Biomedical subjects

M M Moore

Publications and source records attributed to M M Moore.

At least 19 recordsLinked to original sources

PubMed1992

In vitro mammalian mutagenesis as a model for genetic lesions in human cancer.

Recently in vitro assays of mutagenesis have been criticized as being poorly predictive of long-term in vivo rodent assays of carcinogenicity. Questions have also been raised concerning the relevance of rodent assays to human risk. In vitro assays using mammalian cells can detect most types of genetic lesions thought to be important in human malignant disease. Molecular and cytogenetic analyses of mutations induced by a variety of genotoxic compounds at the heterozygous thymidine kinase locus in mouse lymphoma cells indicate that this in vitro assay does indeed register the range of genetic lesions recently found in a wide variety of human tumors. The types and complexity of the induced lesions are reflected in mutant colony phenotype in a compound-specific fashion. These studies point to the use of appropriate in vitro mammalian mutagenesis assays as new model systems for dissecting the genetic lesions important in human carcinogenesis, and as a means of determining the potential for compounds to induce such lesions.

Animals↗

PubMed1992

Large deletions are tolerated at the hprt locus of in vivo derived human T-lymphocytes.

A cloning assay was used to recover hprt- T-lymphocytes from adult human males. Analysis of crude cellular extracts by polymerase chain reactions (PCRs) demonstrated that 7% (16/218) of the hprt mutations were due to total deletion of the hprt gene. 14 of the 16 mutants were examined by PCR for the presence of flanking DNA to determine the extent of the deletions. The deletion mutation in 13 mutants was at least 350 kb with 5 of these deletions being at least 700 kb. The largest deletions were greater than 15 times the size of the hprt gene. Therefore, large deletions are tolerated at the hprt locus of human T-lymphocytes.

Adult↗

PubMed1992

V(D)J recombinase-mediated deletion of the hprt gene in T-lymphocytes from adult humans.

The hprt T-cell cloning assay allows the detection of mutations occurring in vivo in the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene of T-lymphocytes. We have shown previously that the illegitimate activity of V(D)J recombinase accounts for about 40% of the hprt mutations in T-lymphocytes of human newborns as measured with umbilical cord blood samples (Fuscoe et al., 1991). This mechanism results in deletion of hprt exons 2 + 3. In this report, we examined a collection of 314 HPRT-deficient clones derived from adult humans for evidence that the mutations were caused by this mechanism by analyzing exons 2 + 3 deletion mutations. DNA sequence analysis of deletion breakpoint junctions showed that 8 of the mutations were the result of V(D)J recombinase activity. The frequency of the recombinase-mediated mutations was similar in the adults and newborns (2-4 x 10(-7). However, since the hprt mutant frequency is about 10-fold higher in the adult than in the newborn, the recombinase-mediated mutations account for only a few percent of the adult mutations. These mutations are likely to have occurred during early development and persist into adulthood. Unregulated expression of V(D)J recombinase activity may be an important mechanism for genomic rearrangements in the genesis of cancer.

Adult↗

PubMed1992

Synthetic trehalose dicorynomycolate and antimicrobials increase survival from sepsis in mice immunocompromised by radiation and trauma.

When mammalian antimicrobial defenses are compromised by radiation, death from sepsis may occur. Tissue trauma in irradiated hosts significantly increases mortality from bacterial infections and makes antimicrobial treatments more difficult than when individuals are subjected to trauma or radiation alone. We determined that postirradiation therapy with the immunomodulator synthetic trehalose dicorynomycolate (S-TDCM) and antimicrobials increases survival in mice after lethal irradiation and tissue trauma. Single agent therapy with systemic oxacillin, gentamicin, ofloxacin, and S-TDCM did not increase survival. Topical treatment of the injury with gentamicin cream in addition to systemic therapy with oxacillin or S-TDCM was necessary to enhance survival. Therapy with gentamicin and S-TDCM had a synergistic effect on survival. Therapies combining augmentation of non-specific host defense mechanisms with antimicrobials may be valuable in treating irradiated patients also sustaining tissue trauma.

Animals↗

PubMed1991

Considerations in the U.S. Environmental Protection Agency's testing approach for mutagenicity.

