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T G Rossman

Publications and source records attributed to T G Rossman.

At least 37 records · Page 2Linked to original sources
PubMed1994

Induction of arsenite tolerance and thermotolerance by arsenite occur by different mechanisms.

Both V79 and As/R28A cells (an arsenite-resistant Chinese hamster V79 cell variant) show increased resistance to toxic concentrations of arsenite after pretreatment with a nontoxic concentration. The induced tolerance can be completely inhibited by actinomycin D or cycloheximide. Pretreatment with a nontoxic heat shock (45 degrees C, 10 min) resulted in a clear increased thermotolerance in both cell lines but failed to induce arsenite tolerance in either cell line. Pretreatment with arsenite induced a thermotolerance in V79 cells but not in As/R28A cells. These results are consistent with a model whereby the signal for induction of arsenite tolerance involves binding of arsenite to a protein effector which is amplified in the As/R28A line, thereby preventing action of arsenite in the regulation of heat shock factor which induces the heat shock response.

Animals↗
PubMed1994

Large-scale supercoiled plasmid preparation by acidic phenol extraction.

A novel method for large-scale plasmid preparation is described. Crude extracts are subjected to acidic phenol extraction to remove any contaminants present in the aqueous phase. The supercoiled plasmid DNA, which preferentially remains in the organic phase and inter-phase, is extracted back into the aqueous phase with 1.5 M TRIZMA base, from which it is precipitated. The resultant plasmid DNA is highly pure and satisfactory for any subsequent procedures. The method is extremely economical and takes only 3-4 h.

Centrifugation↗
PubMed1993

Mutagenicity of soluble and insoluble nickel compounds at the gpt locus in G12 Chinese hamster cells.

Nickel is an established human and animal carcinogen, but efforts to demonstrate its mutagenicity in a number of cell types have not been successful. In this report we describe the mutational response to nickel compounds in the G12 cell line, an hprt deficient V79 cell line containing a single copy of the E. coli gpt gene. This cell line has a low spontaneous background, making it suitable for assessment of mutagenic responses to environmental contaminants. When G12 cells were treated with insoluble particles of crystalline nickel sulfide < 5 microns in diameter, a strong, dose-dependent mutagenic response was observed up to 80 times the spontaneous background. Of 48 mutant gpt(-) clones isolated that were induced by insoluble nickel, all were capable of DNA amplification of the gpt sequences by polymerase chain reaction (PCR). The ability to produce full-length PCR products is an indication that large deletions of gene sequences have not occurred. When G12 cells were treated with soluble nickel sulfate, the mutational response was not significantly increased over the spontaneous background. This difference in mutagenic response reflects a large difference in the mutagenic potential of soluble and insoluble nickel compounds, which reflects the carcinogenic potential of these forms of nickel.

Animals↗
PubMed1993

Stable and inducible arsenite resistance in Chinese hamster cells.

A number of Chinese hamster V79 cell sublines that are arsenite resistant or arsenite sensitive were isolated. After more than 6 months of growth in the absence of arsenite, these sublines still maintain their arsenite-resistant or -sensitive phenotypes. At least some arsenite-resistant cell lines are also cross-resistant to sodium arsenate and potassium antimonyl tartrate. Wild-type or arsenite-resistant sublines show a further increase in resistance to toxic concentrations of arsenite after pretreatment with a nontoxic concentration. Pretreatment of cells with a nontoxic dose of arsenite also increased their survival to toxic doses of antimonite. Likewise, pretreatment with antimonite increased survival to arsenite as well as to antimonite. The inducible arsenite resistance increases with pretreatment time, reaching a plateau after 8 hr of pretreatment. Fusion of arsenite-resistant cells with arsenite-sensitive cells demonstrated a clear dominance of arsenite resistance. These results suggest that mammalian cells contain an arsenite/antimonite pump whose activity may be modulated by prior exposure to arsenite or antimonite.

Animals↗
PubMed1992

Differential susceptibility to carcinogen-induced amplification of SV40 and dhfr sequences in SV40-transformed human keratinocytes.

