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Deep-level transient spectroscopy measurements on β-Ga 2 O 3 crystals reveal the presence of three defect signatures labeled E 2 a, E 2 b, and E 3 with activation energies at around 0.66 eV, 0.73 eV, and 0.95 eV below the conduction band edge. Using secondary ion mass spectrometry, a correlation between the defect concentration associated with E 3 and the Ti concentration present in the samples was found. Particularly, it is found that E 3 is the dominant Ti-related defect in β-Ga 2 O 3 and is associated with a single Ti atom. This finding is further corroborated by hybrid functional calculations that predict Ti substituting on an octahedral Ga site, denoted as Ti GaII, to be a good candidate for E 3. Moreover, the deep level transient spectroscopy results show that the level previously labeled E 2 and attributed to Fe substituting on a gallium site (Fe Ga) consists of two overlapping signatures labeled E 2 a and E 2 b. We tentatively assign E 2 a and E 2 b to Fe substituting for Ga on a tetrahedral or an octahedral site, respectively.

Deep level transient spectroscopy was used to study the defects induced by alpha-particle irradiation from an Am241 source in antimony doped n-type germanium. Previous investigations of the well know Sb-vacancy defect have led to the discovery of a second defect with very similar emission properties, referred to as the E′. Although both defects have similar emission rates, they have very different annealing properties. In this study we further investigated these properties of the E′ in Sb doped samples irradiated at 270 K with alpha particles from an Am241 source. Laplace deep level transient spectroscopy was used to determine the concentration of each defect. An isothermal annealing study of the E′ was carried out in the temperature range 300 K to 325 K in 5 K increments, while the Sb-vacancy was annealed out completely at 410 K onwards, long after the E′ was completely annealed out. The annealing activation energy was determined through isothermal annealing profiles for both the Sb-Vacancy and the E′ as 1.05 eV and 0.73 eV respectively with a prefactor of 2.05 × 109 s−1 and 2.7 × 108 s−1.

Alpha particle irradiation was used to study the radiation-induced defects in n-type germanium (Ge). Investigation of the well-known antimony (Sb)-vacancy complex (commonly known as the E-center) in Ge, with an activation energy of 0.37 eV (E 0.37 ), has led to the discovery of another defect with a DLTS signature virtually indistinguishable from the E-center, but with different annealing characteristics. We shall refer to this new defect as the E-prime. Although the two defects are easily distinguishable by annealing, the DLTS signal produced by the E-center and E-prime were not distinguishable through conventional deep level transient spectroscopy (DLTS). Separation of the two peaks was only possible through the use of low noise equipment in conjunction with high resolution Laplace-DLTS. The activation energy of the Sb-vacancy and the E-prime was determined to be 0.370 ± 0.005 eV and 0.375 ± 0.005 eV. Depth profiles showed uniform distributions of both defects below the Schottky junction.

Centchroman (CC), a non-steroidal oral contraceptive and a candidate drug for breast cancer, has been reported to exhibit partial to complete remission of lesions in 40.5% of breast cancer patients. The potent anti-oestrogenic activity, negligible side-effects and anti-breast cancer activity of CC prompted us to evaluate the antimutagenic effects of this compound in a bacterial mutagenicity assay and CHO/HPRT and AS52/GPT mutation assays in vitro and in vivo in female Swiss albino mice as measured by both sister chromatid exchange (SCE) and chromosome aberrations (CA) against three known positive mutagen compounds, dimethylbenz[a]anthracene (DMBA), cyclophosphamide (CP) and mitomycin C (MMC). Antimutagenicity assays in Salmonella strains TA97a, TA100, TA98 and TA102 were carried out against commonly used known positive mutagens, sodium azide, 4-nitro-o-phenylenediamine, cumine hydroperoxide, 2-aminofluorene and danthron. A significantly reduced number of bacterial histidine revertant colonies was observed in the plates treated with 0.1, 1, 5 and 10 microg/plate CC and a positive compound when compared with bacterial plates treated with the respective positive compound alone. Ethyl methanesulfonate (EMS), a commonly used positive mutagen for CHO/HPRT and AS52/GPT gene mutation assays, was used for antimutagenicity assay in these cells. CC exhibited protective effects against the mutagenicity of EMS in these two mammalian cell mutation assays, CHO/HPRT and AS52/GPT. In the in vivo studies, pretreatment with CC reduced DMBA-induced SCE and CA and CP- and MMC-induced CA when compared with the group treated only with the positive compounds. These results indicate that CC can reduce the mutagenic effects of known genotoxic compounds.

Coumarin (1,2-benzopyrone), a natural dietary constituent and drug currently under evaluation for treatment of certain cancers and lymphedema, reduces polycyclic aromatic hydrocarbon-induced neoplasms in rodents. Because most rodents metabolize coumarin through 3,4-epoxidation, whereas 7-hydroxylation predominates in humans, their suitability as a model for coumarin effects in humans has been questioned. We examined coumarin chemoprotection against the promutagen and dietary contaminant aflatoxin B1 with human liver S9 bioactivation in the Chinese hamster ovary cell/hypoxanthine-guanine phosphoribosyltransferase mutation assay. Coumarin in the absence of aflatoxin B1 was not mutagenic or cytotoxic up to 500 microM. When included with either 1 or 10 microM aflatoxin B1, coumarin produced a dose-dependent increase in mutant frequency and cytotoxicity. At concentrations greater than 50 microM, coumarin stimulated human liver S9 bioactivation of aflatoxin B1 to the mutagenic 8,9-epoxide. This increase was 12- and fivefold at 500 microM coumarin with 1 and 10 microM aflatoxin B1, respectively, compared with incubations with aflatoxin B1 alone. These findings differ from previous results with liver S9 from other species, and indicate that coumarin co-mutagenicity with aflatoxin B1 and human liver S9 is through increased aflatoxin B1 bioactivation.

