Field and genetic analysis of Chinese fast-growing rhizobia

This research investigated the symbiotic effectivity and competitiveness of three strains of Chinese fast-growers (Rhizobium fredii) in the greenhouse and in the field, and evaluated the competitive ability of siderophore over-producing mutants of Chinese fast-growers (developed through transposon-mediated mutation) in an alkaline soil of Iowa. In the greenhouse study on effectiveness, three Chinese fast-growers were tested along with two USDA slow-growers (Bradyrhizobium japonicum) on four soybean cultivars. There was a significant strain-by-cultivar interaction, and two of the fast-growers were as effective as USDA 123 in N fixation on most of the cultivars, but less effective than USDA 110 on all cultivars tested. In the competition studies, the competitiveness of the fast-growers was evaluated in the greenhouse against native bradyrhizobia in six different soils. Nodule occupancy by fast-growers ranged from 1.5 to 38.8%, and there were significant interactions among strains, soils, and cultivars. For field testing, two separate sites each year were selected in the summers of 1987 and 1988, and each of the three fast-growers was introduced into soil at approximately 10[superscript]6 viable cells cm[superscript]-1~ row. Nodule occupancy over the four site-years on the cultivars Corsoy 79 and Williams 82 ranged from 3.8 to 13.3%. Both cultivars responded to N fertilizer with higher yields than the noninoculated controls, which indicated that the native slow-growers were not fixing all the N needed for maximum growth of the plants. The fast-growers persisted reasonably well as saprophytes over a 3-yr period based on nodule occupancy in the greenhouse of field-collected soil. In the siderophore study, only USDA 135 of the three slow-growers tested produced siderophore in liquid culture, which could explain their dominance in alkaline soils where availability of iron is presumed low. In an alkaline soil, two siderophore over-producing mutants, which had retained their symbiotic characteristics and tested positive on Southern hybridization with a Tn5 probe, occupied only one-sixth as many nodules as the wild-type. The symbiotic effectivity of the mutants, however, was not affected by mutation induced by Tn5 insertion. Further characterization of the mutants is necessary to understand why the mutants occupied fewer nodules

Opportunities to integrate new approaches in genetic toxicology: An ILSI-HESI workshop report

Genetic toxicity tests currently used to identify and characterize potential human mutagens and carcinogens rely on measurements of primary DNA damage, gene mutation, and chromosome damage in vitro and in rodents. The International Life Sciences Institute Health and Environmental Sciences Institute (ILSI-HESI) Committee on the Relevance and Follow-up of Positive Results in In Vitro Genetic Toxicity Testing held an April 2012 Workshop in Washington, DC, to consider the impact of new understanding of biology and new technologies on the identification and characterization of genotoxic substances, and to identify new approaches to inform more accurate human risk assessment for genetic and carcinogenic effects. Workshop organizers and speakers were from industry, academe, and government. The Workshop focused on biological effects and technologies that would potentially yield the most useful information for evaluating human risk of genetic damage. Also addressed was the impact that improved understanding of biology and availability of new techniques might have on genetic toxicology practices. Workshop topics included (1) alternative experimental models to improve genetic toxicity testing, (2) Biomarkers of epigenetic changes and their applicability to genetic toxicology, and (3) new technologies and approaches. The ability of these new tests and technologies to be developed into tests to identify and characterize genotoxic agents; to serve as a bridge between in vitro and in vivo rodent, or preferably human, data; or to be used to provide dose response information for quantitative risk assessment was also addressed. A summary of the workshop and links to the scientific presentations are provided.International Life Sciences Institute/Health and Environmental Sciences Institute Committe

DNA damage and pathway-focused gene expression profiling in the heart of F344 rats exposed to doxorubicin

Doxorubicin (DOX) is an antineoplastic drug effective against many human malignancies. DOX’s clinical efficacy is greatly limited because of severe cardiotoxicity. To evaluate if DOX is genotoxic in the heart, approximately 7- week-old, male F344 rats were administered intravenously 1, 2 and 3 mg/kg bw DOX at 0, 24, 48 and 69 hr and the Comet assays in heart, liver, kidney, and testis were conducted. Rats were euthanized at 72 hr and single cells were isolated from multiple tissues for the Comet assays. None of the doses of DOX induced a significant DNA damage in any of the tissues examined by the alkaline Comet assay. Contrastingly, the glycosylase enzymes-modified Comet assay showed a significant dose dependent increase in the oxidative DNA damage in the cardiac tissue (P ≤ 0.05). In the liver, only the top dose induced significant increase in the oxidative DNA damage (P ≤ 0.05). The histopathology showed no severe cardiotoxicity but non-neoplastic lesions were present in both untreated and treated samples. A severe toxicity likely occurred in the bone marrow because no viable reticulocytes could be screened for the MN assay. Gene expression profiling of the heart tissues showed a significant alteration in the expression of 11 DNA damage and repair genes. These results suggest that DOX is genotoxic in the heart and the DNA damage may be induced primarily via the production of reactive oxygen species

