Gene Expression over Time during Cell Transformation Due to Non-Genotoxic Carcinogen Treatment of Bhas 42 Cells

The Bhas 42 cell transformation assay (Bhas 42 CTA) is the first Organization for Economic Cooperation and Development (OECD)-certificated method used as a specific tool for the detection of the cell-transformation potential of tumor-promoting compounds, including non-genotoxic carcinogens (NGTxCs), as separate from genotoxic carcinogens. This assay offers the great advantage of enabling the phenotypic detection of oncotransformation. A key benefit of using the Bhas 42 CTA in the study of the cell-transformation mechanisms of tumor-promoting compounds, including non-genotoxic carcinogens, is that the cell-transformation potential of the chemical can be detected directly without treatment with a tumor-initiating compound since Bhas 42 cell line was established by transfecting the v-Ha-ras gene into a mouse fibroblast cloned cell line. Here, we analyzed the gene expression over time, using DNA microarrays, in Bhas 42 cells treated with the tumor-promoting compound 12-O-tetradecanoylphorbol-13-acetate (TPA), and NGTxC, with a total of three repeat experiments. This is the first paper to report on gene expression over time during the process of cell transformation with only a tumor-promoting compound. Pathways that were activated or inactivated during the process of cell transformation in the Bhas 42 cells treated with TPA were related not only directly to RAS but also to various pathways in the hallmarks of cancer.     

Effect of process distillation on mutagenicity and cell-transformation activity of solvent-refined,coal-derived liquids

Blended SRC-II process streams, representing a full boiling range distillate material, were fractionally distilled into non-overlapping 50 °F cuts with boiling points between 300 and 850 °F. Another set of 18 distillate cuts were obtained with boiling points ranging between 138 and 1055 °F. Distillate cuts were assayed for mutagenic activity using the histidine reversion assay with Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537, as well as for mammalian-cell transformation activity in the Syrian hamster embryo test, and DNA damage in the prophage induction assay. Samples were also separated into chemical class fractions by alumina column chromatography and analysed by high resolution gas chromatography so that the chemical composition of the cuts could be related to their relative activity in the different assays. In the mammalian cell transformation and microbial mutagenicity assays, significant activity was found almost exclusively in distillate cuts with components boiling > 700 °F, with the highest activity in the transformation assay observed for cuts > 800 °F. All of the distillate cuts showed increased levels of DNA damage as expressed by lambda prophage induction in Escherichia coli 8177. However, the greatest activity was associated with distillate cuts with boiling points in the 800 °F + range. Chemical analysis of the 50 °F distillate cuts showed a variety of polycyclic aromatic hydrocarbons (PAH) and amino-PAH compounds to be present in the distillate cuts boiling > 700 °F and essentially absent from cuts boiling < 700 °F. The sample set of non-overlapping (50 °F) cuts were reblended according to the proportions of each cut found in the original blend material. These reblended composites were then assayed to compare their activity with that predicted from the activities of the component distillate cuts. The reblending experiments indicated the microbial mutagenicity response was essentially additive. Mammalian cell transformation activities were non-additive, indicating a compositional effect on the expression of transforming agents in the complex mixture.     

Mutagenic potential of ammonia-related aflatoxin reaction products in cottonseed meal

In a joint research effort, the Food and Drug Administration, the National Toxicology Program and the US Department of Agriculture studied the mutagenic potential of aflatoxin reaction products following ammoniation of contaminated cottonseed meal under conditions approximating those approved for commercial ammoniation of nonaflatoxin-contaminated meal. Uniformly ring-labeled¹⁴C-aflatoxin B₁ was added to cottonseed meal that contained ca. 4000 μg naturally incurred aflatoxin B₁/kg. Distribution of the radiolabeled compound was used to trace the modification of aflatoxin B₁ after treatment with ammonia. The radioactivity-to-weight ratio of the fractions isolated by solvent extractions and chemical and enzymic treatments was used to measure the relative concentration of an aflatoxin decontamination product. All extract fractions having a radioactivity-to-weight ratio ≥1 were tested for mutagenic activity using theSalmonella/microsome mutagenicity test (Ames test). Purified aflatoxin B₁ was mutagenic at a concentration of ca. 0.005 μg/plate. The methylene chloride extract of the ammoniated meal after Pronase digestion exhibited a similar response when 180 μg of this fraction was applied to each plate. This fraction represented 0.16% of the original added radioactivity. The other fractions produced no detectable mutagenic response at the concentrations tested (10–1000 μg/plate) onSalmonella tester strain TA100. Ammonia treatment of aflatoxin-contaminated cottonseed meal significantly decreased aflatoxin levels, and the aflatoxin decontamination products formed by the treatment had little or no mutagenic potential.     

