Distinguishing between genotoxic and non-genotoxic hepatocarcinogens by gene expression profiling and bioinformatic pathway analysis.

A rapid and sensitive method to determine the characteristics of carcinogens is needed. In this study, we used a microarray-based genomics approach, with a short-term in vivo model, in combination with insights from statistical and mechanistic analyses to determine the characteristics of carcinogens. Carcinogens were evaluated based on the different mechanisms involved in the responses to genotoxic carcinogens and non-genotoxic carcinogens. Gene profiling was performed at two time points after treatment with six training and four test carcinogens. We mapped the DEG (differentially expressed gene)-related pathways to analyze cellular processes, and we discovered significant mechanisms that involve critical cellular components. Classification results were further supported by Comet and Micronucleus assays. Mechanistic studies based on gene expression profiling enhanced our understanding of the characteristics of different carcinogens. Moreover, the efficiency of this study was demonstrated by the short-term nature of the animal experiments that were conducted.

Stouffer's test in a large scale simultaneous hypothesis testing.

In microarray data analysis, we are often required to combine several dependent partial test results. To overcome this, many suggestions have been made in previous literature; Tippett's test and Fisher's omnibus test are most popular. Both tests have known null distributions when the partial tests are independent. However, for dependent tests, their (even, asymptotic) null distributions are unknown and additional numerical procedures are required. In this paper, we revisited Stouffer's test based on z-scores and showed its advantage over the two aforementioned methods in the analysis of large-scale microarray data. The combined statistic in Stouffer's test has a normal distribution with mean 0 from the normality of the z-scores. Its variance can be estimated from the scores of genes in the experiment without an additional numerical procedure. We numerically compared the errors of Stouffer's test and the two p-value based methods, Tippett's test and Fisher's omnibus test. We also analyzed our microarray data to find differentially expressed genes by non-genotoxic and genotoxic carcinogen compounds. Both numerical study and the real application showed that Stouffer's test performed better than Tippett's method and Fisher's omnibus method with additional permutation steps.

B cell increases and ex vivo IL-2 production as secondary endpoints for the detection of sensitizers in non-radioisotopic local lymph node assay using flow cytometry.

Non-radioisotopic local lymph node assay (LLNA) using 5-bromo-2'-deoxyuridine (BrdU) with flow cytometry (FCM) is gaining attention since it is free from the regulatory issues in traditional LLNA (tLLNA) accompanying in vivo uses of radioisotope, (3)H-thymidine. However, there is also concern over compromised performance of non-radioisotopic LLNA, raising needs for additional endpoints to improve the accuracy. With the full 22 reference substances enlisted in OECD Test Guideline No. 429, we evaluated the performance of LLNA:BrdU-FCM along with the concomitant measurements of B/T cell ratio and ex vivo cytokine production from isolated lymph node cells (LNCs) to examine the utility of these markers as secondary endpoints. Mice (Balb/c, female) were topically treated with substances on both ears for 3 days and then, BrdU was intraperitoneally injected on day 5. After a day, lymph nodes were isolated and undergone FCM to determine BrdU incorporation and B/T cell sub-typing with B220+ and CD3e+. Ex vivo cytokine production by LNCs was measured such as IL-2, IL-4, IL-6, IL-12, IFN-γ, MCP-1, GM-CSF and TNFα. Mice treated with sensitizers showed preferential increases in B cell population and the selective production of IL-2, which matched well with the increases in BrdU incorporation. When compared with guinea pig or human data, BrdU incorporation, B cell increase and IL-2 production ex vivo could successfully identify sensitizers with the accuracy comparable to tLLNA, suggesting that these markers may be useful for improving the accuracy of LLNA:BrdU-FCM or as stand-alone non-radioisotopic endpoints.

Identification of biomarkers of chemically induced hepatocarcinogenesis in rasH2 mice by toxicogenomic analysis.

