Comparative potency analysis of whole smoke solutions in the bacterial reverse mutation test.

Short-term in vitro genotoxicity assays are useful tools to assess whether new and emerging tobacco products potentially have reduced toxicity. We previously demonstrated that potency ranking by benchmark dose (BMD) analysis quantitatively identifies differences among several known carcinogens and toxic chemicals representing different chemical classes found in cigarette smoke. In this study, six whole smoke solution (WSS) samples containing both the particulate and gas phases of tobacco smoke were generated from two commercial cigarette brands under different smoking-machine regimens. Sixty test cigarettes of each brand were machine-smoked according to the International Organization for Standardization (ISO) puffing protocol. In addition, either 60 or 20 test cigarettes of each brand were machine-smoked with the Canadian Intense (CI) puffing protocol. All six WSSs were evaluated in the bacterial reverse mutation (Ames) test using Salmonella typhimurium strains, in the presence or absence of S9 metabolic activation. The resulting S9-mediated mutagenic concentration-responses for the four WSSs from 60 cigarettes were then compared using BMD modelling analysis and the mutagenic potency expressed as number of revertants per µl of the WSS. The quantitative approaches resulted in a similar rank order of mutagenic potency for the Ames test in both TA98 and TA100. Under the conditions of this study, these results indicate that quantitative analysis of the Ames test data can discriminate between the mutagenic potencies of WSSs on the basis of smoking-machine regimen (ISO vs. CI), and cigarette product (differences in smoke chemistry).

A Host-Guest Supercapacitor Electrode Material Based on a Mixed Hexa-Transition Metal Sandwiched Arsenotungstate Chain and Three-Dimensional Supramolecular Metal-Organic Networks with One-Dimensional Cavities.

The mixed hexa-transition metal (hexa-TM) sandwiched arsenotungstate derivative, [CuI3(pz)2(phen)3]2[CuI(phen)2][{Na(H2O)2}{(VIV5CuIIO6)(AsIIIW9O33)2}]·6H2O (1) (pz = pyrazine; phen = 1,10-phenanthroline), has been hydrothermally synthesized and structurally characterized. In compound 1, two {AsW9O33} clusters are connected by mixed hexa-TM ring unit {VVI5CuIIO6} to form a sandwich-type dimer, which are further bonded in "ABAB" mode by the {Na(H2O)2} linker resulting in pure inorganic chains. The unique "L-shaped" trinuclear complex {Cu3(phen)3(pz)2} is supported together via staggered π-π interactions to generate extending waveform two-dimensional supramolecular layers, which are further aggregated with their adjacent analogues by complexes {Cu(phen)2} via H-bonding interaction to yield an unprecedented three-dimensional (3D) metal-organic networks with one-dimensional (1D) cavities. The pure inorganic 1D sandwich chains are implanted in the cavities as guest units via supramolecular interactions to form a POMOF 3D framework. Compound 1, as the electrode of the supercapacitor, exhibits higher specific capacitances (825 F g-1 at a current density of 2.4 A g-1), better rate capability, more durable cyclic stability (91.4% of cycle efficiency after 3000 cycles), and improved conductivity and electroactivity compared to those of parent polyoxometalate (POM) Na9[AsW9O33]·19H2O (2) and 6-Cu-substituted POM [Cu6(imi)6{AsIIIW9O30Cl3}2]·6H2O (3), which may be attributed to the introduction of V4+, the unique host-guest structure, and the rich π electron system. In addition, compound 1 exhibits dual-function electrocatalytic behavior in reducing inorganic salt IO3- and oxidizing the organic molecule dopamine.

Low-frequency KRAS mutations are prevalent in lung adenocarcinomas.

AIM: This study quantified low-frequency KRAS mutations in normal lung and lung adenocarcinomas, to understand their potential significance in the development of acquired resistance to EGFR-targeted therapies. MATERIALS & METHODS: Allele-specific Competitive Blocker-PCR was used to quantify KRAS codon 12 GAT (G12D) and GTT (G12V) mutation in 19 normal lung and 21 lung adenocarcinoma samples. RESULTS: Lung adenocarcinomas had KRAS codon 12 GAT and GTT geometric mean mutant fractions of 1.94 × 10-4 and 1.16 × 10-3, respectively. For 76.2% of lung adenocarcinomas, the level of KRAS mutation was greater than the upper 95% confidence interval of that in normal lung. CONCLUSION: KRAS mutant tumor subpopulations, not detectable by DNA sequencing, may drive resistance to EGFR blockade in lung adenocarcinoma patients.