Magnetic microparticulate carriers with immobilized selective ligands in DNA diagnostics

Magnetic poly(2-hydroxyethyl methacrylate)- and poly(glycidyl methacrylate)-based microparticles were prepared by dispersion polymerization in the presence of iron oxide nanoparticles, both commercial and laboratory-made. The polymerization was highly sensitive to even subtle changes in the various reaction parameters involved in the process. The size of the final microparticles was determined by the composition of the dispersion medium (e.g. water/ethanol ratio, monomer concentration at the moment of phase separation, stabilizer concentration, initiator type and concentration, polymerization temperature). Several DNA applications of developed microparticles were described, among others RNA and DNA degradation and Salmonella cell magnetic separation by RNase A and DNase I and anti-Salmonella or proteinase K immobilized on developed magnetic carriers. The sensitivity of polymerase chain reaction (PCR) in cell detection was negatively affected by some magnetic carriers and compounds used in their preparation. Carboxyl group-containing magnetic microparticles were prepared for isolation of genomic DNA from cell lysate in the presence of poly(ethylene glycol) and sodium chloride.     

Ozonation of Dyestuffs and Intermediates and Further Decrease in Dissolved Organic Carbon by Post-Biodegradation

Four each of water-soluble dyestuffs, intermediates and reference compounds were examined to determine the effect of the combined use of ozonation and post-biodegradation on the decrease in the amount of dissolved organic carbon (DOC) and the synergistic effect induced by ozonation. The amount of DOC removed by ozonation was increased initially with increasing ozonation time, and showed a plateau thereafter. The amount of ozone required to remove 1 mg of DOC (ΔO₃/ΔDOC) ranged from 5.2 to 18.6 mgO₃/mgC for the dyestuffs and the intermediates. The DOC concentrations of all the ozonized solutions were decreased with incubation time. In the case of the dyestuffs and the intermediates, the total amounts of DOC removed were increased with increasing ozonation time and showed a plateau thereafter. The synergistic effect (the ozonation-induced increase in the amount of DOC removed by the biological process) was dependent on the initial biodegradability, and was observed in all the dyestuffs and the intermediates in the range of 0.2 to 42.7 mgDOC. On the other hand, no synergistic effect was observed in the reference compounds of high biodegradability.     

Comparison of oxidation efficiency of disperse dyes by chemical and photoelectrocatalytic chlorination and removal of mutagenic activity

Degradation of Disperse Orange 1, Disperse Red 1 and Disperse Red 13 dyes has been performed using electrochemical oxidation on Pt electrode, chemical chlorination and photoelectrochemical oxidation on Ti/TiO₂ thin film electrodes in NaCl or Na₂SO₄ medium. 100% discoloration was obtained for all tested methods after 1 h of treatment. Faster color removal was obtained by photoelectrocatalytic oxidation in 0.1 mol L⁻¹ NaCl pH 4.0 under UV light and an applied potential of +1.0 V (vs SCE reference electrode), which indicates also values around 60% of TOC removal. The conventional chlorination method and electrochemical oxidation on Pt electrode resulted in negligible reduction of TOC removal. All dyes showed positive mutagenic activity in the Salmonella/microsome assay with the strain TA98 in the absence and presence of S9 (exogenous metabolic activation). Nevertheless, there is complete reduction of the mutagenic activity after 1 h of photoelectrocatalytic oxidation, suggesting that this process would be good option to remove disperse azo dyes from aqueous media.     