OPP: This paper provides the rationale and support for the decisions the OPP will make in requiring and reviewing mutagenicity information. The regulatory requirement for mutagenicity testing to support a pesticide registration is found in the 40 CFR Part 158. The guidance as to the specific mutagenicity testing to be performed is found in the OPP's Pesticide Assessment Guidelines, Subdivision F, Hazard Evaluation: Human and Domestic Animals (referred to as the Subdivision F guideline). A revised Subdivision F guideline has been presented that becomes the current guidance for submitters of mutagenicity data to the OPP. The decision to revise the guideline was the result of close examination of the version published in 1982 and the desire to update the guidance based on developments since then and current state-of-the-science. After undergoing Agency and public scrutiny, the revised guideline is to be published in 1991. The revised guideline consists of an initial battery of tests (the Salmonella assay, an in vitro mammalian gene mutation assay and an in vivo cytogenetics assay which may be either a bone marrow assay for chromosomal aberrations or for micronuclei formation) that should provide an adequate initial assessment of the potential mutagenicity of a chemical. Follow-up testing to clarify results from the initial testing may be necessary. After this information as well as all other relevant information is obtained, a weight-of-evidence decision will be made about the possible mutagenicity concern a chemical may present. Testing to pursue qualitative and/or quantitative evidence for assessing heritable risk in relation to human beings will then be considered if a mutagenicity concern exists. This testing may range from tests for evidence of gonadal exposure to dominant lethal testing to quantitative tests such as the specific locus and heritable translocation assays. The mutagenicity assessment will be performed in accordance with the Agency's Mutagenicity Risk Assessment Guidelines. The mutagenicity data would also be used in the weight-of-evidence consideration for the potential carcinogenicity of a chemical in accordance with the Agency's Carcinogen Risk Assessment Guidelines. In instances where there are triggers for carcinogenicity testing, mutagenicity data may be used as one of the triggers after a consideration of available information. It is felt that the revised Subdivision F guideline will provide appropriate, and more specific, guidance concerning the OPP approach to mutagenicity testing for the registration of a pesticide. It also provides a clearer understanding of how the OPP will proceed with its evaluation and decision making concerning the potential heritable effects of a test chemical.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1991

Genotoxicity of 2-amino-6-N-hydroxyadenine (AHA) to mouse lymphoma and CHO cells.

2-Amino-6-N-hydroxyadenine (AHA) treated L5178Y/TK (+/-)-3.7.2C mouse lymphoma cells were evaluated for mutations at the tk, hgprt, and Na+/K+ ATPase loci, as well as for gross chromosome aberrations and induction of micronuclei. In addition, AHA was evaluated for its ability to induce HGPRT mutants in CHO cells. AHA was found to induce mutations at all evaluated loci and in both cell types. The TK mutants were primarily large colonies although a few small colonies were also induced, particularly at the higher concentrations. Preliminary cytogenetic analysis of AHA-treated mouse lymphoma cells indicated that some gross aberrations but not micronuclei were induced. The 20 small-colony TK mutants evaluated by banded karyotype indicate that only a small fraction (2 of 20) showed chromosome 11 abnormalities. From these studies, it appears that AHA may be one of a very few chemicals that is capable of inducing multi-locus point mutations, with only slight clastogenic activity. Particularly at the higher concentrations, some of the mutants may contain multi-locus point mutations that result in slow growth.

Adenine↗

PubMed1991

Localization of the mouse thymidine kinase gene to the distal portion of chromosome 11.

We report the cytogenetic mapping of the thymidine kinase (tk-1) gene in the mouse using two complementary and independent analyses: (1) investigation of chromosome aberrations associated with tk-1 gene inactivation in the L5178Y TK+/- -3.7.2C cell line, and (2) fluorescence in situ molecular hybridization of cloned tk-1 cDNA probes to mitotic chromosomes of this cell line. The consensus location from both analyses is 11E1-E2. Consideration of the mouse tk-1 gene localization, along with evidence that the homologous human TK1 gene is located distally on the large arm of chromosome 17, appears to extend the region of homology between MMU11 and HSA17 to the distal end of both chromosomes.

Animals↗

PubMed1991

Analysis of the genotoxicity of anthraquinone dyes in the mouse lymphoma assay.

