Global gene expression associated with hepatocarcinogenesis in adult male mice induced by in utero arsenic exposure.Our previous work has shown that exposure to inorganic arsenic in utero in utero (in u´ter-o) [L.] within the uterus.
In the uterus.
in utero adv. produces hepatocellular carcinoma hep·a·to·cel·lu·lar carcinoma
A carcinoma derived from parenchymal cells of the liver. Also called hepatocarcinoma, malignant hepatoma. (HCC HCC Hepatocellular Carcinoma (liver cancer)
HCC Hertfordshire County Council (administrative region of south eastern England UK)
HCC Harford Community College (Maryland) ) in adult male mice. To explore further the molecular mechanisms of transplacental transplacental /trans·pla·cen·tal/ (-plah-sen´tal) through the placenta.
Relating to or involving passage through or across the placenta. arsenic hepatocarcinogenesis, we conducted a second arsenic transplacental carcinogenesis car·ci·no·gen·e·sis
The production of cancer.
production of cancer.
viruses and some parasites are capable of initiating neoplasia. study and used a genomewide microarray in profile arsenic-induced aberrant gene expression more extensively. Briefly, pregnant C3H C3H Coumarate 3 Hydroxylase mice were given drinking water drinking water
supply of water available to animals for drinking supplied via nipples, in troughs, dams, ponds and larger natural water sources; an insufficient supply leads to dehydration; it can be the source of infection, e.g. leptospirosis, salmonellosis, or of poisoning, e.g. containing 85 ppm arsenic as sodium arsenite or unaltered water from days 8 to 18 of gestation. The incidence of HCC in adult male offspring was increased 4-fold and tumor multiplicity 3-fold after transplacental arsenic exposure. Samples of normal liver and liver tumors were taken at autopsy for genomic analysis. Arsenic exposure in utero resulted in significant alterations (p < 0.001) in the expression of 2,010 genes in arsenic-exposed liver samples and in the expression of 2,540 genes in arsenic-induced HCC. Ingenuity Pathway Analysis revealed that significant alterations in gene expression occurred in a number of biological networks, and Myc plays a critical role in one of the primary networks. Real-time reverse transcriptase-polymerase chain reaction and Western blot analysis West·ern blot analysis
An electrophoretic procedure for separating proteins. of selected genes/ proteins showed > 90% concordance concordance /con·cor·dance/ (-kord´ins) in genetics, the occurrence of a given trait in both members of a twin pair.concor´dant
n. . Arsenic-altered gene expression included activation of oncogenes oncogenes
1. genes carried by tumor viruses that are directly and solely responsible for the neoplastic transformation of host cells. Many oncogenes function after integration into the DNA of the host cell and some up-regulate normal downstream host cell genes to cause neoplasia. and HCC biomarkers, and increased expression of cell proliferation-related genes, stress proteins, and insulin-like growth factors insulin-like growth factors (IGF),
n a group of polypeptides responsible for the activity of growth hormones, similar in chemical structure to insulin. and genes involved in cell-cell communications. Liver feminization feminization /fem·i·ni·za·tion/ (fem?i-ni-za´shun)
1. the normal development of primary and secondary sex characters in females.
2. the induction or development of female secondary sex characters in the male. was evidenced by increased expression of estrogen-linked genes and altered expression of genes that encode gender-related metabolic enzymes. These novel findings are in agreement with the biology and histology of arsenic-induced HCC, thereby indicating that multiple genetic events are associated with transplacental arsenic hepatocarcinogenesis. Key words: Agilent mouse oligo 22K microarray, arsenic, hepatocellular carcinoma, real-time RT-PCR RT-PCR
reverse transcriptase-polymerase chain reaction. See PCR1. , transplacental exposure, Western blotting. Environ Health Perspect 114:404-411 (2006). doi: 10.1289/ehp.8534 available via http://dx.doi.ong/[Online 6 October 2005]
Inorganic arsenic is an environmental pollutant. Arsenic exposure in humans comes mainly from consumption of drinking water contaminated with inorganic arsenic [National Research Council (NRC NRC
1. National Research Council
2. Nuclear Regulatory Commission
Noun 1. NRC - an independent federal agency created in 1974 to license and regulate nuclear power plants ) 1999], but elevated exposure can occur from burning of coal containing high levels of inorganic arsenic (Liu et al. 2002). Epidemiologic studies have demonstrated that chronic arsenic exposure causes tumors of the skin, urinary bladder, lung, liver, prostate, kidney, and possibly other sites [International Agency for Research on Cancer The International Agency for Research on Cancer (IARC, or CIRC in its French acronym) is an intergovernmental agency forming part of the World Health Organisation of the United Nations.