Studies of in vitro cell transformation and mutagenicity by surfactants and other compounds

Cryopreserved primary cultures of Syrian golden hamster embryo cells were used as the source of target and feeder cells for an in vitro bioassay of carcinogenesis. Three different cultures that gave the best responses in a preliminary test were used for the bioassay. The capacities of ten surfactants and two other compounds to induce morphological transformation were examined using this system. The mutagenic potential of these materials was also tested using Salmonella typhimurium strains TA100 and TA98. None of the ten surfactants tested induced in vitro transformation of hamster embryo cells, neither were they mutagenic in S. typhimurium. Of the two other compounds tested, N-nitrosomethyl-n-dodecylamine was mutagenic, and dimethylglyoxime induced transformation, the results suggest that neither of these two short-term tests for determining carcinogenicity is adequate alone and that combining the two systems will give more useful results.     

Characterization of eluent by hot water extraction of coals in terms of total organic carbon and environmental impacts

Powdered coal samples were extracted by a hot water extraction (HWE) process, and the determination of total organic carbon (TOC) in the HWE eluent was performed by a TOC analyzer. The degree of TOC in the eluent greatly varied with kind of coals, and it tended to increase as the O/C value of coal increased. By use of heat-treated coals, which had somewhat smaller specific surface areas compared to the raw coals, it was shown that the degree of TOC in the eluent was also affected by specific surface area of coal. Almost no leaching was observed for hazardous heavy metals. The environmental impacts of the extract from the HWE eluent were evaluated by the Ames Salmonella mutagenicity assay and the estrogen receptor binding assay. The extracts from the HWE eluent did not show any notable mutagenicity. However, the extract from the HWE eluent for a lignite provided an appreciable affinity to estrogen receptor.     

Looking at Developmental Neurotoxicity Testing from the Perspective of an Invertebrate Embryo

Developmental neurotoxicity (DNT) of chemical compounds disrupts the formation of a normal brain. There is impressive progress in the development of alternative testing methods for DNT potential in chemicals, some of which also incorporate invertebrate animals. This review briefly touches upon studies on the genetically tractable model organisms of Caenorhabditis elegans and Drosophila melanogaster about the action of specific developmental neurotoxicants. The formation of a functional nervous system requires precisely timed axonal pathfinding to the correct cellular targets. To address this complex key event, our lab developed an alternative assay using a serum-free culture of intact locust embryos. The first neural pathways in the leg of embryonic locusts are established by a pair of afferent pioneer neurons which use guidance cues from membrane-bound and diffusible semaphorin proteins. In a systematic approach according to recommendations for alternative testing, the embryo assay quantifies defects in pioneer navigation after exposure to a panel of recognized test compounds for DNT. The outcome indicates a high predictability for test-compound classification. Since the pyramidal neurons of the mammalian cortex also use a semaphorin gradient for neurite guidance, the assay is based on evolutionary conserved cellular mechanisms, supporting its relevance for cortical development.     

Human Hazard Assessment Using Drosophila Wing Spot Test as an Alternative In Vivo Model for Genotoxicity Testing—A Review

Genomic instability, one of cancer’s hallmarks, is induced by genotoxins from endogenous and exogenous sources, including reactive oxygen species (ROS), diet, and environmental pollutants. A sensitive in vivo genotoxicity test is required for the identification of human hazards to reduce the potential health risk. The somatic mutation and recombination test (SMART) or wing spot test is a genotoxicity assay involving Drosophila melanogaster (fruit fly) as a classical, alternative human model. This review describes the principle of the SMART assay in conjunction with its advantages and disadvantages and discusses applications of the assay covering all segments of health-related industries, including food, dietary supplements, drug industries, pesticides, and herbicides, as well as nanoparticles. Chemopreventive strategies are outlined as a global health trend for the anti-genotoxicity of interesting herbal extract compounds determined by SMART assay. The successful application of Drosophila for high-throughput screening of mutagens is also discussed as a future perspective.     

