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Metabolism plays a key role in chemical genotoxicity; however, most mammalian cells used for in vitro genotoxicity testing lack effective metabolizing enzymes. We recently developed a battery of TK6-derived cell lines that individually... more
Metabolism plays a key role in chemical genotoxicity; however, most mammalian cells used for in vitro genotoxicity testing lack effective metabolizing enzymes. We recently developed a battery of TK6-derived cell lines that individually overexpress one of eight cytochrome P450s (CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, and 3A4) using a lentiviral expression system. The increased expression and metabolic function of each individual CYP in each established cell line were confirmed using real-time PCR, Western blotting, and mass spectrometry analysis; the parental TK6 cells and empty vector (EV) transduced cells had negligible CYP levels. Subsequently, we evaluated these cell lines using two prototypical polyaromatic hydrocarbon mutagens, 7,12-dimethylbenz[a]anthracene (DMBA) and benzo[a]pyrene (B[a]P), that require metabolic activation to exert their genotoxicity. DMBA-induced cytotoxicity, phosphorylation of histone H2A.X, and micronucleus formation were significantly increased in TK6 c...
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The Thymidine kinase (Tk) gene forward mutation assay, known as the mouse lymphoma assay (MLA), has been widely used for evaluating the genotoxicity of chemical agents. A striking morphological feature of Tk mutant colonies is the bimodal... more
The Thymidine kinase (Tk) gene forward mutation assay, known as the mouse lymphoma assay (MLA), has been widely used for evaluating the genotoxicity of chemical agents. A striking morphological feature of Tk mutant colonies is the bimodal distribution of their sizes, with cells from the large colonies growing at a normal rate and cells from the small colonies growing at a slower rate than normal. To understand the molecular distinction for the different growth rates, we performed whole genome sequencing (WGS) analysis of the large and small colony mutants generated from the MLA. Three large colony and three small colony mutants generated from cells treated with 4-nitroquinoline 1-oxide (4-NQO) or the vehicle control were selected for analysis. The WGS data were analyzed for loss of heterozygosity (LOH) and chromosome copy number along chromosome 11, where the Tk gene is located. Although there were LOH alterations in both large and small colony mutants, copy number changes near Tk l...
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TEMPO and its derivatives are stable free radical nitroxides widely used in the field of chemistry, biology, and pharmacology. TEMPO was previously found to be mutagenic and to induce micronuclei in mammalian cells. In the present study,... more
TEMPO and its derivatives are stable free radical nitroxides widely used in the field of chemistry, biology, and pharmacology. TEMPO was previously found to be mutagenic and to induce micronuclei in mammalian cells. In the present study, we investigated and quantified the genotoxicity of four structurally similar nitroxides, TEMPO and three of its derivatives (4-hydroxy-TEMPO, 4-oxo-TEMPO, and 4-methoxy-TEMPO), using the mouse lymphoma assay (MLA) and Comet assay in L5178Y Tk+/- cells. The results showed that all tested nitroxides were cytotoxic and mutagenic in the MLA, both in the presence and absence of S9, with metabolic activation significantly enhancing the cytotoxicity and/or mutagenicity. In addition, the four nitroxides caused DNA strand breakage. The mutagenicity and DNA damaging dose-responses of the test articles were compared using the PROAST benchmark dose (BMD) software package. The potency ranking of the four nitroxides for mutagenicity was different from the ranking...
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In vitro genotoxicity dose-response data have been investigated for their utility in modeling and assessing potential differences in mutagenic responses between machine-generated whole smoke solutions (WSSs) from combusted cigarette... more
In vitro genotoxicity dose-response data have been investigated for their utility in modeling and assessing potential differences in mutagenic responses between machine-generated whole smoke solutions (WSSs) from combusted cigarette tobacco products. Our previous study observed that potency ranking by benchmark dose (BMD) analysis was a useful modeling approach for quantitative assessment of differences between the mutagenicity of several structurally diverse chemical constituents of cigarette smoke. To follow-up on these observations, we used the mouse lymphoma assay (MLA) to evaluate the mutagenicity of WSSs prepared from two commercial cigarettes smoked under two different smoking machine regimens. L5178Y cells were exposed to ≥5 concentrations of each WSS for 4 hr ± S9 activation. S9 reduced the cytotoxicity and enhanced the mutagenicity of the WSSs. The resulting S9-mediated mutagenicity dose-responses were compared between test articles using BMD analysis, the lowest dose exce...
