 5 and 10 mg/kg body weight/day of AA or its epoxide metabolite glycidamide (GA) via the drinking water, an AA treatment regimen comparable to those used to produce cancer in rats. After 2 months of dosing, the rats were euthanized and blood was taken for the micronucleus assay; spleens for the lymphocyte Hprt mutant assay; and liver, thyroid, bone marrow, testis (from males), and mammary gland (females) for the cII mutant assay. Neither AA nor GA increased the frequency of micronucleated reticulocytes. In contrast, both compounds produced small (approximately twofold to threefold above background) but significant increases in lymphocyte Hprt mutant frequency (MF, p < 0.05), with the increases having dose-related linear trends (p < 0.05 to p < 0.001). Neither compound increased the cII MF in testis, mammary gland (tumor target tissues), or liver (nontarget tissue), while both compounds induced weak positive increases in bone marrow (nontarget tissue) and thyroid (target tissue). Although the genotoxicity in tumor target tissue was weak, in combination with the responses in surrogate tissues, the results are consistent with AA being a gene mutagen in the rat via metabolism to GA.

Acrylamide, a food contaminant, is carcinogenic in experimental animals, with both genotoxic and nongenotoxic pathways being proposed. To obtain information regarding mechanisms of acrylamide tumorigenesis, we compared the extent of DNA adduct formation and induction of micronuclei and mutations in mice treated neonatally with acrylamide and its electrophilic metabolite glycidamide. Male and female B6C3F1/Tk mice were treated intraperitoneally on postnatal days (PNDs) 1, 8 and 15 or PNDs 1-8 with 0.14 or 0.70 mmol acrylamide or glycidamide per kg body weight per day. One day after the final dose, B6C3F1/Tk(+/+) mice were killed to measure DNA adduct levels and peripheral blood micronuclei. Three weeks after the last treatment, B6C3F1/Tk(+/-) mice were killed to assess the Hprt and Tk mutant frequencies in spleen lymphocytes. The levels of N7-(2-carbamoyl-2-hydroxyethyl)guanine, the major glycidamide-DNA adduct, decreased in the order 0.70 mmol glycidamide > 0.70 mmol acrylamide > 0.14 mmol glycidamide approximately 0.14 mmol acrylamide. Only glycidamide increased the frequency of micronucleated reticulocytes and normochromatic erythrocytes. In mice treated on PNDs 1, 8 and 15, the Hprt mutant frequency was increased by 0.70 mmol glycidamide. In mice dosed on PNDs 1-8, 0.70 mmol glycidamide caused extensive mortality; each of the other treatments increased the Tk mutant frequency, whereas acrylamide increased the Hprt mutant frequency. These data suggest that the mutagenic response in neonatal mice treated on PNDs 1, 8 and 15 is due to glycidamide, whereas mutations resulting from dosing on PNDs 1-8 are due to another mechanism., ((c) 2008 Wiley-Liss, Inc.)

Zidovudine (3'-azido-3'-deoxythymidine; AZT) and lamivudine [(-)2',3'-dideoxy-3'-thiacytidine; 3TC] are nucleoside reverse transcriptase inhibitors used to treat and prevent human immunodeficiency virus-1 infections. In short-term incubations (<48 h), AZT, but not 3TC, has been shown to interfere with cell cycle progression. In the present study, we examined if these alterations persist during long-term incubations in which cells were exposed to AZT (0-1000 microM) or 3TC (0-500 microM) in continuous culture for up to 5 weeks. In addition, we investigated the reversibility of these effects upon removal of the drugs. Both drugs caused concentration- and time-dependent decreases in the number of viable cells, with the effect being more pronounced with AZT. There was only a slight increase in the number of viable cells treated with AZT for 5 weeks and then allowed a 1-week recovery period; cell viability in cells treated with 3TC returned to control levels during the recovery period. The decrease in viable cells was not due to apoptotic or necrotic cell death, but rather was associated with S and G2/M phase cell cycle arrest. Western blot analysis indicated that AZT treatment caused a decrease in checkpoint kinase 1 (Chk1) and checkpoint kinase 2 (Chk2) at all time points. Cyclin-dependent kinase 1 was decreased at later time points, while cyclin A was increased at early times. These data indicate that AZT and, to a lesser extent, 3TC interfere with cell growth by slowing cell cycle progression and that checkpoint proteins Chk1 and Chk2 may play an important role in this delay.

In previous studies, we have shown that zidovudine (3'-azido-3'-deoxythymidine; AZT), but not lamivudine [(-)2',3'-dideoxy-3'-thiacytidine; 3TC], is genotoxic when administered to neonatal mice, and that 3TC when coadministered with AZT does not alter the responses observed with AZT alone (Von Tungeln et al. [2002] Carcinogenesis 23:1427-1432). We now have investigated the transplacental transfer of these drugs and the induction of mutants and micronuclei in the neonatal offspring. From gestational day 12 until parturition, female C57BL/6N and C57BL/6N/Tk(+/-) mice, which had been mated to male C3H/HeNMTV mice, were treated daily by gavage with AZT, 3TC, or a combination of AZT and 3TC. In both dams and fetuses, AZT was found at much higher levels than its metabolites, AZT 5'-glucuronide and 3'-azido-3'-deoxythymidine. In the neonates, AZT and the mixture of AZT and 3TC caused a decrease in the percentage of reticulocytes (RETs) and an increase in the percentage of micronucleated RETs and micronucleated normochromatic erythrocytes. When assessed 3 weeks after birth, AZT and the combination of AZT and 3TC increased the thymidine kinase (Tk) mutant frequency in male mice; at 5 weeks, 3TC increased the Tk mutant frequency in female mice. The increase in Tk mutants in mice treated with AZT and the mixture of AZT and 3TC was associated with loss of the wild-type (Tk(+)) allele (loss of heterozygosity; LOH) and a pattern of discontinuous LOH. These data indicate that AZT, 3TC, and the combination of AZT and 3TC are transplacental mutagens and that the increase in mutants resulting from AZT is due mainly to large-scale genetic alterations., ((c) 2006 Wiley-Liss, Inc.)