Its main offices are in Lyon, France. ([ARC) 1987; Morales et al. 2000; NRC 1999]. Until recently, inorganic arsenic was considered a "paradoxical" human carcinogen carcinogen: see cancer.
Agent that can cause cancer. Exposure to one or more carcinogens, including certain chemicals, radiation, and certain viruses, can initiate cancer under conditions not completely understood. because of strong human evidence but limited evidence for animal carcinogenesis (IARC 1987; Waalkes et al. 2004a, 2004b). Indeed, negative results had been obtained for inorganic arsenic carcinogenesis in mice, rats, hamsters, rabbits, dogs, and monkeys (IARC 1987; Kitchin 2001). Recently, important advances have been made in the development of rodent models of inorganic arsenic carcinogenesis. These include skin cancer models in which inorganic arsenic acts as a co-promoter with 12-O-tetradecanoyl phorbol-13-acetate in Tg.AC mice (Germolec et al. 1998), or as a co-carcinogen with ultraviolet irradiation in hairless mice (Rossman et al. 2001). In addition, our group has developed a transplacental model in which short-term exposure to inorganic arsenic in utero produces a variety of internal tumors in the offspring when mice reach adulthood (Waalkes et al. 2003, 2004b). These tumors include cancers of the liver, lung, ovary ovary, ductless gland of the female in which the ova (female reproductive cells) are produced. In vertebrate animals the ovary also secretes the sex hormones estrogen and progesterone, which control the development of the sexual organs and the secondary sexual , and adrenal adrenal /ad·re·nal/ (ah-dre´n'l)
2. adrenal gland.
3. pertaining to an adrenal gland.
1. induced in adulthood by transplacental exposure to arsenic (Waalkes et al. 2003, 2004b).
The brief period of exposure (10 days) to inorganic arsenic, which was sufficient for it to be a complete transplacental carcinogen in mice, is not only alarming but points to a clear period of high sensitivity to the metalloid metalloid (met´loid),
n a nonmetallic element that behaves as a metal under certain conditions. . Indeed, gestation is a period of high sensitivity to chemical carcinogenesis in general in both rodents and probably humans (Anderson et al. 2000). Inorganic arsenic can readily cross the placenta and enter the fetal system (Chattopadhyay et al. 2002; Concha concha /con·cha/ (kong´kah) pl. con´chae [L.] a shell-shaped structure.
concha of auricle et al. 1998; NRC 1999). Thus, in human populations exposed to inorganic arsenic, it is reasonable to assume that exposure during gestation does occur, and that the carcinogenesis sensitivity observed in rodents may predict similar effects in humans.
Hepatocellular carcinoma (HCC) has been identified as a tumor type associated with arsenic exposure in humans (Centeno et al. 2002; Chen and Ahsan 2004; Chen et al. 1997; Zhou et al. 2002). HCC and nonalcoholic non·al·co·hol·ic
A beverage usually containing less than 0.5 percent alcohol by volume. liver cirrhosis and ascites Ascites Definition
Ascites is an abnormal accumulation of fluid in the abdomen.
Rapidly developing (acute) ascites can occur as a complication of trauma, perforated ulcer, appendicitis, or inflammation of the colon or other are leading causes of mortality in the arsenic endemic area of Guizhou, China (Liu et al. 2002; Zhou et al. 2002). Also, hepatomegaly hepatomegaly /hep·a·to·meg·a·ly/ (hep?ah-to-meg´ah-le) enlargement of the liver.