Tissue-specific microRNA responses in rats treated with mutagenic and carcinogenic doses of aristolochic acid.

Aristolochic acid (AA) is an active component in herbal drugs derived from the Aristolochia species. Although these drugs have been used since antiquity, AA is both genotoxic and carcinogenic in animals and humans, resulting in kidney tumours in rats and upper urinary tract tumours in humans. In the present study, we conducted microarray analysis of microRNA (miRNA) expression in tissues from transgenic Big Blue rats that were treated for 12 weeks with 0.1-10mg/kg AA, using a protocol that previous studies indicate eventually results in kidney tumours and mutations in kidney and liver. Global analysis of miRNA expression of rats treated with 10 mg/kg AA indicated that 19 miRNAs were significantly dysregulated in the kidney, with most of the miRNAs related to carcinogenesis. Only one miRNA, miR-34a (a tumour suppressor), was differentially expressed in the liver. The expression of the two most responsive kidney miRNAs (miR-21, an oncomiR and miR-34a) was further examined in the kidney, liver and testis of rats exposed to 0, 0.1, 1.0 and 10mg/kg AA. Expression of miR-21 was up-regulated in the kidney only, while miR-34a was dose-dependently up-regulated in both the kidney and liver; the expression of miR-21 and miR-34a was unaltered by the AA treatment in the testis. Analysis of cII mutations in the testis of treated rats also was negative. Our results indicate that AA treatment of rats produced dysregulation of a large number of miRNAs in the tumour target tissue and that the up-regulation of miR-21 correlated with the carcinogenicity of AA while the up-regulation of miR-34a correlated with its mutagenicity.

ACB-PCR quantification of somatic oncomutation.

Allele-specific competitive blocker-polymerase chain reaction (ACB-PCR) is a sensitive approach for the selective amplification of an allele. Using the ACB-PCR technique, hotspot point mutations in oncogenes and tumor-suppressor genes (oncomutations) are being developed as quantitative biomarkers of cancer risk. ACB-PCR employs a mutant specific primer (with a 3'-penultimate mismatch relative to the mutant DNA sequence, but a double 3'-terminal mismatch relative to the wild-type DNA sequence) to selectively amplify rare mutant DNA molecules. A blocker primer (having a non-extendable 3'-end and with a 3'-penultimate mismatch relative to the wild-type DNA sequence, but a double 3'-terminal mismatch relative to the mutant DNA sequence) is included in ACB-PCR to selectively repress amplification from the abundant wild-type molecules. Consequently, ACB-PCR is capable of quantifying the level of a single basepair substitution mutation in a DNA population when present at a mutant:wild type ratio of 10(-5) or greater. Quantification of rare mutant alleles is achieved by parallel analysis of unknown samples and mutant fraction (MF) standards (defined mixtures of mutant and wild-type DNA sequences). The ability to quantify specific mutations with known association to cancer has several important applications, including evaluating the carcinogenic potential of chemical exposures in rodent models and in the diagnosis and treatment of cancer. This chapter provides a step-by-step description of the ACB-PCR methodology as it has been used to measure human KRAS codon 12 GGT to GAT mutation.

Genotoxicity of 2-bromo-3'-chloropropiophenone.