Gene amplification contributes to carcinogenesis by enhancing proto-oncogene activity and causing chromosomal instability. The ease of detecting amplified tumor-virus sequences has encouraged use of this system as a surrogate for studying the molecular events involved in endogenous gene amplification. We report here a new system for studying carcinogen-induced amplification of both endogenous and viral sequences in the SV40-transformed human keratinocyte line AG06. Treatment with carcinogens induced a transient dose-dependent amplification of the integrated SV40 sequences. The amplified sequences appeared in the extrachromosomal fraction. Treatment of these cells with carcinogens prior to methotrexate (MTX) selection increased the frequency of MTX-resistant colonies, 67% of which exhibited dihydrofolate reductase (dhfr) amplification. The abilities of five carcinogens with different DNA-damaging activities (the DNA-damaging agents N-methyl-N-nitro-N-nitrosoguanidine, mitomycin C (MMC), ultraviolet light C, and X-rays and the non-DNA-damaging agent arsenite) to induce SV40 and dhfr amplification at concentrations that result in 50% clonal survival were compared. All four DNA-damaging carcinogens (as well as growth arrest) were able to elicit some SV40 amplification, but responses varied markedly, from 1.8-fold for X-rays to sevenfold to eightfold for MMC. There was no correlation between the ability to elicit the two amplification responses. Arsenite, which did not induce SV40 amplification, was the best inducer of MTX resistance. These results point to different controls involved in the induction of viral and dhfr amplification. The signal for amplification of viral genes may be triggered by DNA damage and growth arrest, whereas amplification of dhfr, and perhaps other endogenous sequences, seems to be triggered by other signals as well.

Aphidicolin↗
PubMed1992

Mutagenesis and comutagenesis by lead compounds.

We have previously reported that lead(II) is weakly mutagenic to Chinese hamster V79 cells. A transgenic cell line G12 containing a single copy of the E. coli gpt gene was developed in this laboratory from Chinese hamster V79 cells. The gpt locus in the G12 cells is more mutable by radiation and oxidative agents compared with the endogenous hprt locus of wild-type V79 cells. We have investigated the mutagenicity of two lead compounds at the gpt locus in G12 cells. Only at a toxic dose is lead acetate significantly mutagenic to G12 cells. Lead nitrate is not significantly mutagenic at any dose. Although both compounds are water-soluble, lead acetate, but not lead nitrate, forms a fine white insoluble precipitate upon addition to growth medium. A nick translation assay on cells treated with lead compounds and then permeabilized indicated that lead nitrate and, to a greater extent, lead acetate causes the appearance of nicks in chromosomal DNA. Lead ions in the presence of hydrogen peroxide, but not alone, introduced nicks into supercoiled plasmid DNA in vitro, suggesting that lead ions can partake in a Fenton reaction and thereby damage DNA. At lower nonmutagenic concentrations, lead acetate enhances the mutagenicity of MNNG and ultraviolet light. DNA damage by ultraviolet light is not enhanced by lead ions in vitro. Our data support the concept that non-toxic concentrations of lead(II) can inhibit DNA repair. Thus, at biologically relevant doses, lead(II) could act as a comutagen and possibly a cocarcinogen, but is not likely to act as an initiating genotoxic carcinogen.

Animals↗
PubMed1992

Is cadmium genotoxic?

Previous evidence that cadmium(II) causes gene mutations in bacteria or mammalian cells was weak. However, alterations in protocol have recently led to better evidence for its mutagenicity, especially in bacteria. Mutagenicity results may be confounded by tolerance mechanisms. Exposure of DNA in vitro to Cd2+ or to Cd2+ hydrogen peroxide does not result in strand breaks or alkali-labile sites. The fact that bacterial and mammalian cells appear to sustain some type of repairable DNA damage after exposure to Cd2+ suggests that the damage must be caused in an indirect manner. Recently, the ability of cadmium-metallothionein complex to cause DNA strand breaks has been described. Cd2+ also induces a "pro-oxidant state" by causing a depletion of cellular glutathione. This finding is consistent with the role of Cd2+ as a clastogen and may explain its weak mutagenicity at loci which cannot detect complex mutations. Cd2+ can also inhibit DNA repair, and can therefore act synergistically with certain mutagens and, presumably, carcinogens.

Animals↗
PubMed1991

Comutagenesis of sodium arsenite with ultraviolet radiation in Chinese hamster V79 cells.