Despite their widespread use and potential for significant human exposure, genotoxicity data on anthraquinones and other dyes are limited. In this study, 16 anthraquinones and one azo dye (Solvent Red 1) were selected for testing using the thymidine kinase (tk) locus and micronucleus (MN) analysis in L5178Y/TK(+/-)-3.7.2C mouse lymphoma cells. Six of the dyes were from the same lot used in the NTP rodent bioassay. The dyes used were all production lots and thus varied in their purity. Disperse Blue 7, 2-aminoanthraquinone, 1-amino-2-methylanthraquinone, Disperse Blue 3 and Disperse Red 11 were genotoxic (inducing 1814 mutants/10(6) survivors, 369 MN/1000 cells at 13% survival; 397 mutants/10(6) survivors, 196 MN/1000 cells at 21% survival; 178 mutants/10(6) survivors, 119 MN/1000 cells at 51% survival; 264 mutants/10(6) survivors, 109 MN/1000 cells at 15% survival, respectively). Reactive Blue 19 was weakly mutagenic (inducing 144 mutants/10(6) survivors, but only 8 MN/1000 cells at 13% survival). Vat Yellow 4 and Solvent Red 1, with exogenous activation, were also mutagenic (inducing 300 mutants/10(6) survivors, 18 MN/1000 cells at 57% survival, and 100 mutants/10(6) survivors and 16 MN/1000 cells at 22% survival, respectively). With activation 1-nitro-2-methylanthraquinone was judged to give an equivocal mutagenicity response. The maximum test concentration was limited for some compounds by their solubility. Those chemicals that did not induce mutation or cytotoxicity at the limits of solubility were classified separately. Compounds which were not evaluated without exogenous activation because of insolubility but were evaluated with activation include 1-nitro-2-methylanthraquinone, Solvent Red 1 and Vat Yellow 4. Compounds which were not evaluated either with or without S9 activation because of their insolubility in the culture medium include 1-amino-2,4-dibromoanthraquinone, D&C Green, Disperse Blue 1, Disperse Red 60, Vat Blue 4, Vat Blue 20, Vat Brown 1 and Vat Brown 3.

Animals↗

PubMed1991

Comparison of mutagenicity results for nine compounds evaluated at the hgprt locus in the standard and suspension CHO assays.

The Chinese hamster ovary (CHO) assay, which measures newly induced mutations at the hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus, has been widely used for mutagenesis testing. The insensitivity of the standard assay to some genotoxic agents has been speculated to be due to the relatively small number of cells used in the assay. In the present study, we have compared the standard monolayer assay with a suspension adapted CHO assay that uses cell numbers comparable to that of the L5178Y mouse lymphoma assay. Nine compounds, ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), 2-methoxy-6-chloro-9-[3-(ethyl-2-chloroethyl)-aminopropylamino]-acridine 2HCl (ICR 170), methyl acrylate, ethyl acrylate, tetraethylene glycol diacrylate, trimethylolpropane triacrylate, 2-ethylhexyl acrylate and dicyclopentenyloxyethyl methacrylate were evaluated in the monolayer and suspension assays. Both assays gave the same overall qualitative results for the test compounds. There were some quantitative differences in the mutant frequency for the three compounds found to be mutagenic (EMS, MMS and ICR 170). The acrylates (many of which appear to exert their genotoxic effect through a clastogenic mechanism) were negative in both test systems. The use of the suspension assay did not improve the ability of the hgprt locus to detect the genotoxicity of the acrylates. Thus, increasing the number of cells does not improve the ability of the CHO/HGPRT assay to detect compounds that act primarily by a clastogenic mechanism.

Acrylates↗

PubMed1991

Genotoxicity in mouse lymphoma cells of chemicals capable of Michael addition.