The first step in the activation of the anti-retroviral nucleoside analogue azidothymidine (AZT) involves its conversion to a 5'-monophosphate. In this study, we have evaluated the role of cytosolic thymidine kinase (Tk), the major enzyme involved in phosphorylating thymidine and its analogues, in the nuclear DNA damage produced by AZT in neonatal mice. Tk+/+, Tk+/- and Tk-/- mice were treated intraperitoneally with 200 mg/kg/day of AZT on postnatal days 1 through 8, and micronuclei were measured in peripheral blood 24 h after the last dose. AZT treatment increased the micronucleus (MN) frequencies to similar extents in both the reticulocytes (RETs) and normochromatic erythrocytes (NCEs) of Tk+/+ and Tk+/- mice; AZT did not increase the frequency of micronucleated RETs (MN-RETs) or micronucleated NCEs (MN-NCEs) in Tk-/- mice. Unexpectedly, neonatal Tk-/- mice treated with the vehicle had significantly elevated MN frequencies for both RETs and NCEs relative to Tk+/+ and Tk+/- mice (e.g., approximately 3.4% MN-RETs and approximately 4.8% MN-NCEs in Tk-/- mice versus approximately 0.7 and approximately 0.6% MN-RETs and MN-NCEs in neonatal Tk+/+ mice). Additional assays performed on untreated Tk-/- mice showed that elevated spontaneous MN frequencies persisted until at least 20 weeks of age, which approaches the average lifespan of Tk-/- mice. These results indicate that metabolism by Tk is necessary for the genotoxicity of AZT in neonatal mice; however, the genotoxicity of AZT is not altered by reducing the Tk gene dose by half. The elevated spontaneous MN frequencies in Tk-/- mice suggest the presence of an endogenous genotoxic activity in these mice.

Identifying genes that are differentially expressed in response to DNA damage may help elucidate markers for genetic damage and provide insight into the cellular responses to specific genotoxic agents. We utilized cDNA microarrays to develop gene expression profiles for ionizing radiation-exposed human lymphoblastoid TK6 cells. In order to relate changes in the expression profiles to biological responses, the effects of ionizing radiation on cell viability, cloning efficiency, and micronucleus formation were measured. TK6 cells were exposed to 0.5, 1, 5, 10, and 20 Gy ionizing radiation and cultured for 4 or 24 hr. A significant (P < 0.0001) decrease in cloning efficiency was observed at all doses at 4 and 24 hr after exposure. Flow cytometry revealed significant decreases in cell viability at 24 hr in cells exposed to 5 (P < 0.001), 10 (P < 0.0001), and 20 Gy (P < 0.0001). An increase in micronucleus frequency occurred at both 4 and 24 hr at 0.5 and 1 Gy; however, insufficient binucleated cells were present for analysis at the higher doses. Gene expression profiles were developed from mRNA isolated from cells exposed to 5, 10, and 20 Gy using a 350 gene human cDNA array platform. Overall, more genes were differentially expressed at 24-hr than at the 4-hr time point. The genes upregulated (> 1.5-fold) or downregulated (< 0.67-fold) at 4 hr were those primarily involved in the cessation of the cell cycle, cellular detoxification pathways, DNA repair, and apoptosis. At 24 hr, glutathione-associated genes were induced in addition to genes involved in apoptosis. Genes involved in cell cycle progression and mitosis were downregulated at 24 hr. Real-time quantitative PCR was used to confirm the microarray results and to evaluate expression levels of selected genes at the low doses (0.5 and 1.0 Gy). The expression profiles reflect the cellular and molecular responses to ionizing radiation related to the recognition of DNA damage, a halt in progression through the cell cycle, activation of DNA-repair pathways, and the promotion of apoptosis.

Malachite green, a triphenylmethane dye used in aquaculture as an antifungal agent, is rapidly reduced in vivo to leucomalachite green. Previous studies in which female B6C3F1 mice were fed malachite green produced relatively high levels of liver DNA adducts after 28 days, but no significant induction of liver tumors was detected in a 2-year feeding study. Comparable experiments conducted with leucomalachite green resulted in relatively low levels of liver DNA adducts but a dose-responsive induction of liver tumors. In the present study, we fed transgenic female Big Blue B6C3F1 mice with 450 ppm malachite green and 204 and 408 ppm leucomalachite green (the high doses used in the tumor bioassays) and evaluated genotoxicity after 4 and 16 weeks of treatment. Neither malachite green nor leucomalachite green increased the peripheral blood micronucleus frequency or Hprt lymphocyte mutant frequency at either time point; however, the 16-week treatment with 408 ppm leucomalachite green did increase the liver cII mutant frequency. Similar increases in liver cII mutant frequency were not seen in the mice treated for 16 weeks with malachite green or in female Big Blue rats treated with a comparable dose of leucomalachite green for 16 weeks in a previous study [Mutat. Res. 547 (2004) 5]. These results indicate that leucomalachite green is an in vivo mutagen in transgenic female mouse liver and that the mutagenicities of malachite green and leucomalachite green correlate with their tumorigenicities in mice and rats. The lack of increased micronucleus frequencies and lymphocyte Hprt mutants in female mice treated with leucomalachite green suggests that its genotoxicity is targeted to the tissue at risk for tumor induction.