Solar ultraviolet radiation has been associated with the induction of skin cancer. Recent studies have indicated that near-ultraviolet, especially UVB, is mutagenic. Exposure to trivalent inorganic arsenic compounds has also been associated with increased skin cancer prevalence. Trivalent arsenic compounds are not mutagenic per se, but are comutagenic with a number of cancer agents. Here, we test the hypothesis that arsenite enhances skin cancer via its comutagenic action with solar ultraviolet radiation. Irradiation of Chinese hamster V79 cells with UVA (360 nm), UVB (310 nm) and UVC (254 nm) caused a fluence-dependent increase in mutations at the hprt locus. On an energy basis, UVC was the most mutagenic and UVA the least. However, when expressed as a function of toxicity, UVB was more mutagenic than UVC. Nontoxic concentrations of arsenite increased the toxicity of UVA, UVB and UVC. Arsenite acted as a comutagen at the three wavelengths; however, higher concentrations of arsenite were required to produce a significant (P less than 0.05) comutagenic response with UVB. The increased mutagenicity of UVB and UVA by arsenite may play a role in arsenite-related skin cancers.

Animals↗
PubMed1991

Performance of 133 compounds in the lambda prophage induction endpoint of the Microscreen assay and a comparison with S. typhimurium mutagenicity and rodent carcinogenicity assays.

The Microscreen assay was developed as a means of testing very small samples, as in complex mixture fractionation. It is a multi-endpoint assay which utilizes E. coli WP2s(lambda). Exposure takes place to serial dilutions of the test compound in microtitre wells (250 microliters) followed by sampling from wells in which growth has occurred ('non-toxic wells'). Although a number of different endpoints can be measured, only the prophage induction endpoint (the first one developed) has been extensively tested. Results with 133 compounds are presented. These include 111 compounds which have been tested in the S. typhimurium assay and 66 compounds for which both rodent bioassay and S. typhimurium assay data exists. The concordance for the Microscreen assay and the S. typhimurium assay was 71%. For this group of compounds, the sensitivity of the Microscreen assay in detecting carcinogens was 76% compared with 58% for the S. typhimurium assay. However, the S. typhimurium assay was somewhat more specific (69%) compared with the Microscreen (56%). The overall association between carcinogenicity and Microscreen results was statistically significant (p = 0.029), whereas for the S. typhimurium assay the association with carcinogenicity was non-significant (p = 0.086). The Microscreen assay was able to detect halogenated compounds better than the S. typhimurium assay. The Microscreen assay should prove useful in complex mixture fractionation, or in other situations where sample size is limiting.

Animals↗
PubMed1990

Transgenic Chinese hamster V79 cell lines which exhibit variable levels of gpt mutagenesis.

The Escherichia coli gpt gene coding for xanthine-guanine phosphoribosyl transferase has been stably transfected into HPRT- Chinese hamster V79 cells. Several gpt- cell lines have been established, which retain the sequence(s) even after long-term culture without selection for gpt. Each cell line exhibits a characteristic spontaneous mutation frequency (10(-5) to 10(-2)) in 6-thioguanine (6TG) selection. While spontaneous mutagenesis to gpt- occurs rather frequently for most cell lines, it cannot be correlated with either the number of plasmid integration sites or deletion of the plasmid sequence(s). One transgenic cell line (g12), which continuously maintains a low spontaneous mutation frequency (approximately 3 x 10(-5)), was used in comparative mutagenesis studies with wild-type V79 cells (gpt vs. hprt). Alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and beta-propiolactone (BPL) are shown to be equally toxic and mutagenic in both g12 and V79 cells. UV and X-rays are also equally toxic to both cell lines. The gpt locus of the g12 transfectants, however, is two to three times more sensitive to UV and 2.5-4.5 times more sensitive to X-ray mutagenesis than the endogenous hprt of wild-type V79 cells. The data presented here suggests that g12 cells may be useful to study mammalian mutagenesis by agents which yield limited response at the hprt locus. Future studies with these transgenic cells and other transgenic lines are planned to compare the mutability and repair of the same gene (gpt) at different integration sites in mammalian cells.

Animals↗
PubMed1989

Specific high frequency rearrangements induced by MNNG in SV40-infected human keratinocytes.