Over the past several years, we have been evaluating the mutagenicity and clastogenicity of compounds capable of Michael-type reactions. These compounds, including acrylamide, several acrylate and methacrylate esters, vinyl sulfones, and phorone, have been evaluated using TK+/- -3.7.2C mouse lymphoma cells. Mutagenic chemicals induced increases in the number of small colony tk- deficient mutants. This suggested a clastogenic mechanism which was confirmed by demonstrating increases in aberrations and micronucleus frequencies in cultured cells. Vinyl sulfone was found to be the most effective chemical mutagen with induction of genotoxic effects at concentrations as low as 0.25 microgram/ml. The other compounds also produced positive results, but at higher concentrations. Since these compounds are known to deplete glutathione, phorone, a model glutathione depleter, was examined and found to produce similar effects as the other compounds in mouse lymphoma cells. These results suggest that the direct-acting Michael-type reaction has activity relevant to producing a genotoxic effect. Since acrylamide has been found to be a potent germ cell mutagen, this mechanism may be also relevant in the induction of heritable mutagenic risk.

Acrylates↗

PubMed1991

Treatment of mice with sepsis following irradiation and trauma with antibiotics and synthetic trehalose dicorynomycolate (S-TDCM).

Compromise of antimicrobial defenses by irradiation can result in sepsis and death. Additional trauma can further predispose patients to infection and thus increase mortality. We recently showed that injection of synthetic trehalose dicorynomycolate (S-TDCM) significantly augments resistance to infection and increases survival of mice compromised either by whole-body irradiation with gamma radiation or equal mixtures of fission neutron and gamma radiation. In this study, C3H/HeN mice were given a lethal dose of gamma radiation (8.0 Gy) and an open wound (15% total body surface area [TBSA]) 1 hr later while anesthetized. Irradiated/wounded mice became more severely leukopenic and thrombocytopenic than mice exposed to radiation alone, and died from natural wound infection and sepsis within 7 days. S-TDCM given 1 hr postirradiation increased survival of mice exposed to radiation alone. However, this treatment did not increase survival of the irradiated/wounded mice. Systemic antibiotic therapy with gentamicin or ofloxacin for 10 days significantly increased survival time compared with untreated irradiated/wounded mice (p less than 0.01). Combination therapy with topical gentamicin cream and systemic oxacillin increased survival from 0% to 100%. Treatment with S-TDCM combined with the suboptimal treatment of topical and systemic gentamicin increased survival compared with antibiotic treatment alone. These studies demonstrate that post-trauma therapy with S-TDCM and antibiotics augments resistance to infection in immunocompromised mice. The data suggest that therapies which combine stimulation of nonspecific host defense mechanisms with antibiotics may increase survival of irradiated patients inflicted with accidental or surgical trauma.

Administration, Topical↗

PubMed1991

Therapy of infections in mice irradiated in mixed neutron/photon fields and inflicted with wound trauma: a review of current work.

When host antimicrobial defenses are severely compromised by radiation or trauma in conjunction with radiation, death from sepsis results. To evaluate therapies for sepsis in radiation casualties, we developed models of acquired and induced bacterial infections in irradiated and irradiated-wounded mice. Animals were exposed to either a mixed radiation field of equal proportions of neutrons and gamma rays (n/gamma = 1) from a TRIGA reactor or pure gamma rays from 60[Co sources. Skin wounds (15% of total body surface area) were inflicted under methoxyflurane anesthesia 1 h after irradiation. In all mice, wounding after irradiation decreased resistance to infection. Treatments with the immunomodulator synthetic trehalose dicorynomycolate (S-TDCM) before or after mixed neutron-gamma irradiation or gamma irradiation increased survival. Therapy with S-TDCM for mice irradiated with either a mixed field or gamma rays increased resistance to Klebsiella pneumoniae-induced infections. Combined therapy with S-TDCM and ceftriaxone for K. pneumoniae infections in mice exposed to a mixed radiation field or to gamma rays was more effective than single-agent therapy. In all irradiated-wounded mice, single therapy of acquired infections with an antibiotic or S-TDCM did not increase survival. Survival of irradiated-wounded mice after topical application of gentamicin sulfate cream suggested that bacteria colonizing the wound disseminated systemically in untreated irradiated mice, resulting in death from sepsis. In lethal models of acquired infections in irradiated-wounded mice, significant increases in survival were achieved when systemic treatments with S-TDCM or gentamicin were combined with topical treatments of gentamicin cream. Therapies for sepsis in all mice exposed to a mixed field were less effective than in mice exposed to gamma rays. Nonetheless, the data show a principle by which successful therapy may be provided to individuals receiving tissue trauma in conjunction with radiation injury.