The abnormal enlargement of the liver. Also called megalohepatia. and liver diseases are commonly associated with arsenic exposure through the drinking water in West Bengal, India (Santra et al. 2000). In accord with human data, transplacental exposure to inorganic arsenic in mice induced a marked, dose-related increase in HCC formation in male offspring in both an initial (Waalkes et al. 2003) and a subsequent followup study (Waalkes et al. 2004a, 2004b). In utero arsenic exposure also caused a variety of hepatic genes to be aberrantly expressed, including genes probably critical to the carcinogenic carcinogenic
having a capacity for carcinogenesis. process, as evidenced by analysis of liver samples taken at autopsy during the initial transplacental study with sodium arsenite (Liu et al. 2004; Waalkes et al. 2004a). This initial genomic analysis was limited to individual genes of interest (Waalkes et al. 2004a) or to a limited number of genes (588) in a custom array (Liu et al. 2004).
Our second transplacental arsenic carcinogenesis study in mice (Waalkes et al. 2004b) confirmed the hepatocarcinogenic potential of gestational arsenic exposure seen in the initial study (Waalkes et al. 2003). It also provided a unique opportunity to perform a genomewide analysis through the National Center for Toxicogenomics using the Agilent 22K chip arrays. Changes in expression of specific genes of interest were confirmed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis. This study confirmed and extended our initial observations, but it also revealed a number of novel pathways and gene expression alterations potentially associated with hepatocarcinogenesis induced by transplacental arsenic exposure.
Materials and Methods
Chemicals. Sodium arsenite (NaAs[O.sub.2]) was obtained from Sigma Chemical Co. (St. Louis, MO) and dissolved in the drinking water at 85 mg arsenic/L (85 ppm). The Agilent 22K mouse oligo array was obtained from Agilent Technologies (Palo Alto, CA). Monoclonal antibodies against [alpha]-fetoprotein (sc-8399), K-ras (sc-30), c-myc (sc-42), estrogen receptor (ER)-[alpha] (sc-8002), [beta]-actin (sc-1616) and polyclonal antibodies against cyclin cy·clin
A class of proteins that fluctuate in concentration at specific points during the cell cycle and that regulate the cycle by binding to a kinase. DI (sc-753), cdk4 (sc-601), and endothelial endothelial /en·do·the·li·al/ (-the´le-al) pertaining to or made up of endothelium.
A layer of cells that lines the inside of certain body cavities, for example, blood vessels. growth factor receptor A growth factor receptor is a receptor which binds to growth factor. External links
• • (EGFR EGFR Epidermal Growth Factor Receptor (a kinase enzyme)
EGFR Estimated Glomerular Filtration Rate , sc-03) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibodies against proliferating cell nuclear antigen (PCNA PCNA Proliferating Cell Nuclear Antigen
PCNA Preventive Cardiovascular Nurses Association
PCNA Pepsi Cola North America
PCNA Post Conflict Needs Assessment (United Nations)
PCNA Pudelpointer Club of North America , 610664), cytokeratin 5/8 (550505), and plasminogen activator inhibitor-1 (PAI-1, 612024) were purchased from BD Biosciences (San Jose, CA). All other chemicals were commercially available and of reagent grade.