Microarray analysis is a powerful tool to identify the biological effects of drugs or chemicals on cellular gene expression. In this study, we compare the relationships between traditional measures of genetic toxicology and mutagen-induced alterations in gene expression profiles. TK6 cells were incubated with 0.01, 0.1, or 1.0 microM +/-anti-benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide (BPDE) for 4 h and then cultured for an additional 20 h. Aliquots of the exposed cells were removed at 4 and 24 h in order to quantify DNA adduct levels by 32P post-labeling and measure cell viability by cloning efficiency and flow cytometry. Gene expression profiles were developed by extracting total RNA from the control and exposed cells at 4 and 24 h, labeling with Cy3 or Cy5 and hybridizing to a human 350 gene array. Mutant frequencies in the Thymidine Kinase and Hypoxanthine Phosphoribosyl Transferase genes were also determined. The 10alpha-(deoxyguanosin-N(2)-yl)-7alpha,8beta,9beta-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene (dG-N(2)-BPDE) adduct increased as a function of dose and was the only adduct identified. A dose-related decrease in cell viability was evident at 24 h, but not at 4 h. Cell death occurred by apoptosis. At 4 h, analysis of the gene expression profiles revealed that Glutathione Peroxidase and Gadd45 were consistently upregulated (greater than 1.5-fold and significantly (P < 0.001) greater than the control in two experiments) in response to 1.0 microM BPDE exposure. Fifteen genes were consistently down-regulated (less than 0.67-fold and significantly (P < 0.001) lower than the control in two experiments) at 4 h in cultures exposed to 1.0 microM BPDE. Genes with altered expression at 4 h included genes important in the progression of the cell-cycle and those that inhibit apoptosis. At 24 h post-exposure, 16 genes, involved in cell-cycle control, detoxification, and apoptosis were consistently upregulated; 10 genes were repressed in cultures exposed to the high dose of BPDE. Real-time quantitative PCR confirmed the differential expression of selected genes. These data suggest that changes in gene expression will help to identify effects of drugs and chemicals on molecular pathways in cells, and will provide useful information about the molecular responses associated with DNA damage. Of the endpoints evaluated, DNA adduct formation was the most sensitive indicator of DNA damage. DNA adduct formation was clearly evident at low doses, but the number of genes with significantly altered expression (P < 0.001) was minimal. Alterations in gene expression were more robust at doses associated with cellular toxicity and induction of mutations.

A novel approach was developed for rapidly estimating the frequency of specific mutations in genetically engineered Chinese hamster ovary (CHO) cells. We designed double-transgenic CHO cell lines that contain a transgene consisting of the sequence coding for green fluorescent protein under the control of a tetracycline (Tet) responsive promoter and a second transgene coding for the constitutively expressed Tet repressor. Cultures of these CHO cells were treated with gamma-radiation, N-methyl-N-nitrosourea or methyl methanesulfonate, and the fluorescence of individual cells from both control and treated cultures was measured by flow cytometry. The treatments increased the number of highly fluorescent cells, those with presumed mutations in the Tet-repressor gene. Mutant cells from gamma-radiation-exposed cultures were isolated by fluorescence-activated cell sorting, cultured, and individual clones expanded. A PCR-based analysis indicated that the highly fluorescent expanded cells had lost the transgene coding for the Tet repressor, suggesting that the system mainly detects large genetic alterations. A similar approach may be useful for making high-throughput in vivo models for mutation detection.

Coumestrol, a phytoestrogen found in high levels in alfalfa and red clover, is of concern since endocrine disorders have been observed in farm animals exposed to high levels of phytoestrogens. Previous studies found that coumestrol was an effective inducer of DNA strand breaks, micronuclei, and mutations in the Hypoxanthine phosphoribosyl transferase (HPRT) gene of Chinese hamster ovary cells. In the experiments presented here, we extended the previous studies to examine the effect of coumestrol exposure on AHH-1 TK(+/-) human lymphoblastoid cells. Micronuclei were induced with the highest frequency occurring at day 2 after exposure. Flow cytometric analysis of annexin V-FITC-7-aminoactinomycin D stained cells indicated that the primary pathway of cell death was by apoptosis. Mutations were induced in the Thymidine Kinase (TK) gene and were due primarily to the induction of clones with the slow-growth phenotype. Subsequent molecular analysis revealed the loss of exon 4 in the coumestrol-induced clones, indicative of loss-of heterozygosity and consistent with a proposed inhibition of topoisomerase-II activity as a mechanism of action for coumestrol. Taken together, these results suggest that coumestrol exhibits both mutagenic and clastogenic properties in cultured human lymphoblastoid cells.