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Acetaminophen (APAP) overdose is the leading cause of acute liver failure. Yet the mechanisms underlying adaptive tolerance toward APAP-induced liver injury are not fully understood. To better understand molecular mechanisms contributing... more
Acetaminophen (APAP) overdose is the leading cause of acute liver failure. Yet the mechanisms underlying adaptive tolerance toward APAP-induced liver injury are not fully understood. To better understand molecular mechanisms contributing to adaptive tolerance to APAP is an underpinning foundation for APAP-related precision medicine. In the current study, the mRNA and microRNA (miRNA) expression profiles derived from next generation sequencing data for APAP-treated (5 and 10 mM) HepaRG cells and controls were analyzed systematically. Putative miRNAs targeting key dysregulated genes involved in APAP hepatotoxicity were selected using in silico prediction algorithms, un-biased gene ontology, and network analyses. Luciferase reporter assays, RNA electrophoresis mobility shift assays, and miRNA pull-down assays were performed to investigate the role of miRNAs affecting the expression of dysregulated genes. Levels of selected miRNAs were measured in serum samples obtained from children wi...
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4-Methoxy-TEMPO, a derivative of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), is a stable nitroxide radical and is generally used in organic and pharmaceutical syntheses for the oxidation of alcohols. Previously, we reported the... more
4-Methoxy-TEMPO, a derivative of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), is a stable nitroxide radical and is generally used in organic and pharmaceutical syntheses for the oxidation of alcohols. Previously, we reported the involvement of reactive oxygen species (ROS) and c-Jun N-terminal kinases (JNK) in TEMPO-induced apoptosis in mouse L5178Y cells. In this study, we investigated 4-methoxy-TEMPO induced toxicity in human HepG2 hepatoma cells and its underlying mechanisms. Treatments with 4-methoxy-TEMPO (0.5-5 mM for 2-6 h) caused oxidative stress as demonstrated by increased intensity of the ROS indicator H2DCF-DA, decreased levels of glutathione. 4-Methoxy-TEMPO treatment also induced DNA damage as characterized by increased levels of DNA tail intensity in the Comet assay, increased phosphorylation of related proteins including γ-H2A.X, p-Chk1, and p-Chk2, and activation of MAPK signaling pathways. In addition, 4-methoxy-TEMPO also induced autophagy as demonstrated by the ...
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Ginkgo biloba has been used as a traditional herbal remedy for many thousand years and its leaf extract has been consumed as a botanical dietary supplement for decades. Ginkgo biloba extract is a complex mixture with numerous components,... more
Ginkgo biloba has been used as a traditional herbal remedy for many thousand years and its leaf extract has been consumed as a botanical dietary supplement for decades. Ginkgo biloba extract is a complex mixture with numerous components, including flavonol glycosides and terpene lactones, and is one of the most widely sold botanical dietary supplements worldwide. Concerns about potential health risks for the general population have been raised because of the widespread human exposure to Ginkgo biloba and its potential toxic and carcinogenic activities in rodents. The National Toxicology Program conducted 2-year gavage studies and concluded that there was clear evidence of carcinogenic activity of Ginkgo biloba leaf extract in mice based on an increased incidence of hepatocellular carcinoma and hepatoblastoma. Recently, Ginkgo biloba leaf extract has been classified as a possible human carcinogen (Group 2B) by the International Agency for Research on Cancer. This review presents upda...