Bleomycin is one of the radiomimetic antibiotics which induces DNA double-strand breaks by highly specific free radical attack on deoxyribose moieties in DNA. Earlier, we have shown that bleomycin induces a high proportion of large deletions involving one or more exons in the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in a Chinese hamster ovary (CHO) cell line CHO K1-BH4, in which no spontaneously occurring large deletions were detected by a polymerase chain reaction (PCR)-based deletion screening assay. Here we report the molecular nature of another class of mutants in which we did not observe any abnormal exon pattern. We refer to these mutants as the “nondeletion” type. Since bleomycin is a reactive oxygen species (ROS)-generating agent, we also studied whether the change of intracellular levels of ROS may affect the bleomycin-induced mutation spectra. We therefore also investigated the hprt mutation spectra induced by bleomycin with pretreatment by TRIEN (triethylenetetramine), a superoxide dismutase (SOD) inhibitor, and TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl), a SOD mimic. Analysis of these three bleomycin-induced “nondeletion” mutation spectra revealed that 5′-GTC-3′ or 5′-GCC-3′ sequences were the hot spots for single basepair deletions. Other types of mutation include abnormal cDNA or no cDNA amplification on the hprt locus. Due to the small sample size, we are unable to draw a definitive conclusion about the effects of TRIEN and TEMPOL on bleomycin-induced spectrum of “nondeletion” type hprt mutations. Environ. Mol. Mutagen. 32:244–250, 1998 © 1998 Wiley-Liss, Inc.

Coumarin (1,2-benzopyrone), a natural food constituent, prevents polycyclic aromatic hydrocarbon-induced neoplasms in rats and mice, but has not been studied with other chemical carcinogens. We examined coumarin chemoprotection against aflatoxin B1 using the 6-thioguanine resistance mutation assay in two different Chinese hamster ovary cell lines (K1BH4 and AS52) with liver S9 from rats and 19-day-old chick embryos for aflatoxin B1 bioactivation. Laboratory rodents metabolize coumarin through 3-hydroxylation, whereas 7-hydroxylation predominates in chick embryos and humans. Chick embryo liver S9 was approximately 25-fold more effective in activating aflatoxin B1 to the mutagenic and cytotoxic metabolite(s) than rat liver S9. Coumarin added at 50 and 500 microM with chick embryo liver S9 reduced the mutant frequency of 1 microM aflatoxin B1 by 40 and 85%, respectively. Coumarin up to 500 microM had no effect on aflatoxin B1 mutagenicity with rat liver S9. When liver S9 from chick embryos pretreated with coumarin was used for aflatoxin B1 bioactivation, mutant frequency and cytotoxicity were decreased compared to liver S9 from vehicle-treated controls. Liver S9 from coumarin-treated rats did not significantly affect mutant frequency or cytotoxicity. HPLC analysis of chick embryo liver S9 incubated with 1 microM aflatoxin B1 showed a dose-dependent decrease by coumarin of aflatoxin B1 activation to the 8,9-epoxide ranging from 70% of controls at 5 microM coumarin to 4% of controls at 500 microM coumarin. In contrast, coumarin produced a dose-dependent increase in 20 microM aflatoxin B1 activation by rat liver S9, reaching twice the control levels at 500 microM coumarin. These findings, using a mammalian cell system as a mutagenic endpoint, demonstrate marked species differences in chemoprotection by coumarin.

Conjugation and detoxification of mixed function oxidase (MFO)-mediated benzo(a)pyrene [B(a)P] metabolites with glucuronic acid and glutathione (GSH) are major pathways of B(a)P elimination and ultimately excretion in vivo. We have studied the effects of uridine diphosphate α-D-glucuronic acid (UDPGA) and GSH, a cofactor for the synthesis of glucuronide and GSH conjugates, respectively, on B(a)P-induced cytotoxicity and mutagenicity in mammalian cells. The S9-mix used in the Chinese hamster ovary cell/hypoxanthine-guanine phosphoribosyltransferase (CHO/HPRT) mutational assay was supplemented with either UDPGA, GSH, or GSH plus purified GSH-S-transferases (GSHTs), to study modulation of glucuronide and GSH detoxification mechanisms on B(a)P-induced cytotoxic and mutagenic effects. We found that the addition of UDPGA to S9-mix reduces cytotoxicity induced by either B(a)P or B(a)P 6-OH but not by B(a)P 7,8-diol [B(a)P-diol]. The reduction of B(a)P and B(a)P 6-OH-induced cytotoxicity by glucuronide conjugation is likely due to elimination of cytotoxic phenols and quinones. The addition of GSH to the S9-mix resulted in a reduction of B(a)P- and B(a)P-diol-induced cytotoxicity. GSH plus GSHT reduced B(a)P-induced cytotoxicity and mutagenicity. GSH inhibited the mutagenicity at low concentrations of B(a)P-diol. GSH plus GSHTs inhibited the cytotoxicity and mutagenicity of B(a)P-diol at concentrations not affected by GSH alone. These studies demonstrate that mechanisms of detoxification can affect the biological activity of B(a)P and B(a)P-diol as profoundly as bioactivation by the MFO system. Future research should address studies of mutagenicity modulation by metabolic effectors at both the molecular (DNA sequence) and cellular (quantitative mutagenesis) level.

Bleomycin-induced, 6-thioguanine-resistant, "non deletion" mutants pretreated with or without either TRIEN (triethylenetetramine), a superoxide dismutase (SOD) inhibitor, or TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl), a SOD mimic, were analyzed by polymerase chain reaction (PCR)-directed DNA sequencing in a Chinese hamster ovary (CHO) cell derivative, AS52. Among the 23 bleomycin-induced mutants, six have 3-bp 5'-TGA-3' deletions in the region of 366-371, five have single-base deletions, seven have base substitutions, three have insertions, and two have possible translocations. Among the 16 bleomycin-induced mutants pretreated with TRIEN, six have the 5'-TGA-3' deletion (366-371), two have single-base deletions, one has a 13-bp deletion, four have single-base substitutions, one has a double-base substitution, and two have insertions. Among the 17 bleomycin-induced mutants pretreated with TEMPOL, six have the same TGA deletions, two have single-base deletions, two have single-base insertions, four have single-base substitutions, one mutant has a 12-bp deletion, one has a 13-bp deletion, and one mutant shows no detectable change in its coding region in the DNA sequence. A possible shift from a ROS-mediated mutational spectrum to a spontaneous mutational spectrum by TRIEN further indicates that reactive oxygen species play an important role in bleomycin mutagenesis in mammalian cells.