Toxicogenomic approaches have been applied to chemical-induced heptocarcinogenesis rodent models for the identification of biomarkers of early-stage hepatocarcinogenesis and to help clarify the underlying carcinogenic mechanisms in the liver. In this study, we used toxiciogenomic methods to identify candidate biomarker genes associated with hepatocarcinogenesis in rasH2 mice. Blood chemical, histopathologic, and gene expression analyses of the livers of rasH2 mice were performed 7 and 91 days after the administration of the genotoxic hepatocarcinogens 2-acetylaminofluorene (AAF) and diethylnitrosoamine (DEN), the genotoxic carcinogen melphalan (Mel), and the nongenotoxic noncarcinogen 1-naphthylisothiocynate (ANIT). Histopathologic lesions and a rise in accompanying serum marker levels were found in the DEN-treated rasH2 mice, whereas no neoplastic lesions were observed in the rasH2 mice. However, biological functional analysis using Ingenuity Pathways Analysis (IPA) software revealed that genes with comparable molecular and cellular functions were similarly deregulated in the AAF- and DEN-treated rasH2 mice. We selected 68 significantly deregulated genes that represented a hepatocarcinogen-specific signature; these genes were commonly deregulated in both the AAF- and DEN-treated rasH2 mice on days 7 and 91. Hierarchical clustering analysis indicated that the expression patterns of the selected genes in the hepatocarcinogen (AAF and DEN) groups were distinctive from the patterns in the control, Mel, and ANIT groups. Biomarker filter analysis using IPA software suggested that 28 of the 68 signature genes represent promising candidate biomarkers of cancer. Quantitative real-time PCR analysis confirmed that the deregulated genes, which exhibited sustained up- and down-regulation up to day 91, are likely involved in early-stage hepatocarcinogenesis. In summary, the common and significant gene expression changes induced by AAF and DEN may reflect early molecular events associated with hepatocarcinogenesis, and these "signature" genes may be useful as biomarkers of hepatocarcinogenesis in mice.

Effects of repeated seafood consumption on urinary excretion of arsenic species by volunteers.

Arsenic (As) is a known human carcinogen and widely distributed in the environment. The main route of As exposure in the general population is through food and drinking water. Seafood harvested in Korea contains high-level organoarsenics such as arsenobetaine, arsenocholine, and arsenosugars, which are much less harmful than inorganic arsenics. However, for those who eat large amounts of seafood it is important to understand whether seafood consumption affects urinary levels of inorganic As metabolites such as arsenite, arsenate, monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA). In this study we investigated urinary As metabolites (inorganic As, MMA[V], DMA[V]) and some biological indexes such as AST, GSH, GPX, lipid peroxidation, and uric acid in volunteer study subjects (seven males and nine females). Total urinary As metabolites were analyzed by the hydride generation method, followed by arsenic speciation using HPLC with ICP-mass spectrometry. Study subjects refrained from eating seafood for 3 days prior to the first urine collection and then ingested seafood daily for 6 consecutive days. The first voided urine of the morning was collected from each subject the first day of the consecutive 6 days of seafood ingestion but prior to the first seafood meal. The first voided urine of the morning was also collected on days 1, 2, 3, 4, 5, 6, 7, 10, and 14 after seafood ingestion. The daily mean intake of total As was 6.98 mg, comprised of 4.71 mg of seaweed (67%), 1.74 mg of flat fish (25%), and 0.53 mg of conch (8%). We observed a substantial increase in total urinary As metabolites for subjects consuming seafood from day 1, which recovered to control level at day 10. The increase in total urinary As metabolites was attributed to the increase in DMA, which is a more harmful metabolite than organoarsenics. However, no significant changes in response biological indexes were observed. These results suggest that it is necessary to evaluate As metabolism when assessing the exposure to inorganic As and potential chronic health effects of seafood consumption in Korea.

Profiling of transcripts and proteins modulated by K-ras oncogene in the lung tissues of K-ras transgenic mice by omics approaches.