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A growing number of studies suggest that isoflavones found in soybeans have estrogenic activity and may safely alleviate the symptoms of menopause. One of these isoflavones, genistein, is commonly used by postmenopausal women as an... more
A growing number of studies suggest that isoflavones found in soybeans have estrogenic activity and may safely alleviate the symptoms of menopause. One of these isoflavones, genistein, is commonly used by postmenopausal women as an alternative to hormone replacement therapy. Although sex hormones have been implicated as an important risk factor for the development of hepatocellular carcinoma, there are limited data on the potential effects of the estrogens, including phytoestrogens, on chemical mutagenesis in liver. Because of the association between mutation induction and the carcinogenesis process, we investigated whether endogenous estrogen and supplemental genistein affect 7,12-dimethylbenz[a]anthracene (DMBA)-induced mutagenesis in rat liver. Intact and ovariectomized female Big Blue rats were treated with 80 mg DMBA/kg body weight. Some of the rats also received a supplement of 1,000 ppm genistein. Sixteen weeks after the carcinogen treatment, the rats were sacrificed, their livers were removed, and mutant frequencies (MFs) and types of mutations were determined in the liver cII gene. DMBA significantly increased the MFs in liver for both the intact and ovariectomized rats. While there was no significant difference in MF between the ovariectomized and intact control animals, the mutation induction by DMBA in the ovariectomized groups was significantly higher than that in the intact groups. Dietary genistein did not alter these responses. Molecular analysis of the mutants showed that DMBA induced chemical-specific types of mutations in the liver cII gene. These results suggest that endogenous ovarian hormones have an inhibitory effect on liver mutagenesis by DMBA, whereas dietary genistein does not modulate spontaneous or DMBA-induced mutagenesis in either intact or ovariectomized rats.
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The physicochemical characteristics of silver nanoparticles (AgNPs) may greatly alter their toxicological potential. To explore the effects of size and coating on the cytotoxicity and genotoxicity of AgNPs, six different types of AgNPs,... more
The physicochemical characteristics of silver nanoparticles (AgNPs) may greatly alter their toxicological potential. To explore the effects of size and coating on the cytotoxicity and genotoxicity of AgNPs, six different types of AgNPs, having 3 different sizes and 2 different coatings, were investigated using the Ames test, mouse lymphoma assay (MLA), and in vitro micronucleus assay. The genotoxicities of silver acetate and silver nitrate were evaluated to compare the genotoxicity of nanosilver to that of ionic silver. The Ames test produced inconclusive results for all types of the silver materials due to the high toxicity of silver to the test bacteria and the lack of entry of the nanoparticles into the cells. Treatment of L5718Y cells with AgNPs and ionic silver resulted in concentration-dependent cytotoxicity, mutagenicity in the Tk gene and the induction of micronucleus from exposure to nearly every type of the silver materials. Treatment of TK6 cells with these silver materia...
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Comfrey has been consumed by humans as a vegetable and a tea and used as an herbal medicine for more than 2000 years. Comfrey, however, produces hepatotoxicity in livestock and humans and carcinogenicity in experimental animals. Comfrey... more
Comfrey has been consumed by humans as a vegetable and a tea and used as an herbal medicine for more than 2000 years. Comfrey, however, produces hepatotoxicity in livestock and humans and carcinogenicity in experimental animals. Comfrey contains as many as 14 pyrrolizidine alkaloids (PA), including 7-acetylintermedine, 7-acetyllycopsamine, echimidine, intermedine, lasiocarpine, lycopsamine, myoscorpine, symlandine, symphytine, and symviridine. The mechanisms underlying
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Research Interests: Genetics, Genetic Toxicology, Environmental Biotechnology, Liver, Mutation, and 15 moreMice, Female, Animals, Micronucleus, Analysis of Variance, Antifungal Agents, Base Sequence, Lymphocytes, Erythrocytes, aniline Compounds, Liver Tumor, Mutagenicity tests, Pharmacology and pharmaceutical sciences, Peripheral blood, and Malachite green
Many usnic acid-containing dietary supplements have been marketed as weight loss agents, although severe hepatotoxicity and acute liver failure have been associated with their overuse. Our previous mechanistic studies revealed that... more
Many usnic acid-containing dietary supplements have been marketed as weight loss agents, although severe hepatotoxicity and acute liver failure have been associated with their overuse. Our previous mechanistic studies revealed that autophagy, disturbance of calcium homeostasis, and ER stress are involved in usnic acid-induced toxicity. In this study, we investigated the role of oxidative stress and the Nrf2 signaling pathway in usnic acid-induced toxicity in HepG2 cells. We found that a 24-h treatment with usnic acid caused DNA damage and S-phase cell cycle arrest in a concentration-dependent manner. Usnic acid also triggered oxidative stress as demonstrated by increased reactive oxygen species generation and glutathione depletion. Short-term treatment (6 h) with usnic acid significantly increased the protein level for Nrf2 (nuclear factor erythroid 2-related factor 2), promoted Nrf2 translocation to the nucleus, up-regulated antioxidant response element (ARE)-luciferase reporter ac...