We have investigated the roles of reactive oxygen species (ROS) in bleomycin (BLM)-induced gene mutations in Chinese hamster ovary (CHO) cells using a superoxide dismutase (SOD) inhibitor, triethylenetetramine (TRIEN), and a SOD mimic, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEM-POL), to lower and increase intracellular 'SOD activity', respectively. Pretreatment of CHO cells with TRIEN (1 mM) for 1 h enhanced the mutagenic response of BLM (5-50 micrograms/ml, 1 h treatment) in the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in CHO cell clone K1-BH4 (CHO/HPRT assay) and the xanthine-guanine phosphoribosyltransferase (gpt) gene in a CHO-K1 cell derivative AS52 (AS52/GPT assay). Pretreatment with TEMPOL (1 mM) for 1 h decreased the BLM (20-100 micrograms/ml, 1 h treatment) mutagenicity in the AS52/GPT assay. The mutagenic response of BLM appears to be modulated by the intracellular level of 'SOD activity' and hence the intracellular level of ROS. These data provide further evidence for the involvement of ROS in bleomycin mutagenesis in mammalian cells.

A series of ionic η5-monocyclopentadienyl–metal compounds possessing p-substituted benzonitrile ligands has been studied by hyper-Rayleigh scattering at the fundamental wavelength of 1.064 µm. Upon systematic variation of the metal ion, the first hyperpolarizability β was found to increase along the sequence Co, Ni, Ru, Fe, with about a three-fold increase from Ru to Fe. This yields very high values for the iron complexes, e.g., β=410×10–30 esu for [Fe(η5-C5H5)(dppe)( p-NCC6H4NO2)]+ [PF6 ]– dissolved in methanol. The high β values are attributed to π back-donation resulting in an extension of the conjugated π-system from the FeII organometallic fragment, acting as a good donor group, via the nitrile to the acceptor group NO2 . Complexes with single phenyl rings as conjugated chains perform better than their biphenyl analogues, which is explained in terms of non-planarity of the coordinated biphenyl ligands in solution. By comparing complexes with electron donor and acceptor substituted ligands it is demonstrated that the organometallic moiety can be used as an extremely effective donor group but not as a good acceptor group.

We have isolated independent Chinese hamster ovary (CHO) cell mutants at the hypoxanthine guanine phosphoribosyltransferase (hprt) locus from untreated, 60Co gamma-ray-exposed, and 212Bi alpha-exposed cells and identified the molecular changes underlying the mutation determined by multiplex polymerase chain reaction (PCR)-based exon deletion analysis. Both the parental CHO-K1 cells and the X-ray-sensitive mutant xrs-5 cells were studied. The radiosensitive xrs-5 cells are defective in DNA double-strand break rejoining ability and in V(D)J recombination, which can be complemented by Ku protein. Of the 71 spontaneous CHO-K1 hprt mutants analyzed, 78% showed no change in exon number or size, 20% showed loss of one to eight exons (partial deletion), and 3% showed loss of all nine hprt exons (total deletion). Exposure of CHO-K1 cells to 6 Gy of gamma rays, which reduced survival levels to 10%, produced a high deletion spectrum with 45% of the 20 mutants analyzed showing a loss of one to eight exons and 30% showing total deletion. Exposure to an equitoxic dose of alpha radiation from 212Bi, a 220Rn daughter, resulted in a spectrum similar to the gamma-ray spectrum in that 75% of the 49 mutants analyzed were deletions. To alpha radiation, however, tended to produce larger intragenic deletions than gamma radiation. Of the 92 spontaneous xrs-5 mutants analyzed for deletions, 43% showed a loss of one to eight exons and 14% showed total deletion. This suggests that, in certain regions of the hprt gene, base alterations can be converted into large deletions and alteration in the Ku protein complex can influence this type of mutational process. Exposure to alpha radiation (10% survival) to xrs-5 cells resulted in a deletion spectrum similar to that seen in CHO-K1 cells. Of the 49 mutants analyzed, 43% showed on change in exon number or size, 16% showed a loss of one to eight exons, and 41% showed total deletion. While the defect in xrs-5 cells has a profound effect on spontaneous mutant spectra, this defect does not appear to affect alpha-induced mutation spectra.

The radiosensitive mutant xrs-5, a derivative of the Chinese hamster ovary (CHO) K1 cell, is defective in DNA double-strand break rejoining ability and in V(D)J recombination. The radiosensitivity and defective repair phenotype are complemented by the 80-kDa subunit of the Ku protein. We determined the nature of the mutations that develop spontaneously at the hprt locus in this cell line using both multiplex PCR deletion screening and DNA sequencing. Ninety-two independent spontaneous mutants were analyzed and the results were compared to the mutation spectrum of 64 previously analyzed hprt spontaneous mutants isolated from the parental CHO-K1 cell line. More than 50% of the spontaneous xrs-5 mutants had lost one or more exons while less than 25% of spontaneous CHO-K1 mutants had lost one or more exons. Most of the deletions in xrs-5 cells involved the loss of multiple exons while single exon deletions predominated in CHO-K1. There was also a nonrandom distribution of breakpoints in both CHO-K1 and xrs-5. Most of the deletion breakpoints were 3' to exon 9, around exons 4-6, or near exon 1. Although the frequency of base substitutions was lower in xrs-5, the spectrum of base substitutions was qualitatively similar to that of CHO-K1. There was no significant difference in the spontaneous mutant frequency in xrs-5 and CHO-K1. The results suggest that in certain regions of the hprt gene, base alterations can be converted to large deletions, and that alterations in the Ku protein complex can influence this process.