Impurities are present in any drug substance or drug product. They can be process-related impurities that are not completely removed during purification or are formed due to the degradation of the drug substance over the product shelf-life. Unlike the drug substance, impurities generally do not have beneficial effects and may present a risk without associated benefit. Therefore, their amount should be minimized. 2-Bromo-3'-chloropropiophenone (BCP) is an impurity of bupropion, a second-generation antidepressant and a smoking cessation aid. The United States Pharmacopeia recommends an acceptable level for BCP that is not more than 0.1% of the bupropion. Because exposure to genotoxic impurities even at low levels is of significant concern, it is important to determine whether or not BCP is genotoxic. Therefore, in this study the Ames test and the in vitro micronucleus assay were conducted to evaluate the genotoxicity of BCP. BCP was mutagenic with S9 metabolic activation, increasing the mutant frequencies in a concentration-dependent manner, up to 22- and 145-fold induction over the controls in Salmonella strains TA100 and TA1535, respectively. BCP was also positive in the in vitro micronucleus assay, resulting in up to 3.3- and 5.1-fold increase of micronucleus frequency for treatments in the absence and presence of S9, respectively; and 9.9- and 7.4-fold increase of aneuploidies without and with S9, respectively. The addition of N-acetyl-l-cysteine, an antioxidant, reduced the genotoxicity of BCP in both assays. Further studies showed that BCP treatment resulted in induction of reactive oxygen species (ROS) in the TK6 cells. The results suggest that BCP is mutagenic, clastogenic, and aneugenic, and that these activities are mediated via generation of reactive metabolites.

Targeted deletion of the antisilencer/enhancer (ASE) element from intron 1 of the myelin proteolipid protein gene (Plp1) in mouse reveals that the element is dispensable for Plp1 expression in brain during development and remyelination.

Myelin proteolipid protein gene (Plp1) expression is temporally regulated in brain, which peaks during the active myelination period of CNS development. Previous studies with Plp1-lacZ transgenic mice demonstrated that (mouse) Plp1 intron 1 DNA is required for high levels of expression in oligodendrocytes. Deletion-transfection analysis revealed the intron contains a single positive regulatory element operative in the N20.1 oligodendroglial cell line, which was named ASE (antisilencer/enhancer) based on its functional properties in these cells. To investigate the role of the ASE in vivo, the element was deleted from the native gene in mouse using a Cre/lox strategy. Although removal of the ASE from Plp1-lacZ constructs profoundly decreased expression in transfected oligodendroglial cell lines (N20.1 and Oli-neu), the element was dispensable to achieve normal levels of Plp1 gene expression in mouse during development (except perhaps at postnatal day 15) and throughout the remyelination period following cuprizone-induced (acute) demyelination. Thus, it is possible that the ASE is non-functional in vivo, or that loss of the ASE from the native gene in mouse can be compensated for by the presence of other regulatory elements within the Plp1 gene.

Discovery of novel microRNAs in rat kidney using next generation sequencing and microarray validation.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate a variety of biological processes. The latest version of the miRBase database (Release 18) includes 1,157 mouse and 680 rat mature miRNAs. Only one new rat mature miRNA was added to the rat miRNA database from version 16 to version 18 of miRBase, suggesting that many rat miRNAs remain to be discovered. Given the importance of rat as a model organism, discovery of the completed set of rat miRNAs is necessary for understanding rat miRNA regulation. In this study, next generation sequencing (NGS), microarray analysis and bioinformatics technologies were applied to discover novel miRNAs in rat kidneys. MiRanalyzer was utilized to analyze the sequences of the small RNAs generated from NGS analysis of rat kidney samples. Hundreds of novel miRNA candidates were examined according to the mappings of their reads to the rat genome, presence of sequences that can form a miRNA hairpin structure around the mapped locations, Dicer cleavage patterns, and the levels of their expression determined by both NGS and microarray analyses. Nine novel rat hairpin precursor miRNAs (pre-miRNA) were discovered with high confidence. Five of the novel pre-miRNAs are also reported in other species while four of them are rat specific. In summary, 9 novel pre-miRNAs (14 novel mature miRNAs) were identified via combination of NGS, microarray and bioinformatics high-throughput technologies.

Aristolochic acid-induced carcinogenesis examined by ACB-PCR quantification of H-Ras and K-Ras mutant fraction.