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The Aloe plant is employed as a dietary supplement in a variety of foods and as an ingredient in cosmetic products. The widespread human exposure and its potential toxic and carcinogenic activities raise safety concerns. Chemical analysis... more
The Aloe plant is employed as a dietary supplement in a variety of foods and as an ingredient in cosmetic products. The widespread human exposure and its potential toxic and carcinogenic activities raise safety concerns. Chemical analysis reveals that the Aloe plant contains various polysaccharides and phenolic chemicals, notably anthraquinones. Ingestion of Aloe preparations is associated with diarrhea, hypokalemia, pseudomelanosis coli, kidney failure, as well as phototoxicity and hypersensitive reactions. Recently, Aloe vera whole leaf extract showed clear evidence of carcinogenic activity in rats, and was classified by the International Agency for Research on Cancer as a possible human carcinogen (Group 2B). This review presents updated information on the toxicological effects, including the cytotoxicity, genotoxicity, carcinogenicity, and adverse clinical effects, of Aloe vera whole leaf extract, gel, and latex.
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Both the Aloe vera whole leaf extract and decolorized whole leaf extract were mutagenic in cultured cells with different genetic damage.
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Cytochrome P450 2C19 (CYP2C19) is involved in the metabolism of many drugs. Extensive studies have demonstrated that genetic variants and endogenous and environmental factors play important roles in the expression of CYP2C19. However, the... more
Cytochrome P450 2C19 (CYP2C19) is involved in the metabolism of many drugs. Extensive studies have demonstrated that genetic variants and endogenous and environmental factors play important roles in the expression of CYP2C19. However, the role of microRNAs (miRNAs) in controlling CYP2C19 expression has not been investigated completely. In the present study, we performed in silico analysis to rank putative miRNA/CYP2C19 hybrids with regards to the predicted stabilities of their duplexes and then we applied a series of biochemical and molecular assays to elucidate the underlying functional mechanisms for the regulation of CYP2C19 by miRNAs. In silico analysis indicated that hsa-miR-23a-3p and hsa-miR-29a-3p target the coding region of CYP2C19 with hybrid stabilities of -27.5kcal/mol and -23.3kcal/mol, respectively. RNA electrophoresis mobility shift assays showed that both hsa-miR-23a-3p and hsa-miR-29a-3p miRNAs were able to bind directly to their cognate targets in the CYP2C19 trans...
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Cytochrome P450 2C19 (CYP2C19) is involved in the metabolism of many drugs. Extensive studies have demonstrated that genetic variants and endogenous and environmental factors play important roles in the expression of CYP2C19. However, the... more
Cytochrome P450 2C19 (CYP2C19) is involved in the metabolism of many drugs. Extensive studies have demonstrated that genetic variants and endogenous and environmental factors play important roles in the expression of CYP2C19. However, the role of microRNAs (miRNAs) in controlling CYP2C19 expression has not been investigated completely. In the present study, we performed in silico analysis to rank putative miRNA/CYP2C19 hybrids with regards to the predicted stabilities of their duplexes and then we applied a series of biochemical and molecular assays to elucidate the underlying functional mechanisms for the regulation of CYP2C19 by miRNAs. In silico analysis indicated that hsa-miR-23a-3p and hsa-miR-29a-3p target the coding region of CYP2C19 with hybrid stabilities of -27.5kcal/mol and -23.3kcal/mol, respectively. RNA electrophoresis mobility shift assays showed that both hsa-miR-23a-3p and hsa-miR-29a-3p miRNAs were able to bind directly to their cognate targets in the CYP2C19 transcript. Further, a significant inverse correlation was found between chemically-induced up-regulation of hsa-miR-29a-3p and CYP2C19 expression in HepaRG cells. In addition, inverse correlations were also observed in human liver tissue samples between the level of CYP2C19 mRNA expression and both hsa-miR-23a-3p and hsa-miR-29a-3p levels. All these results demonstrated the suppressing role of hsa-miR-29a-3p on CYP2C19 expression.