Bleomycin-induced 6-thioguanine-resistant mutants pretreated with or without TRIEN (triethylene-tetramine), a superoxide dismutase (SOD) inhibitor, or TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl), an SOD mimic, were analyzed by polymerase chain reaction (PCR)-based deletion screening in a Chinese hamster ovary (CHO) clone K1-BH4 and its derivative AS52 cells. As we proposed earlier, TRIEN would decrease and TEMPOL would increase the intracellular level of hydroxyl radical leading to a higher and lower recovery of deletion mutants. We found that the proportion of the deletion mutants induced by bleomycin at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in K1-BH4 cells was 45.5% ( 25 55 ). The proportion of deletion HPRT− mutants induced by bleomycin pretreated with TRIEN was 31.0% ( 9 29 ) and with TEMPOL was 50.0% ( 14 28 ). The proportion of deletion mutants induced by bleomycin on the xanthine-guanine phosphoribosyltransferase (gpt) gene in AS52 cells was 61.0% ( 36 59 ). The proportion of deletion GPT− mutants induced by bleomycin pretreated with TRIEN was 56.8% ( 21 37 ) and with TEMPOL was 61.4% ( 27 44 ). The trend of the change of the proportion of bleomycin-induced deletion mutants as affected by TRIEN and by TEMPOL provides molecular evidence for the involvement of reactive oxygen species (ROS) in bleomycin mutagenesis in mammalian cells, in which deletion is a major type of induced mutation.

2-Methoxyethanol (ethylene glycol monomethyl ether) (EGME), is one of the most commonly used solvents for industrial and consumer products. Although the solvent has been shown to be a reproductive toxin the genotoxic activities of EGME especially its metabolites, have not been adequately investigated. The mutagenicity and cytotoxicity of EGME and its major metabolites, methoxyacetaldehyde (MALD) and methoxyacetic acid (MAA) in Chinese hamster ovary (CHO) cells were therefore examined by us. We have determined the mutagenicity of these compounds at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in CHO-K1-BH4 cells (CHO/HPRT assay) and the xanthine-guanine phosphoribosyl transferase (gpt) locus in CHO AS52 cells (AS52/GPT assay). The results show that these chemicals are not mutagenic to the hprt locus in CHO-K1-BH4 cells either with or without rat liver S9 mix as the metabolic activating system. With AS52 cells, only MALD is mutagenic in the absence of S9. It induced a dose-dependent mutagenic response. A dose-dependent cytotoxicity was induced by all compounds in both cell lines. MALD is the most and EGME is the least cytotoxic compounds. Our study shows that a metabolite of EGME, MALD, is highly cytotoxic and likely induces deletion-type mutations in AS52 cells. The genotoxic effect of EGME is, therefore, dependent upon its metabolism and its detection is dependent upon the assays used.

We have studied the mutagenicity and toxicity of physical and chemical agents in the Chinese hamster ovary (CHO) cell line K1-BH4 and its transformant, AS52. The AS52 cells lack the normal X-linked mammalian hypoxanthine-guanine phosphoribosyltransferase (hprt) gene but instead contain a single autosomally integrated copy of the bacterial equivalent, the xanthine-guanine phosphoribosyltransferase (gpt) gene. We found that X-rays and neutrons appear to be equitoxic to both cell types; however, these physical agents are approximately 10 times more mutagenic to the gpt gene of AS52 cells than to the hprt gene of K1-BH4 cells. We reasoned that if reactive oxygens were to mediate the mutagenic effects of both radiomimetic chemicals and radiation, then reactive oxygen-producing chemicals, such as streptonigrin and bleomycin, and oxidizing agents such as potassium superoxide and hydrogen peroxide, would exhibit similar levels of toxicity but different frequencies of mutants when assayed with the two cell lines. Our experiments fulfill such predictions. We postulate that the apparent hypermutability of AS52 cells probably results from a higher recovery of multi-locus deletion mutants in AS52 cells than in K1-BH4 cells, rather than a higher yield of induced mutants. Preliminary studies, using Southern blot and the polymerase chain reaction to analyze the mutational spectrum of the mutants, support our hypothesis that reactive oxygens induce deletion mutations in mammalian cells.

It has been suggested that the more closely spaced, clustered DNA breaks seen with high-LET radiations are more likely to result in chromosome intrachanges than in chromosome interchanges. We determined whether analysis of the spectra of chromosome aberrations or Hprt gene mutations in CHO-K1 cells could detect a shift to more chromosome intrachanges and therefore distinguish exposure to high-LET radiation from exposure to low-LET radiation, and whether alterations in the processing of DNA breaks would influence this process. Both the frequency and type of chromosome aberrations and Hprt gene mutations were determined in CHO-K1 and xrs-5 cells exposed to 60 Co γ rays or 212 Bi α particles. Xrs-5 cells are a radiosensitive derivative of CHO-K1 cells that are defective in rejoining of DNA double-strand breaks. The ratio of dicentrics to centric rings (F ratio) was significantly lower in CHO-K1 and xrs-5 cells exposed to α particles, consistent with a shift to more chromosome intrachanges with higher LET. In contrast, the frequency of large interstitial deletions at the Hprt locus, determined by multiplex polymerase chain reaction-based exon deletion analysis, was the same for both γ-ray- and α-particle-exposed cells in each of the cell lines. Thus the F ratio can distinguish high-LET from low-LET radiations, and the end point is not influenced by differences in the processing of DNA double-strand breaks. The analysis of the spectrum of Hprt mutations, however, appears unable to discriminate low LET from high LET.

When ethylene dibromide (EtBr2) was assayed with the Chinese hamster ovary/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) system coupled with a rat liver metabolic activation system (S9), which contains Ca2+ (Ca, Mg-S9), the cytotoxicity of EtBr2 was greatly increased over that obtained when NADP was omitted from the Ca, Mg-S9 or when EtBr2 was assayed as a direct-acting agent. However, on a molar basis, the mutagenicity of EtBr2 remained unaffected. The omission of Ca2+ from the Ca, Mg-S9 metabolic activation system (Mg-S9), with either the addition or omission of NADP, caused approximately a 2-fold decrease in the mutagenicity of EtBr2 when compared to the results obtained by using the Ca, Mg-S9 system. The cytotoxicity of EtBr2 was further increased when a purified microsomal fraction, prepared from the S9 fraction, was used in the presence of Ca2+. In the absence of this calcium ion, this metabolic activation system was extremely cytotoxic to Chinese hamster ovary cells even without the presence of a mutagen or promutagen. The cytotoxicity of EtBr2 in the following assay systems decreased in this order: Ca, Mg-microsomes greater than Ca, Mg-S9 greater than S9 greater than direct-acting agent greater than or equal to Ca, Mg-S9 without NADP greater than or equal to Mg-S9 without NADP. Cytotoxicity appears to be NADP-dependent on the presence of NADP in the S9 system, the mutant yield (number of mutants that could be induced) was higher in its absence. Addition of reduced glutathione to Mg-S9 without NADP increased the mutagenicity of EtBr2 to values that did not exceed those obtained when EtBr2 was tested as a direct-acting agent. On a molar basis, ethyl methanesulfonate (EMS) is less cytotoxic but equally as mutagenic as EtBr2. However the mutant yield of EMS was higher than that of EtBr. Inclusion of Ca, Mg-S9 in the assay system had no effect on the biological activities of EMS.