The mutated K-ras gene is involved in approximately 30% of human cancers. In order to search for K-ras oncogene-induced modulators in lung tissues of K-ras transgenic mice, we performed microarray and proteomics (LC/ESI-MS/MS) analysis. Genes (RAB27b RAS family, IL-1RA, IL-33, chemokine ligand 6, epiregulin, EGF-like domain and cathepsin) related to cancer development (Wnt signaling pathway) and inflammation (chemokine/cytokine signaling pathway, Toll receptor signaling) were up-regulated while genes (troponin, tropomodulin 2, endothelial lipase, FGFR4, integrin alpha8 and adenylate cyclase 8) related to the tumor suppression such as p53 pathway, TGF-beta signaling pathway and cadherin signaling pathway were down-regulated by K-ras oncogene. Proteomics approach revealed that up-regulated proteins in lung adenomas of K-ras mice were classified as follows: proteins related to the metabolism/catabolism (increased from 7 to 22% by K-ras gene), proteins related to translation/transcription and nucleotide (from 4 to 6%), proteins related to signal transduction (from 3 to 5%), proteins related to phosphorylation (from 1 to 2%). ATP synthase, Ras oncogene family, cytochrome c oxidase, flavoprotein, TEF 1, adipoprotein A-1 BP, glutathione oxidase, fatty acid BP 4, diaphorase 1, MAPK4 and transgelin were up-regulated by K-ras oncogene. However, integrin alpha1, Ras-interacting protein (Rain), endothelin-converting enzyme-1d and splicing factor 3b were down-regulated. These studies suggest that genes related to cancer development and inflammation were up-regulated while genes related to the tumor suppression were down-regulated by K-ras, resulting in the tumor growth. Putative biomarkers such as cell cycle related genes (Cdc37), cancer cell adhesion (Glycam 1, integrin alpha8, integrin alphaX and Clec4n), signal transduction (Tlr2, IL-33, and Ccbp2), migration (Ccr1, Ccl6, and diaphorase 1 (Cyb5r3) and cancer development (epiregulin) can be useful for diagnosis and as prognosis markers and some of the target molecules can be applied for prevention of cancer.

Genotoxicity Studies on Carrageenan: Short-term In Vitro Assays.

Carrageenan is a naturally-occurring sulfated polygalactan which has been widely used in the dairy industry and a gelling agent in non-dairy products. In this study, four short-term in vitro genotoxicity assays were investigated to evaluate the potential genotoxic effects of carrageenan. The mutagenic-ity of carrageenan was evaluated up to a maximum dose of 5 mg/plate in Ames test. There was no increase in the number of revertant colonies compared to its negative control at any dose in all of strains tested. To assess clastogenic effect, the in vitro chromosomal aberration assay was performed using Chinese hamster lung cells. Carrageenan was not considered to be clastogenic in this assay at up to the highest feasible concentration which could be evaluated. The in vitro comet assay and micronucleus test results obtained on L5178Y cells also revealed that carrageenan has no genotoxicity potential, although there was a marginal increase in micronuclei frequencies and DNA damage in the respective micronucleus and comet assays. Taken together, our results indicate that carrageenan was not genotoxic based on four in vitro genotoxicity results.

Profiling of transcripts and proteins modulated by the E7 oncogene in the lung tissue of E7-Tg mice by the omics approach.

The E6 and E7 oncoproteins of human papilloma virus (HPV) type 16 have been known to cooperatively induce the immortalization and transformation of primary keratinocytes. We established an E7 transgenic mouse model to screen HPV-related biomakers using the omics approach. The methods used to identify HPV-modulated factors were genomics analysis by microarray using the Affymetrix 430 2.0 array to screen E7-modulated genes, and proteomics analysis using nano-LC-ESI-MS/MS to screen E7-modulated proteins with the lung tissue of E7 transgenic mice. According to omics data, cyclin B1, cyclin E2, topoisomerase IIα, calnexin, activated leukocyte cell adhesion molecule CD166, actinin α1, diaphorase 1, gelsolin, platelet glycoprotein, and annexin A2 and A4 were up-regulated in the E7-Tg mice, while proteoglycan 4, sarcolipin, titin, vimentin, drep 1, troponin and cofilin-1 were down-regulated. We further confirmed the significance of differences between the expression levels of the selected factors in E7-Tg and non-Tg mice by real-time PCR. Genes related to cancer cell adhesion, cell cycle and migration, proliferation and apoptosis, as well as to the intermediate filament network and to endoplasmic reticulum proteins, were selected. Taken together, the results suggest that the E7 oncogene modulates the expression levels of cell cycle-related (cyclin B1, cyclin E2) and cell adhesion- and migration-related (actinin α1, CD166) factors, which may play important roles in cellular transformation in cancer. In addition, the solubilization of the rigid intermediate filament network by specific proteolysis mediated via up-regulating gelsolin and down-regulating cofilin-1, as well as increased levels of endoplasmic reticulum protein calnexin with chaperone functions, might also be involved in E7-lung epithelial cells.