In order to study carcinogen-induced changes in integrated viral sequences clonal sublines of SV40-transformed human keratinocytes were exposed to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) at sublethal concentrations ranging up to 10 micrograms/ml for a period of 15 min and then examined by Southern blot hybridization using full-length SV40 DNA as a probe. Of the clonal sublines tested, one (AG34) was found to exhibit certain consistent, dose-dependent changes at 4 days post-treatment: (i) loss of at least two EcoRI fragments of approximately 4.4 and 3 kb, with the concomitant appearance of two bands migrating between 1.8 and 2.3 kb; and (ii) loss of a 1.5-kb fragment and the appearance of 2, 3.8 and 5 kb fragments in KpnI digests. Minor variable changes in restriction patterns were seen at 24 h post-treatment but consistent and pronounced effects were observed only at 4 days post-treatment. The altered restriction fragment patterns indicate that MNNG caused highly specific rearrangements in some subset of the DNA sequences associated with the integrated SV40 sequences in human epithelial cells. This differs from what has been found for SV40 sequences in other cell lines where amplification has been reported. These results suggest that (i) the site of SV40 integration may determine the response of integrated SV40 segments after carcinogen treatment, and (ii) carcinogen treatment can result in the induction of a common genetic event throughout the entire population of exposed cells. Genomic libraries created in a lambda phage vector have been used to isolate BamHI fragments containing SV40 sequences. Isolates have been found which exhibit EcoRI and KpnI restriction patterns consistent with the polymorphisms displayed in Southern blots.

Cell Division↗
PubMed1989

Inhibition of DNA ligase activity by arsenite: a possible mechanism of its comutagenesis.

We have previously shown that the inhibition of MNU-induced DNA repair by arsenite occurs after the incision step in Chinese hamster V79 cells. We now report that nuclear DNA ligase activity is inhibited after arsenite treatment and that the inhibited activity is mostly DNA ligase II. Both constitutive and MNU-inducible levels of DNA ligase II are inhibited. The addition of arsenite in vitro also indicates that DNA ligase II is more sensitive to arsenite inhibition than DNA ligase I. Since DNA ligase II is reported to be involved in the ligation step of excision repair, its inhibition by arsenite is a likely mechanism for the inhibition of DNA repair by arsenite and may account for the fact that arsenite acts as a comutagen with a number of different types of mutagens. The carcinogenicity of arsenite may also be a result of ligase inhibition.

Animals↗
PubMed1988

Colony hybridization to identify mammalian cells containing amplified, transfected, or expressed sequences.

The identification of variant cell clones has played an important role in the elucidation of various biochemical pathways. Such clones are typically selected by altering the medium or the growth conditions to give a selective advantage to the desired variant. The availability of cloned DNA probes has made it possible to identify bacterial colonies or viral plaques containing desired sequences, even in the absence of selection. We describe here a new method for the identification and isolation of mammalian cells containing any sequence for which a probe exists. Mammalian cell clones are first replicated onto nylon cloth. The original clones and the replicas are grown for a few days to enlarge the clones. All the clones on the original dish are transferred in situ to a filter and processed for hybridization. By altering the processing, either DNA or RNA sequences in the clones can be identified. When a clone of interest is located by hybridization, the cells can be isolated from the nylon replicas. We expect this method to be of value in the isolation of clones containing amplified, transfected, or mutated genes, as well as those exhibiting altered gene expression.

Animals↗
PubMed1988

Genetic toxicology of lead compounds.

We have investigated the activity of insoluble and soluble lead compounds in inducing mutagenesis, cell transformation and sister chromatid exchange in mammalian cells. Insoluble lead sulfide, readily phagocytized, was more than four times as toxic to V79 cells on a microM basis, than two moderately soluble lead compounds although the exposure time for the soluble salts was five times longer. These findings demonstrate the importance of different cellular mechanism(s) of metal uptake and bioavailability. Both insoluble lead sulfide and more soluble lead nitrate were mutagenic at the HPRT locus in V79 cells. Although less mutagenic at the higher concentrations, lead nitrate at a concentration of 500 microM enhanced the mutation frequency greater than 6-fold above background following a 5-day exposure. Although the mechanism(s) by which lead induces mutations is unknown, failure of both compounds to induce SCE and DNA single-strand breaks, detectable by alkaline elution, suggests that lead-induced mutations may not be a result of direct damage to DNA but may occur via indirect mechanisms including disturbances in enzyme functions important in DNA synthesis and/or repair, or in DNA-helical structure. Lead acetate also transformed SHE cells in a dose-response fashion following a 48-h exposure. Our results indicate that lead compounds may be genotoxic by an indirect mechanism, and lend support to the view that lead is a carcinogen.