Biologically reactive metabolites of benzo[ a ]pyrene (BP)and benzo[ a ]-pyrene 7,8-diol (BP-diol), formed by the mixed-function oxidase (MFO) system, are substrates for conjugation and detoxication by glutathione (GSH) when catalyzed by glutathione S -transferases (GSHT). We have investigated the detoxication of BP- and BP-diol-induced cytotoxicity and mutagenicity with GSH by supplementing the S9 mix used in the Chinese hamster ovary cells/hypoxanthine-guanine phosphoribosyltransferase (CHO/HGPRT) assay with GSH (6.5 mM) of GSH plus GSHT. The addition of GSH to the S9 mix resulted in a reduction of BP- and BP-diol induced cytotoxicity. GSH plus GSHT eliminated BP-induced cytotoxicity and reduced the mutagenicity of BP. GSH inhibited the mutagenicity at low (essentially non-lethal) concentrations of BP-diol, but did not do so at toxic concentrations. GSH plus GSHT inhibited the cytotoxicity and mutagenicity of BP-diol at concentrations not affected by GSH alone. These studies indicate that biochemical mechanisms of detoxication can affect the biological activity of a carcinogen, such as BP or BP-diol as profoundly as bioactivation by the MFO system.

The frequency of ethyl methanesulfonate (EMS)-induced mutations to 6-thioguanine resistance in a Chinese hamster ovary cells clone K1-BH4 was studied at many EMS doses including the minimally lethal range (0-100 microng/ml) as well as the exponential killing portion (100-800 microng/ml) of the survival curve. The mutation frequency increases approximately in proportion with increasing EMS concentration at a fixed treatment time. The pooled data for the observed mutation frequency, f(X), as a function of EMS dose X, is adequately described by a linear function f(X)=10(-6)(8.73+3.45 X), where 0 less than or equal to X less than or equal to 800 microng/ml. One interpretation of the linear dose-response is that, as a result of EMS treatment, ethylation of cellular constituents occurs, which is directly responsible for the mutation. Biochemical analyses demonstrate that most of the randomly isolated 6-thioguanine-resistant variants possess a highly reduced or undetectable level of HGPRT activity suggesting that the EMS-induced mutations to 6-thioguanine resistance affect primarily, if not exclusively, the HGPRT locus.

Ethylene oxide (EO) is made on an industrial scale (2.5 billion pounds per year) and is used not only to prepare a number of commercially important compounds but also as a widely used fumigant to sterilize foodstuffs, textiles, and medical instruments. EO is an alkylating agent that has been shown to interact with proteins and nucleic acids and is mutagenic in a number of test systems, eg, rodents, bacteria, rice, and Drosophila. Reported is the cytotoxicity and mutagenicity of EO in a mammalian cel culture system, the Chinese hamster ovary cell/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) system.

Preparative chromatography of protein mixtures was carried out by tandem separation schemes involving frontal chromatography followed by stepwise desorption or displacement. In this way, with columns and instruments generally employed in analytical high-performance liquid chromatography, proteins could be purified in quantities similar to those typically separated by a preparative-scale system. A mixture of β-lactoglobulin A and B was loaded onto an anion-exchange column, and, in the process, a large fraction of the less-retained β-lactoglobulin B was recovered in pure form. The column was then flushed with the carrier, and subsequent desorption of the substances bound on the stationary phase was carried out by single-step desorption, two-step desorption, or displacement. With this mixture, the last two methods yielded approximately the same results in terms of the amount of product obtained per unit column volume. Whereas stepwise desorption is a simpler technique than displacement, the latter is required for the separation of components having similar adsorption behavior. In another set of experiments, a protein mixture obtained by heat treatment of human growth hormone was fractionated on a reversed-phase column. After loading the column by frontal chromatography, which separated a large fraction of the main product from the other components retained by the column, four desorption steps were applied to recover the individual components. These separation schemes offer an approach to preparative chromatography of proteins that is superior to conventional linear elution in terms of column load capacity, low mobile phase consumption, simultaneous separation and concentration, as well as enrichment of trace components.

Incubation of Chinese hamster ovary (CHO) cells with N6,O2′-dibutyryl cyclic adenosine 3′,5′-phosphate (db-cAMP) causes the epithelial-like cells to assume a fibroblast-like morphology. We have analysed this morphological conversion in cells synchronized by the mitotic shake-off method. Here, we report that only cells in the G I phase of the cell cycle initiate elongation when incubated with db-cAMP.

Host-mediated assays have been developed to allow determination of the mutagenic potential of promutagens and procarcinogens which require metabolic activation to exert their effects on indicator organisms. We report here the development of the host-(mouse)-mediated CHO/HGPRT system using the procarcinogen dimethylnitrosamine (DMN) as a model agent. Using a 2-h treatment time, we observed a linear dose—response relationship up to 250 mg of DMN per kg body weight. At 100 and 500 mg/kg DMN, mutation induction increased with time up to at least 6 h. DMN was not mutagenic when tested in vitro. Athymic (nude) mice, their phenotypically normal littermates, or BALB/c mice of both sexes were found to be suitable as hosts. A time- and dose-dependency of induced mutation frequency by a direct-acting agent, ethyl methanesulfonate (EMS), was observed in both the in vitro and the host-mediated assays.

An assay is described for the measurement of mutation induction at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells utilizing resistance to 6-thioguanine (TG). Optimal selection conditions are defined for such parameters as phenotypic expression time prior to selection, and TG concentration and cell density which permits maximum mutant recovery. The nature of the TG-resistant mutants is characterized by several physiological and biochemical methods. The data demonstrate that more than 98% of the mutant clones isolated by this selection procedure contain altered HGPRTase activity. The CHO/HGPRT system thus shows the specificity necessary for a specific gene locus mutational assay.