Synthesis and mutagenic properties of 4,4'-diamino-p-terphenyl and 4,4'-diamino-p-quaterphenyl

4,4'-Diamino- p -terphenyl and 4,4'-diamino- p -quaterphenyl were synthesised from readily available chemical intermediates. These compounds, along with 1,4-diaminophenylene and benzidine were examined in the Salmonella microsome assay with strains TA98 and TA100. The results of this study indicate that the mutagenicity of these diamines was the greatest for 4,4'-diamino- p -terphenyl, followed by diaminophenylene and benzidine, with 4,4'-diamino- p -quaterphenyl showing no mutagenicity.     

Clinical Breakpoint of Apramycin to Swine Salmonella and Its Effect on Ileum Flora

The purpose of this study was to establish the clinical breakpoint (CBP) of apramycin (APR) against Salmonella in swine and evaluate its effect on intestinal microbiota. The CBP was established based on three cutoff values of wild-type cutoff value (CO_WT), pharmacokinetic-pharmadynamic (PK/PD) cutoff value (CO_PD) and clinical cutoff value (CO_CL). The effect of the optimized dose regimen based on ex vivo PK/PD study. The evolution of the ileum flora was determined by the 16rRNA gene sequencing and bioinformatics. This study firstly established the CO_WT, CO_PD in ileum, and CO_CL of APR against swine Salmonella, the value of these cutoffs were 32 µg/mL, 32 µg/mL and 8 µg/mL, respectively. According to the guiding principle of the Clinical Laboratory Standards Institute (CLSI), the final CBP in ileum was 32 µg/mL. Our results revealed the main evolution route in the composition of ileum microbiota of diarrheic piglets treated by APR. The change of the abundances of Bacteroidetes and Euryarchaeota was the most obvious during the evolution process. Methanobrevibacter, Prevotella, S24-7 and Ruminococcaceae were obtained as the highest abundance genus. The abundance of Methanobrevibacter increased significantly when APR treatment carried and decreased in cure and withdrawal period groups. The abundance of Prevotella in the tested groups was significantly lower than that in the healthy group. A decreased of abundance in S24-7 was observed after Salmonella infection and increased slightly after cure. Ruminococcaceae increased significantly after Salmonella infection and decreased significantly after APR treatment. In addition, the genera of Methanobrevibacter and Prevotella were defined as the key node. Valine, leucine and isoleucine biosynthesis, D-Glutamine and D-glutamate metabolism, D-Alanine metabolism, Peptidoglycan and amino acids biosynthesis were the top five Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in the ileum microbiota of piglets during the Salmonella infection and APR treatment process. Our study extended the understanding of dynamic shift of gut microbes during diarrheic piglets treated by APR.     

Viscoelastic behavior of NBR/phenolic compounds

NBR/phenolic interpenetrating networks (IPNs) offer a wide variety of mechanical and physical properties at moderately high temperature. This temperature stability along with oil and fuel resistance property has made IPNs appropriate candidates for various applications. In the present work, NBR compounds containing 5, 7 and 12 phr of Novolac, as a curable phenolic resin was formulated using a two-roll mill. Low and high acrylonitrile NBR; KNB 35L and Europrene N4560 were selected in the compound and the same condition of mixing was applied in the blend preparation stage. Curing test, followed by a cooling period and the stress relaxation test were carried out consecutively and automatically in a rubber process analyzer. The samples presented various relaxation times. The relaxation curves were well estimated by Maxwell model and the Prony coefficients were determined. Furthermore, compression test was performed on the samples, so that the set or permanent deformation of each sample was measured. The results of both tests have indicated that by adding phenolic resin into the NBR matrices, the viscoelastic behavior of the compounds become more elastic, to the detriment of the viscous component. This phenomenon would be due to IPN formation in the compounds. In addition, by increasing the phenolic resin content in the compounds, the difference between maximum and minimum torque (M _H ? M _L) value became greater, which is an indicator of higher cross-link density and IPN formation. Swelling test results confirmed more extensive cross-links in the compounds by addition of more resin into the compound.     