Adjuvants, Immunologic↗

PubMed1990

Molecular dissection of mutations at the heterozygous thymidine kinase locus in mouse lymphoma cells.

The mouse lymphoma L5178Y TK+/- 3.7.2C cell line allows quantitation of induced TK(+/-)----TK-/- mutations at the heterozygous thymidine kinase (Tk) locus. TK-/- mutant colonies show a bimodal size distribution, reflecting a difference in the growth rates of the two size classes that is hypothesized to result from different degrees of genetic damage. The two homologous chromosomes 11 containing the alleles of the Tk gene in L5178Y 3.7.2C TK+/- cells are distinguishable at the cytogenetic level. We find, in addition, that the two alleles are distinguishable at the molecular level because of an Nco I restriction fragment length polymorphism at the 3' end of the gene. In a set of 51 large-colony and 48 small-colony TK-/- mutants induced by ionizing radiation or by chemical mutagens, we find that 78, including all except one of the small-colony mutants, have lost the Tk+ allele and that some of these have two to four copies of the remaining Tk- allele. Nineteen of the large-colony TK-/- mutants that do not show Tk+ allele loss show no other structural changes detectable at the level of Southern blot analysis. One shows a partial deletion. The variety of mutagenic lesions recorded at the heterozygous Tk locus may be representative of events observed in human malignancy and may include a wider range of mutagenic events than can be observed at hemizygous test loci.

Alleles↗

PubMed1990

Genotoxicity of inhibitors of DNA topoisomerases I (camptothecin) and II (m-AMSA) in vivo and in vitro.

The present study was designed to determine and compare the clastogenicity of m-AMSA and camptothecin (CAMP) in vivo in mouse bone marrow and peripheral blood lymphocytes (PBLs), and in vitro in mouse lymphoma L5178Y cells. m-AMSA interferes with topoisomerase II to induce double-strand DNA breaks. CAMP interferes with topoisomerase I to induce single-strand DNA breaks. Thus, we expected the two drugs to induce different types of chromosomal aberrations (CAs). However, both drugs produced quantitatively and qualitatively similar numbers and types of aberrations under similar experimental conditions. In mouse bone marrow exposed over and 18-h period, both drugs (3 mg/kg) induced approximately 30 damaged cells, with an average of 0.4 chromatid breaks per cell (in 100 cells analyzed/mouse). In addition, both drugs induced only chromatid-type aberrations in mouse bone marrow in vivo when exposure occurred during G2. Cell cycle specificity was indicated by the absence of CAs when exposure to the drugs occurred in vivo in mouse PBLs during G0. In L5178Y cells, m-AMSA was considerably more potent for the induction of mutations and somewhat more potent for the induction of CAs than CAMP was. In contrast to the in vivo bone marrow results, the drugs induced high levels of both chromatid- and chromosome-type aberrations in vitro. The ultimate types of chromosomal damage induced by m-AMSA and CAMP result from a complex interaction of (i) cell cycle specific variations in topoisomerase enzyme levels, (ii) the abilities of these drugs to interfere with the orderly DNA breakage/reunion associated with topoisomerase activity, and (iii) the processing of the damage resulting from these interactions.

Amsacrine↗

PubMed1990

Comparison of chromosome aberration frequency and small-colony TK-deficient mutant frequency in L5178Y/TK(+/-)-3.7.2C mouse lymphoma cells.

The L5178Y/TK(+/-)-3.7.2C mouse lymphoma assay is used to quantitate the induction of thymidine kinase (TK)-deficient mutants. The mutants detected in the assay form colonies that can be distinguished as large or small. The induction of small-colony mutants has been associated with the induction of chromosome mutations. In the present paper, we compare the analysis of induced small-colony TK mutants with gross aberration analysis (the more classical approach to analyzing chromosomal damage). Data are presented for 34 mutagens. As expected, we find that while the induction of gross aberrations and the induction of small-colony TK mutants is correlated, there is no simple mathematical relationship between the two endpoints. The two markers evaluate different subpopulations of chromosome mutations. While either endpoint can be used to detect chromosomal mutations, it should be remembered that the small-colony TK mutants represent genetic events which are compatible with cell viability. Only those alterations compatible with cell viability are a significant risk for human carcinogenicity or mutagenicity.