In Vitro Studies on the Genotoxic Effects of Wood Smoke Flavors.

Smoke flavors based on the thermal decomposition of wood have been applied to a variety of food products as an alternative for traditional smoking. Despite its increasing use, the available genotoxicity data on wood smoke flavors (WSF) are still controversial. Thus, potential genotoxic effects of WSF in four short-term in vitro genotoxicity assays were investigated, which included the Ames assay, chromosomal aberration assay, micronucleus test and the alkaline comet assay. WSF did not cause any mutation in the Ames assay using five tester strains at six concentrations of 0.16, 0.31, 0.63, 1.25, 2.5 and 5 µl/plate. To assess clastogenic effect, the in vitro chromosomal aberration assay was performed using Chinese hamster lung cells. No statistically significant increase in the number of metaphases with structural aberrations was observed at the concentrations of 1.25, 2.5, and 5 µl/ml. The in vitro comet assay and micronucleus test results obtained on L5178Y cells also revealed that WSF has no genotoxicity potential, although there was a marginal increase in micronuclei frequencies and DNA damage in the respective micronucleus and comet assays. Taken together, based on the results obtained from these four in vitro studies, it is concluded that WSF is not a mutagenic agent in bacterial cells and causes no chromosomal and DNA damage in mammalian cells in vitro.

Update on implementation of GHS in Korea and revision of Industrial Safety and Health Act in relation to GHS.

To implement the globally harmonized system of classification and labelling of chemicals (GHS) in Korea, an inter-ministerial GHS committee, involving 8 ministries and an expert working group composed of 9 experts from relevant organizations and one private consultant, have made some progress towards implementation by 2008. As such, the first revision of the official Korean translated version of the GHS in accordance with the GHS purple book revision 1 in 2005, including annexes, started in August, 2006, was completed in December, 2006. The Ministry of Labor also finally revised the Industrial Safety and Health Act (ISHA) relating to the GHS and the detailed notification was announced on Dec 12, 2006 and became effective immediately. The revised ISHA will allow continued use of the existing hazard communication system until Jun 30, 2008. Other revisions of chemical-related regulations will follow soon to facilitate the implementation of the GHS by 2008. Besides, inter-ministerial collaborative efforts on harmonizing regulations and disseminating the GHS in Korea will continue to avoid any confusion or duplication and for the effective use of resources.

Subchronic toxicity studies of the aqueous extract of Aristolochiae fructus in Sprague-Dawley rats.

The subchronic toxicity of Aristolochiae fructus containing aristolochic acids (AAs), a natural component in the Aristolochiaceae family, was investigated. The A. fructus was daily administered by gavage to male and female rats for 90 d at dose levels of 21.35, 213.5, and 2135 mg/kg (equivalent to 0.05, 0.5, and 5 mg/kg as AAs, respectively). During the test period, clinical signs, mortality, body weights, food and water consumption, hematology, serum biochemistry, organ weights, and histopathology were examined. Significant decreases in body weight gain were noted in the high-dose group receiving both the aqueous extract of A. fructus and AAs. Decreases in food consumption were noted beginning at 50 d and did not recover in the high-dose group of aqueous extract of A. fructus and AAs. Irrespective of dose, water consumption was not affected. There was no mortality or adverse clinical signs, hematology, or serum biochemistry in the treatment groups versus control. Nephrotoxicity and hyperplasia of epithelial cells in the forestomach were observed in rats receiving the highest dose of aqueous extract of A. fructus and at doses of >or= 0.5 mg/kg/day AAs. For both genders, the no-observed-adverse-effect level (NOAEL) for A. fructus based on this subchronic study in rats was considered to be 21.3 mg/kg/d.

Identification of glutathione conjugates of 2, 3-dibromopropene in male ICR mice.