The development and validation of EpiComet-Chip, a modified high-throughput comet assay for the assessment of DNA methylation status

DNA damage and alterations in global DNA methylation status are associated with multiple human diseases and are frequently correlated with clinically relevant information. Therefore, assessing DNA damage and epigenetic modifications, including DNA methylation, is critical for predicting human exposure risk of pharmacological and biological agents. We previously developed a higher-throughput platform for the single cell gel electrophoresis (comet) assay, CometChip, to assess DNA damage and genotoxic potential. Here, we utilized the methylation-dependent endonuclease, McrBC, to develop a modified alkaline comet assay, “EpiComet,” which allows single platform evaluation of genotoxicity and global DNA methylation [5-methylcytosine (5-mC)] status of single-cell populations under user-defined conditions. Further, we leveraged the CometChip platform to create an EpiComet-Chip system capable of performing quantification across simultaneous exposure protocols to enable unprecedented speed and simplicity. This system detected global methylation alterations in response to exposures which included chemotherapeutic and environmental agents. Using EpiComet-Chip on 63 matched samples, we correctly identified single-sample hypermethylation (≥1.5-fold) at 87% (20/23), hypomethylation (≥1.25-fold) at 100% (9/9), with a 4% (2/54) false-negative rate (FNR), and 10% (4/40) false-positive rate (FPR). Using a more stringent threshold to define hypermethylation (≥1.75-fold) allowed us to correctly identify 94% of hypermethylation (17/18), but increased our FPR to 16% (7/45). The successful application of this novel technology will aid hazard identification and risk characterization of FDA-regulated products, while providing utility for investigating epigenetic modes of action of agents in target organs, as the assay is amenable to cultured cells or nucleated cells from any tissue

Mutagenicity of Acrylamide and Glycidamide in the Testes of Big Blue Mice

Acrylamide (AA) is an industrial chemical, a by-product of fried starchy foods, and a mutagen and rodent carcinogen. It can also cause damage during spermatogenesis. In this study, we investigated whether AA and its metabolite glycidamide (GA) induce mutagenic effects in the germ cells of male mice. Male Big Blue transgenic mice were administered 1.4 or 7.0mM of AA or GA in the drinking water for up to 4 weeks. Testicular cII mutant frequency (MF) was determined 3 weeks after the last treatment, and the types of the mutations in the cII gene were analyzed by DNA sequencing. The testes cII MFs in mice treated with either the low or high exposure concentrations of AA and GA were increased significantly. There was no significant difference in the cII MFs between AA and GA at the low exposure concentration. The mutation spectra in mice treated with AA (1.4mM) or GA (both 1.4 and 7.0mM) differed significantly from those of controls, but there were no significant differences in mutation patterns between AA and GA treatments. Comparison of the mutation spectra between testes and livers showed that the spectra differed significantly between the two tissues following treatment with AA or GA, whereas the mutation spectra in the two tissues from control mice were similar. These results suggest that AA possesses mutagenic effects on testes by virtue of its metabolism to GA, possibly targeting spermatogonial stem cells, but possibly via different pathways when compared mutations in liver

Mutagenicity of Acrylamide and Glycidamide in the Testes of Big Blue Mice

Acrylamide (AA) is an industrial chemical, a by-product of fried starchy foods, and a mutagen and rodent carcinogen. It can also cause damage during spermatogenesis. In this study, we investigated whether AA and its metabolite glycidamide (GA) induce mutagenic effects in the germ cells of male mice. Male Big Blue transgenic mice were administered 1.4 or 7.0mM of AA or GA in the drinking water for up to 4 weeks. Testicular cII mutant frequency (MF) was determined 3 weeks after the last treatment, and the types of the mutations in the cII gene were analyzed by DNA sequencing. The testes cII MFs in mice treated with either the low or high exposure concentrations of AA and GA were increased significantly. There was no significant difference in the cII MFs between AA and GA at the low exposure concentration. The mutation spectra in mice treated with AA (1.4mM) or GA (both 1.4 and 7.0mM) differed significantly from those of controls, but there were no significant differences in mutation patterns between AA and GA treatments. Comparison of the mutation spectra between testes and livers showed that the spectra differed significantly between the two tissues following treatment with AA or GA, whereas the mutation spectra in the two tissues from control mice were similar. These results suggest that AA possesses mutagenic effects on testes by virtue of its metabolism to GA, possibly targeting spermatogonial stem cells, but possibly via different pathways when compared mutations in liver