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The effects of chemical modifiers of hypoxic radiosensitizer, a 3-nitrotriazole derivative AK-2123 (200 mg/kg) before treatment, and vasodilator of hydralazine (HDZ; 5.0 mg/kg) after treatment on tumor growth of SCCVII of mice were... more
The effects of chemical modifiers of hypoxic radiosensitizer, a 3-nitrotriazole derivative AK-2123 (200 mg/kg) before treatment, and vasodilator of hydralazine (HDZ; 5.0 mg/kg) after treatment on tumor growth of SCCVII of mice were investigated in the radio-thermotherapy combined with mitomycin C (MMC; 2.0 mg/kg) or adriamycin (ADM; 3 mg/kg). The tumor treated by 10 Gy alone (tumor doubling time = 7.5 days), MMC alone (6.9 days), and hyperthermia (43 degrees C, 30 min; HT) alone (8.0 days) showed a slight growth delay (control: 5.6 days). Prolonged growth delay (23.2 days) was observed by MMC-radio-thermotherapy (MMC-10Gy/HT) than that (12.4 days) by 10 Gy/HT. The modification of MMC-radio-thermotherapy by HDZ administered between 10 Gy and HT (MMC-10 Gy/HDZ/HT) resulted in the significant prolongation of tumor growth delay (31.7 days). AK-2123 administration before this treatment, (MMC-AK-2123)-10 Gy/HDZ/HT), enhanced a further tumor growth delay (37.6 days) which is equal to that by 50 Gy alone and resulted in the highest dose modifying factor (DMF) of 5.2. While modification of ADM-radio-thermotherapy by AK-2123 and HDZ, (ADM-AK-2123)-10 Gy/HDZ/HT, gave the equal tumor growth delay to that by 30 Gy alone (DMF = 3.1). These high efficacies of radio-thermo-chemotherapy modified by AK-2123 and HDZ may be caused by tumor blood flow reduction.
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Research Interests: Aging, Adolescent, DNA repair, Humans, T lymphocytes, and 5 moreMiddle Aged, Adult, X chromosome, Cell Survival, and Mutagens
Ginkgo biloba leaf extract has been shown to increase the incidence in liver tumors in mice in a 2-year bioassay conducted by the National Toxicology Program. In this study, the DNA damaging effects of Ginkgo biloba leaf extract and many... more
Ginkgo biloba leaf extract has been shown to increase the incidence in liver tumors in mice in a 2-year bioassay conducted by the National Toxicology Program. In this study, the DNA damaging effects of Ginkgo biloba leaf extract and many of its constituents were evaluated in human hepatic HepG2 cells and the underlying mechanism was determined. A molecular docking study revealed that quercetin, a flavonoid constituent of Ginkgo biloba, showed a higher potential to interact with topoisomerase II (Topo II) than did the other Ginkgo biloba constituents; this in silico prediction was confirmed by using a biochemical assay to study Topo II enzyme inhibition. Moreover, as measured by the Comet assay and the induction of γ-H2A.X, quercetin, followed by keampferol and isorhamnetin, appeared to be the most potent DNA damage inducer in HepG2 cells. In Topo II knockdown cells, DNA damage triggered by Ginkgo biloba leaf extract or quercetin was dramatically decreased, indicating that DNA damage...