Animal treatment and sample collection. We conducted the present study using liver tumors and nontumorous normal liver samples collected in the second of a series of transplacental arsenic carcinogenesis studies in mice for which pathological results have already been reported (Waalkes et al. 2004b). In this study timed pregnant C3H mice were given drinking water ad libitum containing 85 ppm arsenic as sodium arsenite or unaltered water from days 8-18 of gestation. Offspring were weaned at 4 weeks, then randomly placed into separate groups (n = 25 from 10 dams/group) according to maternal exposure level and observed for up to 104 weeks. Animal care was provided in accordance with the Guide on the Care and Use of Animals (Institute of Laboratory Animal Resources 1996), and the Institutional Animal Care and Use Committee Institutional Animal Care and Use Committees are of central importance to the application of laws to animal research in the United States. Most research involving laboratory animals is funded by the United States National Institutes of Health or other federal agencies. approved the study proposal. Animals used in this study were treated humanely and with regard for the alleviation of suffering. Samples of large tumors, later confirmed as HCC, and normal surrounding liver tissues were taken at autopsy, snap frozen in liquid nitrogen, and compared with normal liver samples from contemporaneous controls. A total of 17 samples from male mice were analyzed, including 5 inorganic arsenic-induced HCC samples (designated As-HCC), 5 samples of surrounding nontumorous, normal liver tissues from arsenic-treated mice (As-Normal), and 7 samples of age-matched control mouse liver (Control).
Microarray analysis. We isolated total RNA RNA: see nucleic acid.
in full ribonucleic acid
One of the two main types of nucleic acid (the other being DNA), which functions in cellular protein synthesis in all living cells and replaces DNA as the carrier of genetic from liver samples with TRIzol agent (Invitrogen, Carlsbad, CA), followed by purification and DNase-I digestion with RNeasy columns (Qiagen, Valencia, CA). We evaluated the quality of the RNA using an Agilent 2100 Bioanalyzer (Agilent Technologies). Total RNA was amplified using the Agilent Low RNA Input Fluorescent Linear Amplification Kit protocol. Starting with 500 ng of total RNA, cyanine cy·a·nine
Any of various blue dyes, used to sensitize photographic emulsions to a greater range of light. (Cy)3- or Cy5-labeled cRNA was produced according to the manufacturer's protocol. For each two-color comparison, 750 ng each of Cy3- and Cy5-labeled cRNA was mixed and fragmented using the Agilent In Situ Hybridization in situ hybridization A method for localizing a sequence of DNA, mRNA, or protein in a cell or tissue; the use of a DNA or RNA probe to detect a cDNA sequence in chromosome spreads or in interphase nuclei or an RNA sequence of cloned bacterial or cultured Kit protocol (Agilent Technologies). We performed hybridizations on Agilent mouse 22K oligo assay for 17 hr in a rotating hybridization hybridization /hy·brid·iza·tion/ (hi?brid-i-za´shun)
1. crossbreeding; the act or process of producing hybrids.
2. molecular hybridization
3. oven using the Agilent 60-mer oligo microarray processing protocol. Slides were washed as indicated in this protocol and then scanned with an Agilent Scanner. Data were obtained using the Agilent Feature Extraction software (version 7.5), employing defaults for all parameters. Two hybridizations with fluor reversals were performed for each RNA sample from each group.
Rosetta Resolver. Images and GEML GEML Gene Expression Markup Language (Rosetta Inpharmatics, Inc.) (gene expression markup language) files, including error and p-values, were exported from the Agilent Feature Extraction software and deposited into the Rosetta Resolver system (version 4.0, build 126.96.36.199.7.RSPLIT) (Rosetta Biosoftware, Kirkland, WA). We combined the resultant ratio profiles. Intensity plots were generated for each ratio experiment, and genes were considered "signature genes" if the p-value was < 0.001.
Ingenuity Pathways Analysis. We further analyzed the signature genes with Ingenuity Pathways Analysis (Ingenuity Systems, Redwood City, CA; http://www.ingenuiry. com), a web-delivered application that enables biologists to discover, visualize, and explore relevant networks significant to their experimental results, such as gene expression array data sets. A data set containing gene identifiers and their corresponding expression values such as fold-changes and p-values was uploaded as a tab-delimited text file. Each gene identifier was mapped to its corresponding gene object in the Ingenuity Pathways Knowledge Base. A fold change cutoff of 1.5-fold and p < 0.001 was set to identify genes whose expression was differentially regulated. These genes, called "focus genes," were then used as the starting point for generating biological networks. To start building networks, the application queries the Ingenuity Pathways Knowledge Base for interactions between focus genes and all other gene objects stored in the knowledge base, then generates a set of networks with a network size of approximately 35 genes or proteins. Ingenuity Pathways Analysis then computes a score for each network according to the fit of the user's set of significant genes. The score is derived from a p-value and indicates the likelihood of the focus genes in a network being found together because of random chance. A score of 2 indicates a 1 in 100 change that the focus genes are together in a network because of random chance. Therefore, scores of 2 or higher have at least a 99% confidence level of not being generated by random chance alone. Biological functions are then calculated and assigned to each network.