Fumonisin B1 stimulates apoptosis in a variety of cell types and tissues. We examined the role of sphingolipid changes in fumonisin B1-stimulated apoptosis. Sphinganine accumulated rapidly, sphingosine levels remained unchanged, and ceramides decreased during fumonisin B1 exposure. Increased DNA fragmentation, decreased viability, and apoptotic morphology were observed in cells exposed to fumonisin B1, sphinganine, or N-acetylsphingosine. Co-exposure to N-acetylsphingosine or beta-chloroalanine, which blocks sphinganine accumulation, partially protected cells from fumonisin B1-induced apoptosis. These results illustrate three sphingolipid-dependent mechanisms for inducing apoptosis: accumulation of excess ceramide, accumulation of excess sphinganine, and depletion of ceramide or complex sphingolipids derived from ceramide.

The phytoestrogen, genistein, is a naturally occurring isoflavone found in soy products. On a biochemical basis, genistein is a competitive inhibitor of tyrosine kinases and the DNA synthesis-related enzyme, topoisomerase-II (topo-II). Exposure of mammalian cells to genistein results in DNA damage that is similar to that induced by the topo-II inhibitor and chromosomal mutagen, m-amsa. In order to determine the potential genotoxicity of genistein, human lymphoblastoid cells which differ in the functional status of the tumor suppressor gene, p53, were exposed to genistein and the induction of micronuclei quantified by microscopic analysis. In addition, the mutant fraction at the thymidine kinase (tk) locus (both the normal-growth and slow-growth phenotypes) was determined by resistance to trifluorothymidine (TFT) and at the hypoxanthine phosphoribosyl transferase (hprt) locus by resistance to 6-thioguanine (6-TG). Flow cytometric analysis of the percentage of viable, apoptotic and degenerating cells was utilized to determine the rate and kinetics of cell death after genistein exposure. The detection of micronuclei in both cell lines indicated that genistein-induced damage had occurred in both AHH-1 tk+/- and L3. Linear regression analysis detected a significant increase in the number of 6-TG-resistant clones in both AHH-1 tk+/- (p53+/-) and L3 (p53+/+). A comparison of slopes revealed no difference between the lines. In contrast, a significant, concentration-dependent increase in the number of TFT-resistant clones with the slow-growth phenotype was detected in AHH-1 tk+/- (mutant p53), but not in L3 (wild-type p53). Cell death occurred primarily by apoptosis in both cell lines; however, a concentration-dependent decrease in the percentage of viable cells was detected immediately after exposure in L3, but not until 32 h after exposure in AHH-1 tk+/-. A comparison of the slopes of the concentration-response curves for the percentage of viable cells revealed no difference between the cell lines in the effect of genistein on cell viability. Our results may be interpreted that genistein is a chromosomal mutagen and that p53 functional status affects the recovery of chromosomal mutants, possibly by signalling cells into the apoptosis pathways., (Copyright 1998 Elsevier Science B. V.)

The chromosomal mutagen, bleomycin, is also noted for its toxic properties, although the mechanism of cell death is not fully understood. In order to determine if cell death occurred by apoptosis or necrosis, AHH-1 tk+/- cells were exposed to bleomycin and the percentage of viable, apoptotic and necrotic cells quantified by flow cytometry. Logistic regression analysis indicated that the primary manner of cell death was through the apoptosis pathways, that apoptosis was delayed, and that apoptosis was accompanied by an arrest in the G2 phase of the cell cycle. Once apoptosis was established as a mechanism for cell death, the efficiency with which these pathways removed damaged cells from the population was evaluated with the use of specific-locus mutation assays (tk and hprt) as indicators of cells with DNA damage that maintained viability and clonogenicity. Linear regression analysis detected a significant, concentration-dependent increase in the numbers of TFTr clones with the slow-growth phenotype. This suggests that a proportion of cells with bleomycin-induced DNA damage did not undergo cell death by apoptosis and that apoptosis, a mechanism for the destruction of damaged cells, is not fully efficient in the AHH-1 tk +/- cell line.

Loss-of-function mutations in the p53 tumor suppressor gene result in an altered response to DNA-damaging agents. Included in the mutant p53 phenotype are the loss of the G1 checkpoint and delayed apoptotic cell death, characteristics we have consistently observed in the AHH-1 tk+/- cell line following exposure to DNA-damaging agents. In order to determine the functional status of p53 in the AHH-1 tk+/- cell line, molecular analysis (single-strand conformational polymorphism [SSCP] and sequence analysis) was performed on exons 5-9 of the p53 gene. In addition, the status of the p53 gene in the closely related lymphoblast line, MCL-5, which, in our hands, has a much higher spontaneous rate of apoptosis than AHH-1 tk+/-, was also determined by molecular analysis. Initial SSCP analysis of AHH-1 tk+/- revealed an abnormal migration pattern of exon 8 when compared to a wild-type control. Subsequent sequence analysis indicated that a base-pair substitution (CGG-->TGG) mutation had occurred at codon 282, a reported "hot spot' for 5-methylcytosine mutations in the human p53 gene. Neither SSCP nor sequence analysis of exons 5-9 of MCL-5 indicated any differences from wild-type DNA. These results suggest that the lack of a G1 arrest and the delayed entrance into apoptosis observed in chemically-exposed AHH-1 tk+/- cells are, at least partially, accounted for by a loss-of-function mutation in the p53 gene.