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Quantifying health-related biological effects, like genotoxicity, could provide a way of distinguishing between tobacco products. In order to develop tools for using genotoxicty data to quantitatively evaluate the risk of tobacco... more
Quantifying health-related biological effects, like genotoxicity, could provide a way of distinguishing between tobacco products. In order to develop tools for using genotoxicty data to quantitatively evaluate the risk of tobacco products, we tested five carcinogens found in cigarette smoke, 4-aminobiphenyl (4-ABP), benzo[a]pyrene (BaP), cadmium (in the form of CdCl2), 2-amino-3,4-dimethyl-3H-imidazo[4,5-f]quinoline (MeIQ) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), in the mouse lymphoma assay (MLA). The resulting mutagenicity dose responses were analyzed by various quantitative approaches and their strengths and weaknesses for distinguishing responses in the MLA were evaluated. L5178Y/Tk (+/-) 3.7.2C mouse lymphoma cells were treated with four to seven concentrations of each chemical for 4h. Only CdCl2 produced a positive response without metabolic activation (S9); all five chemicals produced dose-dependent increases in cytotoxicity and mutagenicity with S9. The lowes...
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The biological consequences of exposure to piperidine nitroxides is a concern, given their widespread use in manufacturing processes and their potential use in clinical applications. Our previous study reported that TEMPO... more
The biological consequences of exposure to piperidine nitroxides is a concern, given their widespread use in manufacturing processes and their potential use in clinical applications. Our previous study reported that TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl), a low molecular weight free radical, possesses pro-oxidative activity in L5178Y cells. In this study, we investigated and characterized the role of reactive oxygen species (ROS) in TEMPO-induced toxicity in L5178Y cells. We found that TEMPO induced time- and concentration-dependent intracellular ROS production and glutathione depletion. TEMPO also induced apoptosis as demonstrated by increased caspase-3/7 activity, an increased proportion of annexin V stained cells, and decreased expression of anti-apoptotic proteins including Bcl-2, Bcl-xL and Mcl-1. N-acetylcysteine, a ROS scavenger, attenuated the ROS production and apoptosis induced by TEMPO. Moreover, Western blot analyses revealed that TEMPO activated γ-H2A.X, a hallmar...
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The use of usnic acid as a weight loss agent is a safety concern due to reports of acute liver failure in humans. Previously we demonstrated that usnic acid induces apoptosis and cytotoxicity in hepatic HepG2 cells. We also demonstrated... more
The use of usnic acid as a weight loss agent is a safety concern due to reports of acute liver failure in humans. Previously we demonstrated that usnic acid induces apoptosis and cytotoxicity in hepatic HepG2 cells. We also demonstrated that usnic acid induces autophagy as a survival mechanism against its cytotoxicity. In this study, we investigated and characterized further molecular mechanisms underlying the toxicity of usnic acid in HepG2 cells. We found that usnic acid causes endoplasmic reticulum (ER) stress demonstrated by the increased expression of typical ER stress markers, including CHOP, ATF-4, p-eIF2α, and spliced XBP1. Usnic acid inhibited the secretion of Gaussia luciferase measured by an ER stress reporter assay. An ER stress inhibitor 4-phenylbutyrate attenuated usnic acid-induced apoptosis. Moreover, usnic acid significantly increased the cytosolic free Ca(2+) concentration. Usnic acid increased the expression of calcium release-activated calcium channel protein 1 (...
The field of toxicogenomics, which is becoming an important sub-discipline of toxicology, resulted from the natural convergence of the field of conventional toxicological research and the emergent field of functional genomics. One... more
The field of toxicogenomics, which is becoming an important sub-discipline of toxicology, resulted from the natural convergence of the field of conventional toxicological research and the emergent field of functional genomics. One technology that has played a significant role in the field of toxicogenomics (in addition to many others) is the gene expression microarray. In this chapter, the authors provide an example of the application of gene expression microarrays to the field of toxicogenomics by detailing the strategy that was used for obtaining, analyzing, and interpreting gene expression data generated from RNA isolated from the liver of toxicant-exposed rats.