Real-time RT-PCR analysis. We used real-time RT-PCR analysis to quantify the levels of expression of the selected genes (Liu et al. 2004). The forward and reverse primers for selected genes were designed using ABI Abi (ā`bī) [short for Abijah], in the Bible, King Hezekiah's mother.
(Application Binary Interface) A specification for a specific hardware platform combined with the operating system. Primer Express software (Applied Biosystems, Foster City, CA). Total RNA was reverse transcribed with MuLV reverse transcriptase and oligo-dT primers, then subjected to real-time PCR PCR polymerase chain reaction.
polymerase chain reaction
Polymerase chain reaction (PCR) analysis using SYBR green PCR master mix (Applied Biosystems, Cheshire, UK). The relative differences in expression between groups were determined using cycle time (Ct) values as follows: The Ct values of the genes of interest were first normalized with [beta]-actin of the same sample; then the relative differences between control and treatment groups were calculated and expressed as relative increases, setting the control as 100%. Assuming the Ct value reflects the initial starting copy and there is 100% efficiency, a difference of one cycle is equivalent to a 2-fold difference in starting copy.
Western blot analysis. Tissues were homogenized ho·mog·e·nize
v. ho·mog·e·nized, ho·mog·e·niz·ing, ho·mog·e·niz·es
1. To make homogeneous.
a. To reduce to particles and disperse throughout a fluid.
b. (1:20, w:v) in PER-Tissue Protein Extraction buffer (Pierce, Rockford, IL) containing freshly added protease inhibitor cocktail (Calbiochem, La Jolla, CA) and 100 [micro]M phenylmethylsulfonyl fluoride. Cytosols were prepared by centrifugation Centrifugation
A mechanical method of separating immiscible liquids or solids from liquids by the application of centrifugal force. This force can be very great, and separations which proceed slowly by gravity can be speeded up enormously in centrifugal at 15,000xg for 10 min at 4[degrees]C. Protein concentrations were determined using the dye-binding assay (Bio-Rad, Hercules, CA). Total protein (30 [micro]g) was subjected to electrophoresis on NuPAGE Bis-Tris gels (4-12%) (Invitrogen, San Diego, CA), followed by electrophoretic transfer to nitrocellulose nitrocellulose, nitric acid ester of cellulose (a glucose polymer). It is usually formed by the action of a mixture of nitric and sulfuric acids on purified cotton or wood pulp. membranes at 30 V for 1 hr. Membranes were blocked in 5% dried milk in TBST TBST Tony Backhurst Scuba Travel (UK) (15 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.05% Tween tween
A child between middle childhood and adolesence, usually between 8 and 12 years old.
[Blend of teen1 and between.] 20) for 2 hr, followed by incubation with the primary antibody (1:200 to 1:1,000) in Blotto blot·to
[Perhaps from blot1.]
Brit, Austral & NZ slang (Pierce, Rockford, IL) overnight at 4[degrees]C. After washes with TBST, the membranes were incubated in horse radish peroxidase-conjugated secondary antibody (1:4,000 to 1:10,000) for 1 hr and washed with TBST 3 times. Immunoblots were visualized using SuperSignal chemiluminescent chem·i·lu·mi·nes·cence
Emission of light as a result of a chemical reaction at environmental temperatures.
chem substrate (Pierce, Rockford, IL).