Animals↗

PubMed1989

The role of carotenoids in preventing oxidative damage in the pigmented yeast, Rhodotorula mucilaginosa.

Rhodotorula mucilaginosa is an obligate aerobic yeast which contains a high concentration of carotenoid pigment. To test whether carotenoids are able to protect R. mucilaginosa against oxidative injury, yeast cells in liquid culture were incubated with duroquinone (DQ) (100 microM), a redox-cycling quinone known to generate intracellular O2-. or were grown in a hyperoxic atmosphere (80% O2) under conditions where carotenoid concentrations were altered either intracellularly or extracellularly. Neither of these oxidative challenges affected cell growth unless carotenogenesis was blocked by the addition of diphenylamine (50 microM). In the diphenylamine-treated nonpigmented cells, growth was completely inhibited by DQ and by hyperoxia. In normoxia, however, diphenylamine alone reduced growth by only 30%. The growth inhibition observed in diphenylamine-treated cells exposed to hyperoxia was primarily mycocidal rather than mycostatic since plating of these cells onto solid media revealed that only 25% of the cells were viable after 50 h of incubation when compared to plated control cells. Addition of 10 microM beta-carotene to diphenylamine-treated cells completely prevented the growth inhibition caused by either hyperoxia or DQ. Carotenoids, therefore, are able to prevent oxidant-induced cytotoxicity in R. mucilaginosa. Analysis of the absorption spectra of chloroform extracts of beta-carotene-supplemented cells showed that beta-carotene, not the endogenous carotenoid, torularhodin, was the major carotenoid present in these cells. Superoxide dismutase (SOD) activity in R. mucilaginosa was compared with that of another yeast, Saccharomyces cerevisiae by two methods: (i) activity staining of proteins separated by gel electrophoresis and (ii) measurement of inhibition of ferricytochrome c reduction. By these techniques, the R. mucilaginosa SOD activity had the characteristics of Mn-SOD. No Cu/ZnSOD activity was detected. Thus, the apparent absence of Cu/ZnSOD may make the antioxidant capability of endogenous carotenoids even more critical in preventing oxidative damage in R. mucilaginosa.

Benzoquinones↗

PubMed1989

High-resolution cytogenetic characterization of the L5178Y TK+/- mouse lymphoma cell line.

High-resolution chromosome preparations from L5178Y TK+/- 3.7.2C mouse lymphoma cells were obtained using acridine orange in the cell harvest procedure. With this technique it is possible to visualize over 500 bands in elongated mouse lymphoma cell chromosomes as compared to the approximately 230 bands visualized in metaphase preparations. High-resolution lymphoma cell chromosomes are described, and chromosome rearrangements carried in the cell line are characterized by ideograms representing the position, number, size, and relative staining intensity of the G-band patterns. Use of elongated chromosomes of mouse lymphoma TK+/- mutants should facilitate analysis of the cytogenetic effects associated with TK+/- ----TK-/- mutagenesis.

Animals↗

PubMed1989

High-resolution cytogenetic analysis of L5178Y TK+/- 3.7.2C cells: variation in chromosome 11 breakpoints among small-colony TK-/- mutants.

Since the finding that the mouse lymphoma L5178Y TK+/- ----TK-/- forward mutational assay system can detect and distinguish a range of genetic lesions, including large chromosomal aberrations and smaller, perhaps point mutational events, the chromosomal analysis of these lesions at the highest possible level of band resolution has become increasingly important. We have developed an acridine orange/colcemid/hypotonic treatment for TK-/- mutants to provide high-resolution chromosomes with over 500 G-bands for breakpoint analysis. Using such high-resolution procedures, we find that independently induced small-colony mutants show rearrangements in the distal portion of chromosome 11, with breakpoints occurring between bands B3 and E1.2. This finding of a range of chromosomal breakpoints in different TK-/- mutants complements recent molecular genetic analysis of mutants and is consistent with the hypothesis that chromosomal lesions in small-colony mutants may affect a large portion of the genome in the vicinity of the tk-1 gene.

Animals↗