Fumonisin B1 is associated with various animal and human carcinomas and toxicoses, including leukoencephalomalacia, hepatocarcinoma, pulmonary edema and esophageal carcinoma. We have examined the cellular effects of fumonisin B1 in vitro using cellular model systems relevant to potential human target tissues. Although fumonisin B1 has been described as a mitogen in Swiss 3T3 cells based on stimulation of [3H]thymidine incorporation, in the current work it was found that fumonisin B1 inhibited incorporation of [3H]thymidine by cultured neonatal human keratinocytes and HepG2 human hepatocarcinoma cells at 10(-7) and 10(-4) M respectively. Fumonisin B1 also inhibited clonal expansion of normal human keratinocytes and HET-1A human esophageal epithelial cells at 10(-5) M and growth in mass culture of normal human fibroblasts at 10(-7) M. The clonogenicity of normal human keratinocytes decreased to 45.5% of controls after exposure to 10(-4) M fumonisin B1 for 2 days. However, no differences in the cell cycle distribution of cultured keratinocytes was noted after exposure to 10(-5) M fumonisin B1 for 40 h. The viability of normal human keratinocytes and HET-1A cells decreased as a result of fumonisin B1 treatment, as determined by a fluorescein diacetate/propidium iodide flow cytometric cell viability assay. Fumonisin B1-treated keratinocytes released nucleosomal DNA fragments into the medium 2-3 days after exposure to 10(-4) M fumonisin B1 and increased DNA strand breaks were detected in attached keratinocytes exposed to 0-10(-4) M fumonisin B1 using a terminal deoxynucleotidyl transferase-based immunochemical assay system. Furthermore, fumonisin B1-treated keratinocytes and HET-1A cells developed morphological features consistent with apoptosis, as determined by phase contrast microscopy, fluorescent microscopy of acridine orange stained cells and electron microscopy. These results are consistent with the occurrence of fumonisin B1-mediated apoptosis in vitro.

Background: All-trans retinoic acid is currently used in clinical trials in combination with tamoxifen to treat breast cancer, and 13-cis retinoic acid is used with a-interferon to treat metastatic endometrial cancer. We examined the effects of all-trans retinoic acid and 13-cis RA alone on endometrial adenocarcinoma (RL95-2) cells to investigate the cell biological mechanisms by which retinoic acid may reduce the metastatic phenotype and induce differentiation., Methods: RL95-2 cells were seeded onto 4-chamber plastic slides and treated with 13-cis retinoic acid or all-trans retinoic at 0.5 microM, 1 microM and 5 microM doses for 90 minutes at 37 degrees C and stained for F-actin., Results: Untreated RL95-2 cells exhibited staining of disrupted aggregates of F-actin only near the cell periphery. Cells treated with the three doses of 13-cis retinoic acid exhibited a dramatic reorganization of F-actin throughout the cells. When cells were treated with 0.5 microM all-trans retinoic acid, actin filaments reorganized. Cells treated with 1 microM all-trans retinoic acid and 5 microM all-trans retinoic acid displayed increased organization of F-actin and cell size increased. The percentage of S-phase cells increased at the high doses of retinoic acid treatment. This effect was apparently transient, since retinoic acid did not significantly affect cell growth., Conclusion: An organized cytoskeleton and an increase in cell size are associated with differentiation. We suggest that retinoic acid exerts its effects on these transformed cells by reorganizing actin filaments, and inducing differentiation, thus inducing a more stationary phenotype.

AHH-1 tk +/- cells were exposed to the chemotherapeutic agent, m-amsa, both in complete medium and in medium without serum, subcultured in complete medium, and the effect on the traverse of the cell cycle determined by flow cytometric analysis of bromodeoxyuridine (BrdUrd)-labeled DNA. After exposure to m-amsa (day 0), the percentage of S-phase cells increased significantly (P < 0.0017) with increasing concentration. Cells also accumulated in G2/M as evidenced by the significant (P < 0.0026), concentration-dependent increase in the percentage of cells detected within this phase. Serum deprivation during exposure resulted in significantly (P = 0.024) more cells in S-phase than in cultures exposed to m-amsa in complete medium. After three days in culture, a significant (P = 0.0001) accumulation of cells in G2/M was present; the percentage of cells in G2/M did not differ significantly (P = 0.148) in cultures exposed to m-amsa in complete medium or in serum-free medium. However, a significant (P < 0.001) loss of S-phase cells was found in cultures exposed without serum. At day 7, no significant concentration effects were detected (GO/G1, P = 0.6026; S-phase, P = 0.9773; G2/M, P = 0.8401). These results demonstrate that exposure to m-amsa perturbs the traverse of the cell cycle, initially by inhibiting the completion of S-phase and followed by an accumulation of cells in G2/M. In addition, exposure to m-amsa under conditions of serum deprivation results in an increased percentage of cells in the initial S-phase after exposure, the loss of S-phase cells from the culture after three days, and the appearance of subdiploid peak, consistent with cells undergoing apoptosis.

One role of programmed cell death (apoptosis) is the removal of cells with DNA damage from the population. Certain cells, however, are able to suppress the signals for apoptotic cell death and maintain viability. This suggests that the susceptibility of a cell to either undergo apoptosis or escape from the apoptotic death pathways may be an important factor in chemical mutagenesis. In order to provide insight into the role of apoptosis in the recovery of chemically induced mutants, AHH-1 cells were exposed to the chromosomal mutagen, m-amsa, and the percentage of cells undergoing apoptosis or necrosis quantified by flow cytometry. Logistic regression analysis revealed that the primary manner of cell death was by apoptosis. Two specific-locus mutations assays, the tk and the hprt, were utilized as markers for cells with DNA damage and that retained clonogenicity under conditions known to induce apoptosis. Analysis of variance indicated that the concentration-dependent increase in the mutant fraction at the tk locus was significant and the result of the recovery of clones with the slow-growth phenotype. Because this phenotype is thought to reflect chromosomal mutations, these results are consistent with the survival and clonogenicity of damaged cells. This suggests that the ability to recover mutant cells may be influenced by the suppression of or an escape from the apoptotic death pathways.