Statistics. For microarray analysis, we cross-hybridized samples with Cy3 and Cy5. For Ingenuity Pathways Analysis, we used a Fisher's exact test Fisher's exact test
a statistical test for association in a two-by-two table based on the exact hypergeometric distribution of the frequencies within the table. to calculate a p-value to determine the probability that the biological function assigned to that network could be explained by chance alone. For real-time RT-PCR analysis, means and SEM of individual samples (n = 5-7) were calculated. For the comparisons of gene expression between two groups, Student's t-test was performed. For comparisons among three or more groups, data were analyzed using analysis of variance, followed by Duncan's multiple range test. The level of significance was set at p < 0.05 in all cases.
Microarray analysis of aberrantly expressed genes. The hepatocarcinogenic effects of inorganic arsenic have been verified in a study in which HCC developed in high incidence in male mice after in utero exposure (Waalkes et al. 2004b). Thus, total RNA was isolated from samples of control mouse liver, arsenic-exposed nontumorous normal liver, and arsenic-induced HCC taken at autopsy, and subjected to microarray analysis. Under the criteria of p < 0.001 by the Rosetta Resolver (version 4.0) system, the expression of 2,010 genes was significantly altered in arsenic-exposed normal liver samples compared with control, and 2,540 genes were altered in arsenic-induced HCC. The clustering analysis of these gene alterations clearly showed they generally occur in the same direction (increase or decrease) but are much more pronounced in arsenic-induced tumors than normal surrounding tissue from arsenic exposed mice (Table 1, Figure 1). Examples of a cluster showing increased genes (shown in red) and an example of a gene cluster showing decreased genes (green) are shown in Figure 1. Generally, gene alterations seen in the present study are largely consistent with the previous, more limited gene analysis (Liu et al. 2004) of our first transplacental arsenic carcinogenesis study (Waalkes et al. 2003). The significant gene expression alterations (p < 0.001 and 1.5-fold cutoff) were further selected for Ingenuity Pathway Analysis.
[FIGURE 1 OMITTED]
Ingenuity Pathways Analysis. To further analyze the biological significance of these alterations in gene expression, we used the Ingenuity Pathways Analysis; an example of the highest score in the analysis from arsenic-induced tumors (Figure 2) is depicted. The intensity of the node color indicates the degree of increased (red) or decreased (green) expression in arsenic-induced HCC. Biological functions were assigned to each gene network by using the findings extracted from the published scientific literature and stored in the Ingenuity Pathways Knowledge Base (http://www.ingenuity.com/ products/PathwaysKnowledge.pdf) and are ranked according to the significance of that biological function to the network. The activation of c-myc oncogene oncogene
Gene that can cause cancer. It is a sequence of DNA that has been altered or mutated from its original form, the proto-oncogene (see mutation). Proto-oncogenes promote the specialization and division of normal cells. and its central role in related pathway alterations are consistent with the published literature on arsenic (Chen et al. 2001b), and could be a major component of its hepatocarcinogenic potential.
[FIGURE 2 OMITTED]
Real-time RT-PCR analysis of aberrantly expressed genes. To confirm and extend microarray results, we performed real-time RTPCR RTPCR Reverse Transcriptase Polymerase Chain Reaction analysis of selected genes using individual samples from control mouse liver, arsenic-exposed nontumorous livers, and arsenic-induced HCC. Generally, real-time RT-PCR confirmed microarray results with > 90% concordance but tended to show more pronounced changes than microarray analysis (Table 1). For HCC biomarkers, alpha-fetoprotein (AFP (1) (AppleTalk Filing Protocol) The file sharing protocol used in an AppleTalk network. In order for non-Apple networks to access data in an AppleShare server, their protocols must translate into the AFP language. See file sharing protocol. , 19-fold), plasminogen activator inhibitor-1 (PAI-1, 9-fold), cytokeratin 8 (6-fold), cytokeratin 18 (3-fold) and cytokeratin 19 (11-fold), were dramatically increased in arsenic-induced HCC. The oncogenes c-myc, c-met, k-ras and the neoplastic neoplastic /neo·plas·tic/ (ne?o-plas´tik)
1. pertaining to a neoplasm.