Having a Same Type IIS Enzyme’s Restriction Site on Guide RNA Sequence Does Not Affect Golden Gate (GG) Cloning and Subsequent CRISPR/Cas Mutagenesis

Golden gate/modular cloning facilitates faster and more efficient cloning by utilizing the unique features of the type IIS restriction enzymes. However, it is known that targeted insertion of DNA fragment(s) must not include internal type IIS restriction recognition sites. In the case of cloning CRISPR constructs by using golden gate (GG) cloning, this narrows down the scope of guide RNA (gRNA) picks because the selection of a good gRNA for successful genome editing requires some obligation of fulfillment, and it is unwanted if a good gRNA candidate cannot be picked only because it has an internal type IIS restriction recognition site. In this article, we have shown that the presence of a type IIS restriction recognition site in a gRNA does not affect cloning and subsequent genome editing. After each step of GG reactions, correct insertions of gRNAs were verified by colony color and restriction digestion and were further confirmed by sequencing. Finally, the final vector containing a Cas12a nuclease and four gRNAs was used for Agrobacterium-mediated citrus cell transformation. Sequencing of PCR amplicons flanking gRNA-2 showed a substitution (C to T) mutation in transgenic plants. The knowledge derived from this study could widen the scope of GG cloning, particularly of gRNAs selection for GG-mediated cloning into CRISPR vectors.     

室内轮胎花纹噪声测量方法研究

针对轮胎花纹噪声仿真分析结果与实测数据存在差异的问题，进行了室内不同方位轮胎花纹噪声测量试验。试验发现，轮胎花纹噪声的发声类似扬声器发声，有极强的指向性，若仅用一个测点的噪声声压级评判噪声的大小会导致方案排队和择优上的困难与矛盾。经分析研究，提出了一种全方位测定轮胎花纹噪声的方法，并制定了测量规范。     

Salmonella Infection Causes Hyperglycemia for Decreased GLP-1 Content by Enteroendocrine L Cells Pyroptosis in Pigs

Inflammatory responses have been shown to induce hyperglycemia, yet the underlying mechanism is still largely unclear. GLP-1 is an important intestinal hormone for regulating glucose homeostasis; however, few studies have investigated the influence of digestive tract Salmonella infection on enteroendocrine L cell secretions. In this study, we established a model of Salmonella-infected piglets by oral gavage in order to analyze the effects of Salmonella infection on enteroendocrine L cell function. Furthermore, in vitro lipopolysaccharide (LPS) was administered to STC-1 cells to clarify its direct effect on GLP-1 secretion. The results showed that significantly increased blood glucose in the group of Salmonella-infected piglets was observed, and Salmonella infection decreased blood GLP-1 content. Then, ileal epithelium damage was observed by histological detection, and this was further verified by TUNEL staining. We identified activation of TLR signaling demonstrating up-regulated expressions of TLR4 and nuclear factor-kappa B (NF-ΚB). Furthermore, it was shown that Salmonella induced pyroptosis of enteroendocrine L cells and enhanced the secretion of IL-1β through augmenting gene and protein expressions of NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a carboxyl-terminal CARD (ASC), Caspase 1, and gasdermin D (GSDMD). Meanwhile, in vitro LPS treatment induced the pyroptosis of STC-1 cells and reduced the secretion of GLP-1. Altogether, the results demonstrated that Salmonella infection can reduce secretion of GLP-1 by inducing pyroptosis of intestinal L cells, which may eventually result in hyperglycemia. The results provided evidence for the cause of hyperglycemia induced by inflammation and shed new light on glucose homeostasis regulation.     

Microbiological studies investigating mutagenicity of deep frying fat fractions and some of their components

In this study, the Salmonella/microsome mutagenicity test according to Ames et al. (Mutation Res. 31∶347, 1975) was performed in order to detect possible mutagenicity of oxidized deep frying fat fractions. Furthermore, the mono-, di-, tri- and tetrahydroxyoctadecanoic acids and the hydroperoxide of linoleic acid were investigated as model test substances. The Ames assay was carried out with and without metabolic activation including preincubation and liquid culture procedures as described by Mitchell (Mutation Res. 54∶1, 1978). The results show no mutagenic effects for the oxidized fractions of deep frying fats nor for the model test substances. At higher concentrations, however, limited test reliability resulted from direct toxic effects on bacterial growth.