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It is well known that somatic mutations are induced by ionizing irradiation. We have previously reported the measurement of mutant frequency (MF) on the T-cell receptor (TCR) gene in mouse T-lymphocytes after irradiation by flow... more
It is well known that somatic mutations are induced by ionizing irradiation. We have previously reported the measurement of mutant frequency (MF) on the T-cell receptor (TCR) gene in mouse T-lymphocytes after irradiation by flow cytometry. In this study, we developed an in vitro system using murine EL-4 lymphoma cells and observed frequency of cells defective in TCR gene expression after exposure to ionizing irradiation. EL-4 cells were stained with fluorescein-labeled anti-CD4 and phycoerythrin-labeled anti-CD3 antibodies. They were analyzed with a flow cytometer to detect mutant EL-4 cells lacking surface expression of TCR/CD3 complexes which showed CD3-, CD4+ due to a somatic mutation at the TCR genes. Mutant cells could be observed at 2 days after 3 Gy irradiation. MF of EL-4 cells was 6.7x10(-4) for 0 Gy and the value increased to the maximum level of 39x10(-4) between 4 and 8 days after 3 Gy irradiation and these data were found to be best fitted by a linear-quadratic dose-res...
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Comfrey is a rat liver toxin and carcinogen that has been used as a vegetable and herbal remedy by humans. In order to evaluate the mechanisms underlying its carcinogenicity, we examined the mutagenicity of comfrey in the transgenic Big... more
Comfrey is a rat liver toxin and carcinogen that has been used as a vegetable and herbal remedy by humans. In order to evaluate the mechanisms underlying its carcinogenicity, we examined the mutagenicity of comfrey in the transgenic Big Blue rat model. Our results indicate that comfrey is mutagenic in rat liver and the types of mutations induced by comfrey suggest that its tumorigenicity results from the genotoxicity of pyrrolizidine alkaloids in the plant.
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To improve the analyses of a form of oxidative DNA damage, 8-hydroxyguanine (8-OH-Gua), we treated isolated DNA with formamidopyrimidine DNA glycosylase (Fpg) and analyzed the released 8-OH-Gua by using a high-performance liquid... more
To improve the analyses of a form of oxidative DNA damage, 8-hydroxyguanine (8-OH-Gua), we treated isolated DNA with formamidopyrimidine DNA glycosylase (Fpg) and analyzed the released 8-OH-Gua by using a high-performance liquid chromatography system equipped with an electrochemical detector (HPLC-ECD). The human lung carcinoma cells (A549) and human keratinocyte (HaCaT) were irradiated with gamma-rays. After the isolated DNA was treated with the Fpg protein, we analyzed the released 8-OH-Gua by using an HPLC-ECD. With this method, the background level of 8-OH-Gua in DNA from human lung carcinoma cells was determined to be 3.4 residues per 10(7) guanine (Gua). A similar background level of 8-OH-Gua (3.1 residues per 10(7) Gua) was also detected in human keratinocyte DNA with this method. These background 8-OH-Gua levels in cellular DNA are comparable to that obtained previously by an analysis of 8-OH-dGMP after nuclease P1 digestion of cellular DNA (4.3 residues per 10(7) dCMP). A d...
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We have investigated two assays for measuring the induction of mutations using human T-lymphocytes isolated from leukocyte residue buffy coats obtained from normal donors. Variant cell frequency of T-cells defective in the T-cell receptor... more
We have investigated two assays for measuring the induction of mutations using human T-lymphocytes isolated from leukocyte residue buffy coats obtained from normal donors. Variant cell frequency of T-cells defective in the T-cell receptor (TCR) gene expression was measured using a 2-color flow cytometry, and 6-thioguanine-resistant (TGr) cells were determined using a cloning technique at the HPRT gene after treatment with 250 kVp X-rays or ethylnitrosourea (ENU). The frequencies of TCR mutant cells as well as those of TGr cells increased with increasing doses of X-rays or concentrations of ENU studied. For TCR mutants, the induced mutation frequencies at D37 (giving 37% survival) were 31.7 x 10(-4) and 11.0 x 10(-4) for X-rays and ENU, respectively. For TGr T-cells, the induced mutation frequencies at D37 for the same mutagens were 14.4 x 10(-6) and 75.5 x 10(-6), respectively. Over the dose range studied the relationship appears to be linear between the mutation induction of TCR an...