WI-38 lung diploid fibroblasts repond to protaglandin E1 with increased levels of cyclic adenosine 3′:5′-monophosphate. This increase is affected by cell density in two ways: (a) The initial rate of accumulation of intracellular cyclic AMP increases with increasing cell density. (b) However, the elevated levels of cyclic AMP are more stably maintained in lower-density cells, and this stability decreases with increasing cell density. Cyclic AMP phosphodiesterase activities, as well as the efflux of intracellular cyclic AMP into the medium are simelar at all cell densities.

Monolayer cell cultures grown on coverslips in the presence of radioactive lipid precursors were embedded in silica gel layers for extraction and resolution of the labelled products directly by thin-layer chromatography. The method permits rapid screening of lipid metabolism in tissue cultures with a small number of cells.

About 40 t of chopped sorghum forage (silage variety) was treated with 4% (by weight dry) sodium hydroxide at ensiling in a plastic bag. Another 40 t of the forage was ensiled without treatment in the same bag. Early in fermentation pH was higher in treated forage, but by day 21 pH was about 4.0, and amounts of acid were similar in treated and control silage. In a feeding trial eight cows receiving treated silage after 6-wk ensiling produced 21.2kg milk with 2.83% fat; eight cows fed control silage produced 19.9kg milk containing 2.77% fat. Mean in vitro dry matter disappearance was 58.8 and 52.1% for weekly samples of treated and control silage. Freeze-dried samples of both treated and control silages were treated with an additional 4% sodium hydroxide and 1% nitrogen from urea (by weight dry) in a 2×2 factorial arrangement. Laboratory treatment with alkali increased in vitro dry matter disappearance of dried sorghum silage and interacted significantly with nitrogen supplementation. Apparently treatment of wet sorghum forage with 4% dry sodium hydroxide produced a satisfactory feed but did not achieve maximum improvement in in vitro dry matter disappearance of the silage.

Summary Chinese hamster ovary (CHO) cells in culture were utilized to determine the cytotoxicity, specific-locus mutation induction, and DNA alkylation which result from treatment of the cells with a range of concentrations of N-methyl-N-nitrosourea (MNU) or N-ethyl-N-nitrosourea (ENU). With [3H]MNU over the concentration range 0.43–13.7 mM, methylation of DNA was found to increase linearly, with a mean value of 56.7 pmol residue per μmol nucleoside per mM. With [1-3H]ENU over the concentration range 1.7–26.8 mM, ethylation was linear, with a mean value of 3.8 pmol residue per μmol nucleotide per mM. Mutation induction at the hypoxanthineguanine phosphoribosyl transferase (HGPRT) locus was quantified by determination of the frequency of resistance to 6-thioguanine under stringentlydefined selection conditions. The mutation frequency increased linearly with MNU or ENU concentration (0.01–2.0 mM); mean values were 2800 and 840 mutants per 106 clonable cells per mM, respectively. At equal levels of DNA alkylation, ENU was found to be approx. 4.5 times as mutagenic as MNU.

We have studied the relationship between DNA adducts in Chinese hamster ovary (CHO) cells and mutagenicity as determined in the CHO/hypoxanthine-guanine phosphoribosyltransferase assay. The cells were treated with benzo(a)pyrene 7,8-diol (BP-diol) in the presence of a bioactivation system, S9 mix. DNA binding by bioactivation of BP-diol with S9 mix occurred with both stereoisomers of benzo(a)pyrene diol-epoxide (BPDE) in approximately equal amounts. The number of BPDE-DNA adducts (21–260 adducts/10 6 nucleotide base pairs) increased with increasing treatment concentrations of BP-diol (1.4–7.0 μ m ). A linear relationship was observed between the number of BPDE-DNA adducts and mutagenicity (89–605 mutants/10 6 cloneable cells) over the concentration range of BP-diol assayed.

We have developed a system to study mutations that affect xanthine-guanine phosphoribosyltransferase gene (gpt) expression in hypoxanthine-guanine phosphoribosyltransferase-deficient CHO cells that have been transformed by the plasmid vector pSV2gpt. One isolated transformant, designated AS52, carries a single copy of the Escherichia coli gpt gene stably integrated into the high-molecular-weight DNA and expresses the bacterial gene for the enzyme xanthine-guanine phosphoribosyltransferase. Mutants deficient in this enzyme can be induced in the AS52 cell line by a variety of mutagens, and spontaneous or induced mutants can be selected for resistance to 6-thioguanine (Tgr). Two Tgr clones derived from the AS52 line were analyzed by Southern blot hybridization and were found to contain deletions involving at least a portion of the gpt gene. Because of the small size and stability of the integrated pSV2gpt plasmid, and the well-defined selection protocol for mutant isolation, the AS52 line offers promise as a system suitable for the study of mutation at the molecular level in CHO cells.

When CaCl 2 was added in increasing concentrations to a rat liver metabolic activation system (S9) buffered with sodium phosphate, the mutagenic activity and cytotoxicity of dimethylnitrosamine (DMN) in the Chinese hamster ovary cell/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) system were greatly increased. This effect was not observed with an S9 mix buffered with N -2-hydroxyethylpiperazine- N ′-2-ethanesulfonic acid (HEPES). The calcium phosphate gel precipitate of the S9 mix possessed approximately built1 3 of the total activity of the mix, while the supernatant had only slight activity. However, when the calcium phosphate gel precipitate of a solution of S9 salts (without S9 protein) was added to the supernatant, the remaining 2 3 of the activity was recovered. Commercially obtained calcium phosphate, tricalcium phosphate, and alumina C γ gels could substitute for CaCl 2 in the S9 mix, but diethylaminoethyl cellulose (DEAE cellulose) could not. Alumina C γ gel can exert its effect in the absence of both CaCl 2 and phosphate in the S9 mix. Increasing the time of contact between the S9 protein and the S9 salts increased the efficacy with which the S9 mix activated DMN; this is indicative of an adsorptive process by calcium phosphate gel.