Aristolochic acid (AA) is a strong cytotoxic nephrotoxin and carcinogen associated with the development of urothelial cancer in humans. AA induces forestomach, kidney and urothelial tract tumours in rats and mice. This study was conducted to characterise AA's carcinogenic mechanism of action and compare allele-specific competitive blocker-polymerase chain reaction (ACB-PCR)-based early detection of carcinogenic effect using two different tumour-relevant endpoints. H-Ras codon 61 CAA→CTA mutation was analysed because it is found in rodent forestomach tumours and A:T→T:A transversion is the predominant mutational specificity induced by AA. K-Ras codon 12 GGT→GAT mutation was analysed because it is a common spontaneous mutation present in various rodent tissues and may be a useful generic biomarker for carcinogenic effect. DNA samples from Big Blue rats treated with 0, 0.1, 1.0 or 10.0 mg AA/kg body weight (bw) by gavage, 5 days/week for 12 weeks were used in ACB-PCR in order to examine the induction of the two specific mutations. A significant dose-dependent induction of H-Ras mutant fraction (MF) was observed in liver and kidney. Statistically significant correlations were observed between AA-induced DNA adduct levels or cII mutant frequencies (previously measured in the same rats) and H-Ras MF measurements. No correlation between AA dose and K-Ras MF was found in liver or kidney, although there was a significant induction of K-Ras mutation in kidneys exposed to 0.1 mg/kg bw AA relative to controls. Thus, the data establish a straightforward dose-related increase in H-Ras MF due to fixation of AA-induced DNA adducts, whereas the common spontaneous K-Ras mutation showed a non-monotonic dose-response, consistent with loss of non-targeted mutation at cytotoxic doses.

p53 codon 271 CGT to CAT mutant fraction does not increase in nasal respiratory and olfactory epithelia of rats exposed to inhaled naphthalene.

A 2-year rat tumor bioassay testing whole body exposure to naphthalene (NA) vapor found a significant increase in nasal respiratory epithelial adenomas in male rats and in olfactory epithelial neuroblastomas in female rats. To obtain mechanistic insight into NA-induced nasal carcinogenesis, NA dose-response was characterized in nasal epithelium using a tumor-relevant endpoint. Specifically, levels of p53 codon 271 CGT to CAT mutation were measured in nasal respiratory and olfactory epithelium of NA-exposed male and female rats by allele-specific competitive blocker-PCR (ACB-PCR). Male and female, 8-9 week-old F344 rats (5 rats/group) were exposed to 0, 0.1, 1.0, 10, and 30ppm NA vapor for 13 weeks (6h/day, 5 days/week). The geometric mean p53 mutant fraction (MF) levels in nasal epithelium of control treatment groups ranged between 2.05 × 10(-5) and 3.05 × 10(-5). No significant dose-related changes in p53 mutant fraction (MF) were observed in the olfactory or respiratory epithelia of female rats. However, statistically significant treatment-related differences were observed in male respiratory and olfactory epithelium, with the p53 MF in the respiratory epithelium of male rats exposed to 30ppm NA significantly lower than that in controls. Further, a significant trend of decreasing p53 MF with increasing dose was observed in the male respiratory epithelium. Of the tissue types analyzed, respiratory epithelium is the most sensitive to the cytotoxic effects of NA, suggesting cytotoxicity may be responsible for the loss of p53 mutation. Because ACB-PCR has been used successfully to detect the effects of known mutagenic carcinogens, the absence of any significant increases in p53 MF associated with NA exposure adds to the weight of evidence that NA does not operate through a directly mutagenic mode of action.

Oncomutations as biomarkers of cancer risk.

Cancer risk assessment impacts a range of societal needs, from the regulation of chemicals to achieving the best possible human health outcomes. Because oncogene and tumor suppressor gene mutations are necessary for the development of cancer, such mutations are ideal biomarkers to use in cancer risk assessment. Consequently, DNA-based methods to quantify particular tumor-associated hotspot point mutations (i.e., oncomutations) have been developed, including allele-specific competitive blocker-PCR (ACB-PCR). Several studies using ACB-PCR and model mutagens have demonstrated that significant induction of tumor-associated oncomutations are measureable at earlier time points than are used to score tumors in a bioassay. In the particular case of benzo[a]pyrene induction of K-Ras codon 12 TGT mutation in the A/J mouse lung, measurement of tumor-associated oncomutation was shown to be an earlier and more sensitive endpoint than tumor response. The measurement of oncomutation by ACB-PCR led to two unexpected findings. First, oncomutations are present in various tissues of control rodents and "normal" human colonic mucosa samples at relatively high frequencies. Approximately 60% of such samples (88/146) have mutant fractions (MFs) >10(-5), and some have MFs as high as 10(-3) or 10(-4). Second, preliminary data indicate that oncomutations are present frequently as subpopulations in tumors. These findings are integrated into a hypothesis that the predominant preexisting mutations in particular tissues may be useful as generic reporters of carcinogenesis. Future research opportunities using oncomutation as an endpoint are described, including rodent to human extrapolation, dose-response assessment, and personalized medicine.