Hepatotoxic potential of 2, 3-dibromopropene (2, 3-DBPE) and its conjugation with glutathione (GSH) were investigated in male ICR mice. Treatment of mice with 20, 50, and 100 mg/kg of 2, 3-DBPE for 24 h caused elevation of serum alanine aminotransferase and aspartate aminotransferase activities. The hepatic content of GSH was not changed by 2, 3-DBPE. Meanwhile, the GSH content was slightly reduced when mice were treated with 2, 3-DBPE for 6 h and significantly increased 12 h after the treatment. Subsequently, a possible formation of GSH conjugate of 2, 3-DBPE was investigated in vivo. After the animals were treated orally with 20, 50, and 100 mg/kg of 2, 3-DBPE, the animals were subjected to necropsy 6, 12, and 24 h later. A conjugate of S-2-bromopropenyl GSH was identified in liver and serum treated with 100 mg/kg of 2, 3-DBPE by using liquid chromatography-electrospray ionization tandem mass spectrometry. The protonated molecular ions [M+H]+ of S-2-bromopropenyl GSH were observed at m/z 425.9 and 428.1 in the positive ESI spectrum with a retention time of 6.35 and 6.39 min, respectively. In a time-course study in livers following an oral treatment of mice with 100 mg/kg of 2, 3-DBPE for 6, 12, and 24 h, the 2, 3-DBPE GSH conjugate was detected maximally 6 h after the treatment. The present results suggested that 2, 3-DBPE-induced hepatotoxicity might be related with the production of its GSH conjugate.

In vivo and in vitro immunosuppressive effects of benzo[k]fluoranthene in female Balb/c mice.

Although polycyclic aromatic hydrocarbons (PAHs) have been known to suppress immune responses, few studies have addressed the immunotoxicity of benzo[k]fluoranthene (B[k]F). In this study, we investigated the immunosuppression by B[k]F, both in vivo and in vitro, in female BALB/c mice. To assess the effects of B[k]F on humoral immunity as splenic antibody response to sheep red blood cells (SRBCs), B[k]F was given a single dose or once daily for 7 consecutive days po with 30, 60, and 120 micromol/kg. B[k]F reduced the number of antibody-forming cells (AFCs) in a dose-dependent manner. Subacute treatment with B[k]F caused weight increases in liver and decreases in spleen and thymus. The number of AFCs was dramatically decreased by B[k]F in a dose-dependent manner. In a subsequent study, mice were subacutely exposed to the same doses of B[k]F without an immunization with SRBCs, followed by splenic and thymic lymphocyte phenotypings using a flow cytometry and ex vivo mitogen-stimulated proliferation. B[k]F-exposed mice exhibited reduced splenic and thymic cellularity, decreased numbers of total T cells, CD4(+) cells, and CD8(+) cells in spleen, and immature CD4(+)CD8(+) cells, CD4(+)CD8(-) cells, and CD8(+)CD4(-) cells in thymus. The number of CD4(+) IL-2(+) cells was reduced by about 11%, 31%, and 53% following exposure of mice to 30, 60, and 120 micromol/kg of B[k]F, respectively. In the ex vivo lymphocyte proliferation assay, B[k]F inhibited splenocyte proliferation by LPS and Con A. In the in vitro mitogen-stimulated proliferation by untreated splenic suspensions, B[k]F only suppressed splenocyte proliferation to LPS. These results suggested that B[k]F-induced immunosuppression might be mediated, at least in part, through the IL-2 production, and caused by mechanisms associated with metabolic processes.

Investigation of the bovine leukemia virus proviral DNA in human leukemias and lung cancers in Korea.

The bovine leukemia virus (BLV) is the causative agent of enzootic bovine leucosis. This study investigated the presence of the BLV in leukemia (179 acute lymphoblastic leukemia, 292 acute myeloid leukemia and 46 chronic myelogenous leukemia cases) and 162 lung cancer patients (139 adenocarcinoma, 23 squamous cell carcinoma) to determine if the BLV is a causative organism of leukemia and lung cancer in Koreans. A BLV infection was confirmed in human cells by PCR using a BLV-8 primer combination. All 517 cases of human leukemia and 162 lung cancer were negative for a PCR of the BLV proviral DNA. In conclusion, although meat has been imported from BLV endemic areas, the BLV infection does not appear to be the cause of human leukemia or lung cancer in Koreans. These results can be used as a control for further studies on the BLV in Koreans.