The genotoxic activities of 63, 2-nitronaphthofurans and related molecules were examined using two bacterial short-term tests, the Salmonella mammalian microsome assay test or Mutatest, a mutagenesis assay, and/or the SOS Chromotest, an assay for induction of an SOS function in Escherichia coli. Seven compounds were also investigated in the Chinese hamster ovary cells/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) test, a mammalian gene mutation assay. Our main conclusions are the following: (a) Simple empirical rules relating structure to mutagenic activity in the Mutatest can be derived for some of the compounds. In particular, they account for the extremely high Mutagenic Potency of 7-methoxy-1-methyl-2-nitronaphtho[2,1-b]furan (R7372), approximately 2 X 10(6) mutants/nmol on strain TA100. (b) There is a good quantitative correlation between the Mutagenic Potency in the Salmonella/mammalian microsomes assay and the SOS-inducing potency in the SOS Chromotest. This, and previous evidence, suggests strongly that the 2-nitronaphthofurans derivatives are essentially recA and thus probably umuDC-dependent mutagens. (c) Four out of seven compounds tested in the CHO/HGPRT assay gave responses correlated with the bacterial responses. One of them, 7-methoxy-2-nitronaphtho[2,1-b]furan (R7000), is among, or is, the strongest mutagen described for mammalian cells. We briefly discuss the practical and theoretical implications of these results.

A pSV2gpt-transformed Chinese hamster ovary (CHO) cell line has been used to study mutation at the molecular level. This cell line, designated AS52, was constructed from a hypoxanthine-guanine phosphoribosyl transferase (HPRT)-deficient CHO cell line, and has been previously shown to contain a single, functional copy of the E. coli xanthine-guanine phosphoribosyl transferase (XPRT) gene (gpt) stably integrated into the Chinese hamster genome. In this study, conditions for its use in the study of mammalian cell mutagenesis have been stringently defined. The spontaneous mutation rate (2 X 10(-6)/cell division) and phenotypic expression time (7 days) of the gpt locus compare favorably with those of the hprt locus in wild-type CHO-K1-BH4 cells. While both cell lines exhibit similar cytotoxic responses to ethyl methanesulfonate (EMSO and ICR 191, significant differences in mutation induction were observed. Ratios of XPRT to HPRT mutants induced per unit dose of EMS and ICR 191 are 0.70 and 1.6, respectively. Southern blot hybridization analyses revealed that most XPRT mutant cell lines which arose following treatment with EMS (20/22) or ICR 191 (20/24) exhibited no alterations of the gpt locus detectable by this technique. Similar observations were made for the hprt locus in EMS-(21/21) and ICR 191-induced (22/22) HPRT mutants. In contrast, most spontaneous gpt mutants (14/23) contained deletions, while most spontaneous hprt mutants (18/23) exhibited no detectable alterations. Results of this study indicate that the AS52 cell line promises to be useful for future study of mutation in mammalian cells at the DNA sequence level.

Cyclic nucleotide analogues have been tested for their ability to cause the morphological conversion of Chinese hamster ovary cells in culture, as well as for effects on cyclic AMP-related enzymes. The ability of the analogues to inhibit the cyclic AMP phosphodiesterase activity and to activate the cyclic AMP-dependent protein kinase activity in cell extracts has been measured. Cell cultures were incubated with the analogues and the effects on morphology, intracellular level of cyclic AMP, and in vivo protein kinase activation were determined. All analogues which induced the morphological conversion also caused in vivo activation of the cyclic AMP-dependent protein kinase. Only N6,O2′-dibutryl and N6-monobutyryl cyclic AMp caused caused on increase in intracellular cyclic AMP, presumably through inhibition of the intracellular cyclic AMP phosphodiesterase activity. The increase in cyclic AMP appears to cause the protein kinase activation. However, analogues such as 8-bromo and 8-benzylthio cyclic AMP do not cause any change in intracellular cyclic AMP level and appear to activate the intracellular cyclic AMP-dependent protein kinase directly.

Thirteen continuous lines of Syrian hamster epidermal cells were isolated following direct treatment of epidermal cells with N-methyl-N′-nitro-N-nitrosoguanidine, or from epidermal cells cultured from animals treated transplacentally with 7,12-dimethylbenz(a)anthracene or dimethylnitrosamine. The epidermal origin of the lines was evidenced by the presence of desmosomes with radiating tonofilaments, keratin fibers, and the ability of the cells to use citrulline in place of arginine corresponding with their high argininosuccinate synthetase activity.

We have studied formation and stability of alkylguanines following treatment of Chinese hamster ovary cells with either N-[ 3 H ] methyl -N- nitrosourea (MeNOUr) (applied at 50 ωM and 40 ωM concentrations) or N-[ 3 H ] ethyl -N- nitrosourea (EtNOUr) (applied at 43.1 ωM). Analyses of acid hydrolysates of the methylated DNA revealed that 9.3% and 57.0% of the total DNA were O 6 -methylguanine (m 6 Gua) and 7-methylguanine (m 7 Gua), respectively. Analysis of enzymic hydrolysate resulted in 8.2% m 6 Gua and 50.3% m 7 Gua. For ethylation, the % of ethylated purines identified as O 6 -ethylguanine (e 6 Gua) and 7-ethylguanine (e 7 Gua) were 20.4% and 31.3%, respectively. Half-lives of the main alkylated purines were determined by analysing DNA of dividing cultures over a time interval of 48 h after treatment with carcinogens. Half-lives measured for methylated DNA bases were: m 1 Ade, 20.6 h; m 3 Ade, 25.5 h; m 7 Ade, 0.9 h: m 3 Gua, 1.1 h; m 6 Gua, ∞; m 7 Gua, 39.1 h. Determinations at the level of deoxyribonucleosides resulted in similar half-lives: m 3 dA, 15.2 h; m 7 dA, 2.7 h; m 3 dG, 2.3 h; m 6 dG, 224 h; m 7 dG, 25.6 h. The corresponding values for ethylated purines were: e 3 Ade, 2.9 h; e 7 Ade, 7.1 h; e 3 Gua, 1.4 h; e 6 Gua, ∞; e 7 Gua, 42.6 h. The relatively high yields of the premutagenic m 6 Gua and e 6 Gua, and their long half-lives (≥224 h) are consistent with the suggestion that these adducts play a dominant role in mutation induction at the hypoxanthine-guanine phosphoribosyltransferase ( hgprt ) locus in CHO cells.