Targeting of survivin by nanoliposomal ceramide induces complete remission in a rat model of NK-LGL leukemia.

The natural killer (NK) type of aggressive large granular lymphocytic (LGL) leukemia is a fatal illness that pursues a rapid clinical course. There are no effective therapies for this illness, and pathogenetic mechanisms remain undefined. Here we report that the survivin was highly expressed in both aggressive and chronic leukemic NK cells but not in normal NK cells. In vitro treatment of human and rat NK-LGL leukemia cells with cell-permeable, short-chain C6-ceramide (C6) in nanoliposomal formulation led to caspase-dependent apoptosis and diminished survivin protein expression, in a time- and dose-dependent manner. Importantly, systemic intravenous delivery of nanoliposomal ceramide induced complete remission in the syngeneic Fischer F344 rat model of aggressive NK-LGL leukemia. Therapeutic efficacy was associated with decreased expression of survivin in vivo. These data suggest that in vivo targeting of survivin through delivery of nanoliposomal C6-ceramide may be a promising therapeutic approach for a fatal leukemia.

Measurement of tumor-associated mutations in the nasal mucosa of rats exposed to varying doses of formaldehyde.

This study examined the potential induction of tumor-associated mutations in formaldehyde-exposed rat nasal mucosa using a sensitive method, allele-specific competitive blocker-PCR (ACB-PCR). Levels of p53 codon 271 CGT to CAT and K-Ras codon 12 GGT to GAT mutations were quantified in nasal mucosa of rats exposed to formaldehyde. In addition, nasal mucosa cell proliferation was monitored because regenerative cell proliferation is considered a key event in formaldehyde-induced carcinogenesis. Male F344 rats (6-7 weeks old, 5 rats/group) were exposed to 0, 0.7, 2, 6, 10, and 15 ppm formaldehyde for 13 weeks (6 h/day, 5 days/week). ACB-PCR was used to determine levels of p53 and K-Ras mutations. Although two of five untreated rats had measureable spontaneous p53 mutant fractions (MFs), most nasal mucosa samples had p53 MFs below 10(-5). All K-Ras MF measurements were below 10(-5). No dose-related increases in p53 or K-Ras MF were observed, even though significant increases in bromodeoxyuridine incorporation demonstrated induced cell proliferation in the 10 and 15 ppm formaldehyde-treatment groups. Therefore, induction of tumor-associated p53 mutation likely occurs after several other key events in formaldehyde-induced carcinogenesis.

K-Ras mutant fraction in A/J mouse lung increases as a function of benzo[a]pyrene dose.

K-Ras mutant fraction (MF) was measured to examine the default assumption of low-dose linearity in the benzo[a]pyrene (B[a]P) mutational response. Groups of 10 male A/J mice (7- to 9-weeks old) received a single i.p. injection of 0, 0.05, 0.5, 5, or 50 mg/kg B[a]P and were sacrificed 28 days after treatment. K-Ras codon 12 TGT and GAT MFs in lung DNAs were measured using Allele-specific Competitive Blocker-PCR (ACB-PCR). The K-Ras codon 12 TGT geometric mean MF was 3.88 x 10(-4) in controls, indicating an average of 1 mutation in every approximately 1,288 lung cells. The K-Ras codon 12 TGT geometric mean MFs were as follows: 3.56 x 10(-4); 6.19 x 10(-4); 2.02 x 10(-3), and 3.50 x 10(-3) for the 0.05, 0.5, 5, and 50 mg/kg B[a]P treatment groups, respectively. The 5 and 50 mg/kg dose groups had TGT MFs significantly higher than did controls. Although 10(-5) is considered as the limit of accurate ACB-PCR quantitation, K-Ras codon 12 GAT geometric mean MFs were as follows: 8.38 x 10(-7), 1.47 x 10(-6), 2.19 x 10(-6), 5.71 x 10(-6), and 8.99 x 10(-6) for the 0, 0.05, 0.5, 5, and 50 mg/kg B[a]P treatment groups, respectively. The K-Ras TGT and GAT MFs increased in a B[a]P-dose-dependent manner, with response approximately linear over the 0.05 to 5 mg/kg dose range. K-Ras MF increased with B[a]P adduct burden measured for identical doses in a separate study. Thus, ACB-PCR may be useful in characterizing the shape of a dose-response curve at low doses and establishing relationships between DNA adducts and tumor-associated mutations.