In order to determine the pathway for cell death in alkylating agent-exposed human lymphoblastoid cells, AHH-1 cells were exposed to either ethyl methanesulfonate (EMS) or ethyl nitrosourea (ENU) and the effect on relative cell growth and plating efficiency quantified. Flow cytometric (FCM) assays were utilized to quantify cell viability and to determine if cell death occurred through necrosis or apoptosis. As expected, exposure to the simple ethylating agents resulted in concentration-dependent decreases in plating efficiencies at each time interval after exposure (Days 0, 2, 3 and 7). EMS exposure did not significantly affect the relative cell growth, in contrast to ENU exposure, which inhibited cell growth. The FCM viability assay, based on light scatter characteristics, revealed that exposure to either alkylating agent resulted in a significant reduction in the percentage of viable cells. The results of the FCM dye-exclusion assays revealed that while necrosis occurred in EMS- and ENU-exposed cells, the primary manner of cell death was apoptosis. AHH-1 cells were stained with propidium iodide and fluorescein diacetate, the population of cells sorted electronically and the cell type (necrotic, apoptotic or viable) confirmed morphologically. Our results clearly indicate that exposure to EMS or ENU results in the movement of AHH-1 cells into the pathway for apoptosis and cell death.

The fidelity and progression of DNA synthesis is critically dependent on the correct balance and availability of the deoxynucleoside triphosphate (dNTP) precursors for the polymerases involved in DNA replication and repair. Because folate-derived one-carbon groups are essential for the de novo synthesis of both purines and pyrimidines, the purpose of this study was to determine the effect of folate deprivation on deoxynucleotide pool levels and cell cycle progression. Primary cultures of phytohemagglutin (PHA)-stimulated splenocytes were used as the cellular model. T-cells and macrophages were purified from spleen cell suspensions obtained from F344 rats and recombined in culture. The cells were harvested after a 66-hr incubation with PHA and analyzed for nucleotide levels by reverse-phase HPLC with diode array detection. The proportion of cells in the different phases of the cell cycle was determined by bivariate flow cytometric measurement of bromodeoxyuridine (BrdU) incorporation and DNA content (propidium iodide staining). PHA-stimulated T-cells cultured in medium lacking folate and methionine manifested significant decreases in the deoxynucleotides dCTP, dTMP, dGTP, and dATP relative to cells cultured in complete medium. The reduction in dNTP pools was associated with a decrease in the corresponding ribonucleotide pools. Flow cytometric analysis revealed a 2-fold increase in S and G2/mitosis (G2/M) DNA content in PHA-stimulated cells cultured in the medium lacking folate and methionine, which suggests a delay in cell cycle progression. These alterations in DNA content were accompanied by a 5-fold decrease in BrdU incorporation relative to PHA-stimulated cells cultured in complete medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Current studies in our laboratory are designed to determine the frequency of genotoxic responses induced in lymphocytes isolated from Fischer 344 rats. To evaluate the effect of a model compound, N-ethyl-N-nitrosourea (ENU), on the cell-cycle distribution of spleen lymphocytes, 8-week old, female Fischer 344 rats were injected i.p. with ENU and sacrificed 1, 2, 4, and 6 weeks after exposure. Four replicate cultures per dose per exposure period were established and cells were cultured for 66 hr. Colcemid, an agent which blocks cells in mitosis and induces an accumulation of cells in the G2 + M peak, was added to two of the four cultures as a positive control. After a 3 hr incubation, the cells were harvested, the nuclei stained with propidium iodide, and the DNA content of the individual nuclei was quantified by flow cytometry. As expected, exposure to Colcemid resulted in an accumulation of cells in the G2 + M phase of the cell cycle, which was accompanied by a decrease in the G0 + G1 population. The increase in the G2 + M population was significant (p < 0.05) in cultures of lymphocytes assayed at 4 and 6 weeks after exposure. The effect of increasing ENU concentration was an increase in the percentage of S-phase cells (p = 0.05) and a decrease (p < 0.02) in the percentage of G0 + G1 cells. This finding was observed only in those lymphocytes isolated 1 week after exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

Although sister-chromatid exchange (SCE) analysis is recognized as an indicator of exposure to DNA-damaging agents, the results of these analyses have been confounded by the use of bromodeoxyuridine (BrdUrd) to differentially label the sister chromatids. Not only does BrdUrd itself induce SCE, it also modulates the frequency of SCE induced by certain DNA-damaging agents. In order to examine this effect of BrdUrd on SCE frequency, an indirect method which lends itself to measurements both with and without BrdUrd was employed. Human teratocarcinoma-derived (P3) cells were exposed to ethyl methanesulfonate (EMS) and cultured with increasing concentrations of BrdUrd for lengths of time corresponding to one, two, and three generations of cell growth. At each time point, the distribution of nuclei among the phases of the cell-cycle and cell growth were evaluated for each concentration and chemical. A statistical model was employed which tested both for the main effects of chemicals and culture times and for interactions between these factors. Both EMS and BrdUrd significantly affected the percentages of nuclei within the cell-cycle. Exposure to EMS resulted in decreases in the percentages of nuclei in G0 + G1 and increases in the G2 + M compartment. Exposure to BrdUrd affected the size of the G0 + G1 compartment as well as the percentage of S-phase nuclei. Cell growth was reduced as a consequence of increasing EMS concentration and as a function of BrdUrd concentration; the effects of these chemicals were more readily apparent at the later time points. Most importantly, for both the cell-cycle kinetics data and the cell growth data, no evidence of an interaction between the effects of EMS and the effects of BrdUrd was detected statistically. These results may be interpreted to mean that while both EMS and BrdUrd affect the induction of SCE, under the conditions of this experiment, the effects are additive rather than interactive.