The induction of mutation by certain nitrosamidines and nitrosamides has been quantitated utilizing the hypoxanthine--guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary cells. Dose--response relationships for cytotoxicity and mutagenicity are presented for N-methyl-N-nitrosourea (MNU), N-ethyl-N-nitrosourea (ENU), N-butyl-N-nitrosourea (BNU), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG). Based on the concentration of each agent required to kill 90% of the cells, the following order of cytotoxicity was observed: MNNG greater than ENNG greater than MNU greater than ENU greater than BNU. This is the same order of potency as observed for mutation induction per unit concentration of mutagen.

The first manifestation of reverse transformation, in which a permanent epithelial-like cell is reversibly transformed into a fibroblast-like cell by dibutyryl adenosine cyclic 3′:5′-monophosphate, is the disappearance of a set of violently extending and retracting knobbed structures on the cell membrane with consequent tranquilization of the membrane. The cell then elongates to assume a fibroblast-like morphology. Permanently fibroblast-like cells, which have smooth membranes, can be changed into knobbed epithelial-like cells by the addition of either colcemid or cytochalasin B, agents that disorganize one or the other component, respectively, of the microtubular-microfibrillar system. Permanently smoothmembraned, epithelial-like cells also become knobbed by such treatment. All of these knobbed epithelial-like cells can be converted into smooth-membraned fibroblast-like cells by dibutyryl cAMP alone or with the aid of supporting molecules like testosterone. It is proposed that these are general phenomena and that the knobbed epithelial-like forms and the more highly differentiated smooth-membraned fibroblast-like cells represent extreme situations with respect to the degree of organization of the microtubular-microfibrillar system inside the cell.

Vapor pressure composition data for liquid ozone‐oxygen mixtures were obtained at —195.5°, —183°, and —180°C. The vapor pressure isotherms were used to construct the boiling point‐composition line at one‐atmos pressure. The vapor pressure of pure liquid ozone defines an upper limit for the partial pressure of ozone in the vapor in equilibrium with boiling liquid.The vapor pressure of ozone‐oxygen solutions deviates markedly from Raoult's law in a positive direction. It is suggested that the apparent boiling point maximum reported by Schumacher, which implies negative deviations, may be due to difficulties in analysis of ozone at high concentrations where it is subject to decomposition at ordinary temperatures.Liquid‐liquid equilibrium data are reported which indicate —180°C as an upper bound for the consolute temperature. The region of two liquid phases extends from 9 to 91 percent ozone by weight at —195.5°C, 29.8 to 72.4 percent at —183°.

The data needed for analysis of liquid ozone‐oxygen mixtures by measurement of magnetic susceptibility are presented. Volumetric preparation of liquid ozone‐oxygen mixtures in equilibrium with the vapor phase is described. A permanent magnet at the temperature of the solutions provided the key for use of the Quincke (U‐tube) method for magnetic susceptibility.Results at — 195° and — 183°C show a linear relation between the magnetic susceptibility of the solutions and weight percent oxygen at low oxygen content. Above about 10 percent oxygen the specific susceptibility of oxygen in solution decreases significantly. At — 183°C, the ratio X8/X0 of specific susceptibility of oxygen in solution to that of pure liquid oxygen decreased from 1.30 at low concentrations to 1.13 at 27.6 percent oxygen, the two‐liquid phase boundary. The limiting ratio at — 195°C was 1.42. The discontinuity in magnetic susceptibility was used to establish the compositions at the phase boundaries; 9.2 and 91 percent by weight oxygen a...

Serine dehydratase formation was induced by cyclic 3',5'-AMP and not by other cyclic nucleotides or adenosine monophosphates. A single injection of cyclic 3',5'-AMP (or its dibutyryl derivative) increased the rate of incorporation of 3H-leucine into serine dehydratase. Under the same experimental conditions the rate of synthesis of serum albumin did not change. A single administration of either glucagon or a mixture of amino acids (also an inducer of serine dehydratase) led, within 20 min, to a 40-fold and a 19-fold increase, respectively, in the concentration of cyclic 3',5'-AMP. The concentration of this nucleotide dropped to the original level within 1 hour after the beginning of the inducer administration. The induction of serine dehydratase by N6,O2'-dibutyryl cyclic 3',5'-AMP was fully inhibited by actinomycin D only if the drug was injected 30 to 15 min prior to the injection of the cyclic nucleotide. The uptake of 3H-dibutyryl cyclic 3',5'-AMP by the liver was not inhibited by actinomycin D.

TREATMENT of cultured mammalian cells with cytochalasin B, a metabolite isolated from the mould Helminthosporium dematioideum, yields a population of enucleated cells1–3. Such enucleates are excellent experimental material for the study of nucleus-cytoplasm interactions as certain normal physiological characteristics of the intact cells can be preserved. Indeed, it has been reported that baby hamster kidney cells (BHK-21) and African green monkey kidney cells (BSC-1) enucleated with cytochalasin retain such properties of their intact counterparts as cell shape formation, cell locomotion and contact inhibition4. Furthermore, replication of vaccinia virus5 and polio virus6 can occur in enucleates from mouse L cells and BSC-1 cells, respectively.

As science and technology advance, an extraordinary-quantity of natural and synthetic chemicals is introduced continuously into our environment. Through conventional animal tests, some of these environmental chemicals have been found to be either highly toxic, mutagenic, carcinogenic, or teratogenic. Epidemiological studies have shown that among these harmful chemicals, a few also exhibit such detrimental effects in the human population. Because of the high cost and length of time required for the animal experiments, such tests have been confined to only a very small fraction of these environmental agents. Thus, the biological effects of the great majority of these chemicals, including ingredients of our daily foods and drugs, remain either incompletely tested or unknown.

The CHO/HGPRT system has been developed and defined for quantifying mutation induced by various physical and chemical agents at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells. Various genetic, biochemical, and physiological evidence supports the genetic basis of mutation induction in this system.