Leydig cells express the myelin proteolipid protein gene and incorporate a new alternatively spliced exon.

Although the myelin proteolipid protein gene (Plp1) is highly expressed in the central nervous system encoding the most abundant myelin protein in oligodendrocytes, it is also expressed in other tissues, including testis. Transgenic studies with mice that harbor Plp1-lacZ fusion genes suggest that Leydig cells are the source of Plp1 gene expression in testis. However, virtually nothing is known about Plp1 gene regulation in Leydig cells, which is the focus of this study. The first intron contains both positive and negative regulatory elements that are important in regulating Plp1 gene expression in oligodendrocytes. To test whether these elements are functional in Leydig cells, a battery of Plp1-lacZ fusion genes with partial deletion of Plp1 intron 1 sequence was transfected into the mouse Leydig cell line, TM3. Results presented here suggest that an enhancer, which is very potent in oligodendrocytes, is only nominally active in TM3 cells. The intron also contains several negative regulatory elements that are operative in TM3 cells. Moreover a new exon (exon 1.2) was identified within the first 'intron' resulting in novel splice variants in TM3 cells. Western blot analysis suggests that these splice variants, along with those containing another alternatively spliced exon (exon 1.1) derived from intron 1 sequence, give rise to multiple Plp1 gene products in the mouse testis.

K-RAS mutation in the screening, prognosis and treatment of cancer.

The potential use of K-RAS mutation as a cancer screening biomarker has been investigated for many years. Numerous associations between K-RAS mutation and various cancers have been established, but these associations have not been translated into effective, cost-efficient cancer screening strategies. This lack of progress may be due to the existence of K-RAS mutation in nontumor tissues and/or using detection, rather than quantitation, of K-RAS mutation as the endpoint for cancer risk categorization. K-RAS mutation appears to be a useful prognostic biomarker for colon cancer. Recent progress toward sensitive and quantitative mutation characterization and the successful use of K-RAS mutation in a personalized medicine approach to targeted biological therapy selection are likely to re-direct and expand the use of K-RAS mutation as a cancer biomarker in the near future.

Characterization of an intronic enhancer that regulates myelin proteolipid protein (Plp) gene expression in oligodendrocytes.

The myelin proteolipid protein (Plp) gene is expressed in oligodendrocytes and encodes the most abundant protein (approximately 50%) present in mature myelin from the central nervous system (CNS). Plp gene activity is low to nonexistent early in development but sharply increases, concurrently with the active myelination period of CNS development. Work from our laboratory suggests that the temporal regulation of Plp gene expression in mice is mediated by a positive regulatory element located within Plp intron 1 DNA. We have termed this regulatory element/region ASE (for antisilencer/enhancer). The ASE is situated approximately 1 kb downstream of exon 1 DNA and encompasses nearly 100 bp. To understand the mechanisms by which the ASE augments Plp gene expression in oligodendrocytes, Plp-lacZ constructs were generated and transfected into a mouse oligodendroglial cell line (N20.1). Results presented here demonstrate that upstream regulatory elements in the Plp promoter/5'-flanking DNA are not required for ASE activity; the ASE worked perfectly well when the thymidine kinase (TK) promoter was substituted for the Plp promoter. However, the relative location of the ASE appears to be important. When placed upstream of 2.4 kb of Plp 5'-flanking DNA, or downstream of the lacZ expression cassette, the ASE was no longer effective. Thus, the ASE might have to be in the context of the intron in order to function. To begin to identify the crucial nucleotides within the ASE, orthologous sequences from rat, human, cow, and pig Plp genes were swapped for the mouse sequence. Results presented here demonstrate that the orthologous sequence from rat can substitute for the mouse ASE, unlike those from human, cow, or pig.