The effects of magnesium (Mg) restriction on cell growth and the cell cycle were determined in transformed (TRL-8) and non-transformed (TRL-12-15) epithelial-like rat liver cells. Cells were cultured in RPMI 1640 medium in which the Mg concentration was reduced to 0.5, 0.1, and 0 x the concentration in the regular RPMI 1640 media (100mg/l). Cell growth in the transformed cells was not influenced by the Mg restriction as greatly as in the non-transformed cell line. Transit through the cell cycle also exhibited an independence of the Mg in the medium in the transformed cells. When transformed cells were grown for two generations in Mg-limited medium, the growth rate slowed to a rate similar to that demonstrated by the non-transformed cells. Analysis by flow cytometry showed that transit through the cell cycle was minimally slowed in Mg deficient transformed cells; however, transit through the G1 and S phases in the non-transformed cells was slowed. The TRL-8 cells in Mg-limited medium resulted in fewer nuclei in G1 with subsequent increases in the percentages of S-phase nuclei. The TRL 12-15 cells reacted oppositely with the number of G1 nuclei increased and the number of S-phase nuclei decreased. In respect to growth, these results show that epithelial cells respond in a similar manner to Mg-limitation as do fibroblast cells. The transformed cells exhibited a level of independence from Mg in respect to growth, reproduction, and cell-cycle kinetics.

In order to determine the relationships among the reduction in relative cloning efficiency (RCE), sister-chromatid exchange (SCE) formation, and interference with progression through the cell-cycle, human teratocarcinoma-derived (P3) cells were exposed to either ethyl methanesulfonate or to methyl methanesulfonate. The relationship between SCE and toxicity was quantified, the progression through the cell-cycle was evaluated with flow cytometric methods, and the effects of these chemicals on cell growth and average generation time (AGT) were determined. A strong correlation existed between RCE and SCE (r = -0.978, p less than .001) which was accompanied by an inhibition of growth as evidenced by a significant (p less than .0001) negative linear effect of concentration on the relative cell count from 24 to 72 hours after exposure and by a concentration-dependent increase (p less than .0001) in the AGT. Delays in the transit through S-phase were evident 4 hours after exposure to toxic concentrations of either carcinogen and by 8 to 12 hours post-exposure at the lower concentrations. Increases in the percentage of nuclei in G2 + M, indicative of G2 arrest, occurred from 12 to 24 hours after exposure. One interpretation of these results is that those effects of EMS and MMS exposure which result in S-phase delay and G2 arrest may be those elements common to the induction of SCE and cellular toxicity.

The effect of interleukin-2 (IL-2) on cell proliferation, sister-chromatid exchange (SCE) frequency, and the phosphorylation of nuclear stress proteins was evaluated in phytohemagglutinin (PHA)-stimulated spleen lymphocytes isolated from Fischer 344 rats. In addition, the ability of 2-mercaptoethanol (2-ME) to modulate the induction of these biological responses was characterized. Cell proliferation, as measured by the mitotic index, increased significantly (P less than .003) from a range of 3-4% in PHA-stimulated cultures to a range of 8-11% in PHA-stimulated cultures exposed to IL-2. The average generation time (AGT) did not respond to IL-2 in a concentration-dependent manner and decreased significantly (P less than .05) when 20 microM 2-ME was included with IL-2 in the culture medium. The number of SCE increased significantly (P less than .004) from control frequencies, which ranged from 13.1 to 15.6 SCE per cell, to frequencies of 18.5 to 21.5 SCE per cell as the concentration of IL-2 in the culture medium increased to 50 half-maximal units per ml. A reduction in SCE frequency was observed when cells were cultured with 20 microM 2-ME and IL-2 compared to IL-2 alone. Three nuclear proteins, with relative molecular masses of approximately 13,000-18,000, 20,000, and 80,000, were phosphorylated in IL-2-exposed G1-phase nuclei. Elicitation of these nuclear proteins in IL-2-exposed cells was not affected by exposure to 2-ME.

A single genetic factor may affect the realization of several enzymes. To investigate the extent of pattern pleiotropy in the mouse, the activities of 28 enzymes in livers and brains from an inbred stock of C57BL/6JNctr and five F1 stocks heterozygous for known electrophoretic variants were measured. Five congenic backcross stocks of C57BL/6J, each homozygous for one or more electrophoretic markers, were mated with C57BL/6JNctr to construct the heterozygous variant F1 stocks. One of the five F1 stocks had no enzyme activities significantly different from those of C57BL/6JNctr, while two had one enzyme, one had four enzymes, and another had six enzymes with activities that were significantly different from those of C57BL/6JNctr. The latter two F1 stocks with multiple activity differences were those having the largest proportion of their genome of donor origin. Two of the F1 stocks were different from each other for one enzyme, and two were different for another enzyme. These differences and the relationship of these enzyme activities to the variant genes suggest that several genetic factors may affect an enzyme's realization.

