Lynda J. McGarrity

p53, mutations, and apoptosis in genistein-exposed human lymphoblastoid cells.

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.

The genotoxicity of acrylamide and glycidamide in big blue rats.

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.

DNA adduct formation and induction of micronuclei and mutations in B6C3F1/Tk mice treated neonatally with acrylamide or glycidamide†

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 ∼ 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.

Programmed cell death and mutation induction in AHH-1 human lymphoblastoid cells exposed to m-amsa

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.

Modulation of SCE induction and cell proliferation by 2-mercaptoethanol in phytohemagglutinin-stimulated rat lymphocytes.

Abstract2-Mercaptoethanol (2-ME) is used as a medium supplement to enhance the proliferation of lymphocytes culturedin 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 S to 12.5 µM bromodeoxyuridine (BRd U). 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.

A role for apoptosis in the toxicity and mutagenicity of bleomycin in AHH-1 tk+/− human lymphoblastoid cells

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.

The role of programmed cell death in the toxicity of the mutagens, ethyl methanesulfonate and N-ethyl-N'-nitrosourea, in AHH-1 human lymphoblastoid cells

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.

Evaluation of the genotoxicity of the phytoestrogen, coumestrol, in AHH-1 TK(+/-) human lymphoblastoid cells.

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.

Detection of mutation in transgenic CHO cells using green fluorescent protein as a reporter

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.

Cell cycle traverse in AHH-1 tk +/- human lymphoblastoid cells exposed to the chromosomal mutagen, m-Amsa

AHH‐l 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 (G0/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 a subdiploid peak, consistent with cells undergoing apoptosis.