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The aim of this review is to comprehensively summarize the recent achievements in the field of toxicogenomics and cancer research regarding genetic-environmental interactions in carcinogenesis and detection of genetic aberrations in... more
The aim of this review is to comprehensively summarize the recent achievements in the field of toxicogenomics and cancer research regarding genetic-environmental interactions in carcinogenesis and detection of genetic aberrations in cancer genomes by next-generation sequencing technology. Cancer is primarily a genetic disease in which genetic factors and environmental stimuli interact to cause genetic and epigenetic aberrations in human cells. Mutations in the germline act as either high-penetrance alleles that strongly increase the risk of cancer development, or as low-penetrance alleles that mildly change an individual's susceptibility to cancer. Somatic mutations, resulting from either DNA damage induced by exposure to environmental mutagens or from spontaneous errors in DNA replication or repair are involved in the development or progression of the cancer. Induced or spontaneous changes in the epigenome may also drive carcinogenesis. Advances in next-generation sequencing technology provide us opportunities to accurately, economically, and rapidly identify genetic variants, somatic mutations, gene expression profiles, and epigenetic alterations with single-base resolution. Whole genome sequencing, whole exome sequencing, and RNA sequencing of paired cancer and adjacent normal tissue present a comprehensive picture of the cancer genome. These new findings should benefit public health by providing insights in understanding cancer biology, and in improving cancer diagnosis and therapy.
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The use of usnic acid and usnic acid-containing products is associated with acute liver failure; however, mechanistic studies of hepatotoxicity caused by usnic acid are limited. In this study, we investigated and characterized the... more
The use of usnic acid and usnic acid-containing products is associated with acute liver failure; however, mechanistic studies of hepatotoxicity caused by usnic acid are limited. In this study, we investigated and characterized the possible mechanisms, especially the role of autophagy in usnic acid's toxicity in human HepG2 cells. Usnic acid caused apoptosis as demonstrated by an increased caspase-3/7 activity and an increased subdiploid nucleus formation. Usnic acid-induced autophagy as demonstrated by the conversion of LC3B-I to LC3B-II, degradation of P62, and an increased number of puncta. Inhibition of autophagy by treating cells with autophagy inhibitors (3-methyladenine or chloroquine) or by small interfering RNA against Atg7 aggravated usnic acid-induced apoptosis and decreased cell viability, indicating that autophagy plays a protective role against usnic acid-induced toxicity. Moreover, usnic acid activated the MAPK signaling pathway. Usnic acid-elicited apoptosis was e...
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The incidence of childhood cancer is increasing and recent evidence suggests an association between childhood cancer and environmental exposure to genotoxins. In the present study, the Big Blue transgenic mouse model was used to determine... more
The incidence of childhood cancer is increasing and recent evidence suggests an association between childhood cancer and environmental exposure to genotoxins. In the present study, the Big Blue transgenic mouse model was used to determine whether specific periods in early life represent windows of vulnerability to mutation induction by genotoxins in mouse liver. Groups of mice were treated with single doses of 120 mg N-ethyl-N-nitrosourea (ENU)/kg body weight or the vehicle either transplacentally to the 18-day-old fetus or at postnatal days (PNDs) 1, 8, 15, 42 or 126; the animals were sacrificed 6 weeks after their treatment. The cII mutation assay was performed to determine the mutant frequencies (MFs) in the livers of the mice. Liver cII MFs for both sexes were dependent on the age at which the animals were treated. Perinatal treatment with ENU (either transplacental treatment to the 18-day-old fetus or i.p. injection at PND 1) induced relatively high MFs. However, ENU treatment ...