Chinese hamster ovary cells were exposed to N-hydroxy-2-aminofluorene, N-hydroxy-N'-acetylbenzidine and 1-nitrosopyrene, and the resulting DNA adducts, sister-chromatid exchanges (SCEs) and mutations at the hypoxanthine-guanine phosphoribosyl transferase locus were quantified. Each agent produced a major DNA adduct substituted through the C8 of deoxyguanosine. When the data from all three agents were combined, both mutation and SCE induction correlated strongly with the concentration of DNA adducts. However, significant differences were found in the relationships between adduct formation and the biological responses produced by the individual agents. While N-hydroxy-N'-acetylbenzidine induced the most mutations per adduct, N-hydroxy-2-aminofluorene caused the greatest number of SCEs per adduct. The data support the involvement of C8-deoxyguanosine adducts in mutation and SCE induction, and indicate that the structure of the group adducted to DNA may be an important factor in determining the magnitude of these biological responses. These findings also suggest that SCE and mutation induction are independent expressions of DNA damage.

We have developed methods in our laboratory whereby the effects of toxicant exposure on cell proliferation can be evaluated flow cytometrically. We sought to relate the flow cytometric analyses to other biological response measurements. Thus, we exposed P3 cells to increasing concentrations of bromodeoxyuridine (BRdU) and measured sister-chromatid exchange (SCE) frequency, average generation time (AGT), and relative cloning ability. Each of these is well documented (see introduction) to respond to BRdU exposure in a concentration-dependent manner. In this study, SCE frequency remained constant between the concentrations of 2.5 and 10 microM of BRdU. However, a small, but significant, increase in SCE frequency was observed between the concentrations of 10 microM and 50 microM BRdU. A significant increase in AGT was noted in 50 microM BRdU-exposed cells. Relative cloning efficiency decreased in a concentration-dependent manner when cells were cultured for 24, 48, or 72 hours with BRdU. When cell proliferation was assessed by flow cytometric analysis in cells exposed to 0, 10, or 50 microM BRdU, a statistically significant delay in the cell-cycle was observed in BRdU-exposed cells. These results may be interpreted to mean that inhibition of cell proliferation is detected by this type of analysis at toxicant concentrations that induce other biological endpoints. The inclusion of flow cytometric analysis in a test battery to evaluate toxicant effects is warranted.

We recently identified and confirmed 8 induced mutations in the N2 and N3 progeny of ethyl methanesulfonate (EMS) treated C57BL/6J mice. Each of these mutations altered specific enzyme activities. These separate mutant sublines have been maintained through several generations as heterozygous mutant carriers. The percent decrease of the specific enzyme activity from normal in each subline was calculated for each generation. Additionally, the percentage of breeders within each mutant subline producing abnormal progeny and the fraction of such breeders' total progeny possessing abnormal activity were determined. The aberrant activity values observed in progeny of a confirmed mutant carrier were all lower than normal. 4 of the mutant sublines had decreases in enzyme activities which were constant across the generations analyzed. 3 of the mutant sublines had decreases in activities which were consistent over early generations but changed significantly in later generations. Another subline with decreased enzyme activity was lost. For 7 of the sublines, the number of progeny having altered activity and the number of breeders producing mutant progeny approximated that expected for single gene inheritance. In the remaining subline, a change in the decrease in enzyme activity probably accounts for the deviation from expected inheritance. Although the phenotypes for these quantitative traits are considered to be quasi-continuous, the data indicate that the mutations are probably of major genes.

To determine the relationships between the induction of specific biological responses and exposure to DNA-damaging agents, human teratocarcinoma-derived cells were exposed to either ethyl methanesulfonate or to methyl methanesulfonate, and sister chromatid exchange, cellular proliferation and relative cloning ability measured. SCE increased while cellular proliferation and relative cloning ability each decreased in a concentration-dependent manner. Methyl methanesulfonate was consistently more efficient in inducing biological responses than was ethyl methanesulfonate. When the individual responses were compared, the decrease in cellular proliferation paralleled the reduction in cloning efficiency. A strong correlation was also observed between the reduction in relative cloning ability and sister chromatid exchange frequency. Because these relationships are similar to those previously described in other mammalian cell lines, the observations in our study suggest that the P3 cell line is an appropriate choice for modeling effects of toxicant exposure in human cells.

2-Mercaptoethanol (2-ME) is used as a medium supplement to enhance the proliferation of lymphocytes cultured in vitro. In this study, we have examined the effects of 2-ME on cell growth and on SCE induction in cultures of unstimulated and phytohemagglutinin (PHA)-stimulated Fischer 344 rat lymphocytes. There were virtually no metaphases detected in cells cultured without PHA. In PHA-stimulated cultures, 2-ME decreased SCE frequency but it enhanced SCE frequency in the presence of 5 to 12.5 microM bromodeoxyuridine (BRdU). Both mitotic and replication indices were increased in the PHA/2-ME system. The levels of incorporated exogenous thymidine, in the presence of 2-ME, were relatively low in unstimulated cells, suggesting that 2-ME is not mitogenic for T-cells. However, 2-ME enhanced PHA-induced response of T-cells as evidenced by increased levels of thymidine incorporation into cellular DNA. The growth-promoting effects and the decrease in SCE frequency caused by 2-ME upon PHA stimulation indicate that 2-ME may alter the nature of interaction between PHA and cellular activating properties or the replicative processes.