Animals↗
PubMed1987

Genetic effects of 5-hydroxymethyl-2'-deoxyuridine, a product of ionizing radiation.

Ionizing radiation causes formation of heterogeneous types of damage to DNA. Among those, 5-hydroxymethyl-2'-deoxyuridine (HMdU) was identified as a major thymidine derivative in gamma-irradiated HeLa cells [G.W. Teebor, K. Frenkel and M.S. Goldstein (1984) Proc. Natl. Acad. Sci. (U.S.A.), 81, 318-321]. We report here that HMdU is a strong inducer of lambda prophage in Escherichia coli WP2s(lambda) and is highy mutagenic in Salmonella typhimurium. HMdU causes his+ revertants in strains TA100, which reverts predominantly by base-pair substitution at G-C sites, and TA97, which reverts mainly by frameshift mutation at G-C sites. It does not cause reversion in TA98, another frameshift-sensitive strain, nor in strains TA1535 and TA1537. Of those tested, only the last two strains do not contain pkM101, a plasmid which enhances mutagenic effects of ionizing radiation. HMdU also causes reversion in strains TA102 and TA104, which detect oxidative damage and can revert by base-pair substitution at A-T base pairs at the hisG428 site. We show that HMdU can be incorporated into DNA of TA100 and that, in addition to causing point mutations, it causes suppressor mutations as well. The ability of HMdU to induce lambda prophage and its strong mutagenicity in Salmonella typhimurium provide evidence that the presence of HMdU in DNA is biologically significant and may play a major role in the genetic consequences of ionizing radiation and other types of oxidative damage.

Bacteriophage lambda↗
PubMed1986

The genetic toxicology of metal compounds: II. Enhancement of ultraviolet light-induced mutagenesis in Escherichia coli WP2.

Salts of metals which are carcinogenic, noncarcinogenic, or of unknown carcinogenicity were assayed for their abilities to modulate ultraviolet (UV)-induced mutagenesis in Escherichia coli WP2. In addition to the previously reported comutagenic effect of arsenite, salts of three other compounds were found to enhance UV mutagenesis. CuCl2, MnCl2 (and a small effect by KMnO4), and NaMoO4 acted as comutagens in E coli WP2, which has wild-type DNA repair capability, but were much less comutagenic in the repair deficient strain WP2s (uvrA). The survival of irradiated or unirradiated cells was not affected by these compounds. No effects on UV mutagenesis were seen for 16 other metal compounds. We suggest that the comutagenic effects might occur either via metal-induced decreases in the fidelity of repair replication or (in the case of CuCl2) via metal-induced depurination.

Chlorides↗
PubMed1986

Ascorbate enhances u.v.-mutagenesis in E. coli but inhibits it in Chinese hamster cells.

Ascorbic acid (vitamin C) causes an increase in the mutation frequency of u.v.-irradiated Escherichia coli WP2. The enhancement occurs at all u.v. fluences, and is dependent upon the ascorbate concentration in the medium. A maximum effect (approximately 8- to 13-fold) is seen at 100-150 micrograms/ml, although some enhancement can be seen even at 10 micrograms/ml. The comutagenic effect of ascorbate with u.v. in E. coli is dependent upon peptone, a constituent of nutrient broth. The enhancement of u.v.-mutagenesis by ascorbate is absent in strains WP2s (uvrA) and WP6 (polA), suggesting that ascorbate affects the repair of pyrimidine dimers. The opposite results are observed for u.v.-mutagenesis in Chinese hamster V79 cells. The presence of ascorbate (50 micrograms/ml) during u.v. irradiation does not enhance the u.v. effect, but rather decreases it approximately 30%. These results are discussed with regard to differences in the mechanism of u.v.-mutagenesis and DNA repair in bacterial and mammalian cells.

Animals↗