Protective effect of diallyl disulfide on oxidative stress-injured neuronally differentiated PC12 cells.

The effects of diallyl disulfide (DADS), a garlic-derived compound, on the viability of neuronal cells and cell signals, including phosphatidylinositol 3-kinase (PI3K)/Akt, glycogen synthase kinase-3 (GSK-3), cytochrome c, caspase-3, and poly(ADP-ribose) polymerase (PARP), were investigated in PC12 cells neuronally differentiated by nerve growth factor. To evaluate the toxicity of DADS itself, nPC12 cells were treated with several concentrations of DADS, and 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and trypan blue stain revealed that the viability was not affected by low concentration of DADS, up to 20 microM, but it was decreased at higher than this concentration. The levels of free radicals and membrane lipid peroxidation were significantly increased in nPC12 cells when treated with more than 50 microM DADS, and treatment of PC12 cells with 100 microM DADS killed the cells by inhibiting PI3K/Akt and by promoting activation of GSK-3 and caspase-3, release of cytochrome c, and cleavage of PARP. To evaluate the protective effects of low concentration of DADS on oxidative stress-injured nPC12 cells, the viability of the cells (pretreated with DADS for 2 h vs. not pretreated) was evaluated 24 h after exposure to 100 microM H2O2 for 30 min. Compared to the cells treated with 100 microM H2O2 only, pretreatment of the cells with 20 microM DADS before exposure to 100 microM H2O2 increased the viability and induced activation of PI3K and Akt, inactivation of GSK-3, and inhibition of cytochrome c release, caspase-3 activation, and PARP cleavage. These results indicate that low concentration of DADS has neuroprotective effects by activating PI3K/Akt and by inhibiting GSK-3 activation, cytochrome c release, caspase-3 activation, and PARP cleavage, whereas high concentration is rather cytotoxic. Therefore, some specific optimum concentration of DADS may be a new potential therapeutic strategy for oxidative stress injured in vitro model of neurodegenerative diseases.

Irreversible inhibition of CD13/aminopeptidase N by the antiangiogenic agent curcumin.

CD13/aminopeptidase N (APN) is a membrane-bound, zinc-dependent metalloproteinase that plays a key role in tumor invasion and angiogenesis. Here, we show that curcumin, a phenolic natural product, binds to APN and irreversibly inhibits its activity. The direct interaction between curcumin with APN was confirmed both in vitro and in vivo by surface plasmon resonance analysis and an APN-specific antibody competition assay, respectively. Moreover, curcumin and other known APN inhibitors strongly inhibited APN-positive tumor cell invasion and basic fibroblast growth factor-induced angiogenesis. However, curcumin did not inhibit the invasion of APN-negative tumor cells, suggesting that the antiinvasive activity of curcumin against tumor cells is attributable to the inhibition of APN. Taken together, our study revealed that curcumin is a novel irreversible inhibitor of APN that binds to curcumin resulting in inhibition of angiogenesis.

Betulinic acid inhibits growth factor-induced in vitro angiogenesis via the modulation of mitochondrial function in endothelial cells.

Betulinic acid (BetA), a pentacyclic triterpene, is a selective apoptosis-inducing agent that works directly in mitochondria. Recent study has revealed that BetA inhibits in vitro enzymatic activity of aminopeptidase N (APN, EC 3.4.11.2), which is known to play an important role in angiogenesis, but the anti-angiogenic activity of BetA has not been reported yet. Data presented here show that BetA potently inhibited basic fibroblast growth factor (bFGF)-induced invasion and tube formation of bovine aortic endothelial cells (BAECs) at a concentration which had no effect on the cell viability. To access whether the anti-angiogenic nature of BetA originates from its inhibitory action against aminopeptidase N (APN) activity, the effect of BetA on APN was investigated. Surprisingly, BetA did not inhibit in vivo APN activity in endothelial cells or APN-positive tumor cells. On the other hand, BetA significantly decreased the mitochondrial reducing potential, and treatment with mitochondrial permeability transition (MPT) inhibitors attenuated BetA-induced inhibition of endothelial cell invasion. These results imply that anti-angiogenic activity of BetA occurs through a modulation of mitochondrial function rather than APN activity in endothelial cells.