2. pertaining to neoplasia.
pertaining to neoplasia or a neoplasm. progression protein-3 (Npn3) were also increased 2- to 3-fold in arsenic-induced HCC and nontumorous, normal liver from arsenic exposed mice, while the tumor suppressors BRCA BRCA
One of two genes (designated BRCA1 and BRCA2) that help repair damage to DNA, but when inherited in a defective state increase the risk of breast and ovarian cancer. 1 and BRCA2 decreased 30-50%.
The positive cell cycle regulators cyclin D1 (5-fold), cyclin E (5-fold), cdk2na (14-fold), cdk2nb (6-fold), cdk4 (4-fold), and PCNA (3-fold) were increased as a result of arsenic exposure, while the cell cycle negative regulator p16 decreased 40%. The expression of insulin-like growth factor-1 (IGF-1, 60% of control) was decreased, while the expression of IGF-2 was increased 4-fold. The IGF-binding proteins (IGFBP IGFBP Insulin-Like Growth Factor Binding Protein 1, 9-fold), IGFBP3 (3-fold), and IGFBP5 (2.4-fold) were all increased to some extent.
Arsenic produces oxidative stress within cells as a possible mechanism of its toxicity (Liu et al. 2001a). Stress-related genes, including various glutathione S-transferases (GST-alpha, GST-mu, GST-pi, and GST-theta) and early growth response protein 1 (EGR EGR Engineering
EGR Exhaust Gas Recirculation
EGR Early Growth Response
EGR Extra Grace Required
EGR Enhanced Gas Recovery
EGR Embedded GPS Receiver
EGR Emergency Generator Room 1), increased 2.5- to 4.5-fold. In addition, Cu,Zn-superoxide dismutase (SOD1) and ceruloplasmin ceruloplasmin /ce·ru·lo·plas·min/ (se-roo?lo-plaz´min) an a2-globulin of plasma believed to function in copper transport and its maintenance at appropriate levels in tissue; levels are decreased in Wilson's disease. (Cu-binding protein) were also increased approximately 3-fold by arsenic. In comparison, no major changes occurred in the expression of heme oxygenase-1 (HO-1), a biomarker for arsenic-induced oxidative stress (NRC 1999), and the expression of metallothionein-1 (MT-1) actually decreased 50%.
A feminized pattern of metabolic enzymes has been noted in male livers of mice bearing arsenic-induced HCC after in utero exposure (Waalkes et al. 2004a). Marked elevation in expression of the female predominant cytochtome P450 (CYP CYP
In currencies, this is the abbreviation for the Cyprus Pound.
The currency market, also known as the Foreign Exchange market, is the largest financial market in the world, with a daily average volume of over US $1 trillion. )2A4 (25-fold), and significant increases (2- to 3-fold) of female dominant CYP2B CYP2B Cytochrome P450 2B 9 (2.6-fold) and CYP2D9 were evident. In contrast, expression of the male dominant CYP7B1, CYP2F2, and CYP41 were all decreased by approximately 50% by arsenic exposure. Further evidence for feminization includes increased expression of aldo-keto reducrase lc18 (Akrlc18) and hydroxysteroid 17[beta] dehydrogenase dehydrogenase /de·hy·dro·gen·ase/ (de-hi´dro-jen-as?) an enzyme that catalyzes the transfer of hydrogen or electrons from a donor, oxidizing it, to an acceptor, reducing it.
n. 7 (HSD HSD Human Services Department
HSD High Speed Data
HSD Hillsboro School District (Hillsboro, OR)
HSD Hybrid Synergy Drive (Toyota/Lexus)
HSD High School Diploma
HSD Historical Society of Delaware 17[beta]7), as well as ER-[alpha] linked overexpression of trefoil trefoil (trē`foil) [O.Fr.,=three-leaf], in botany, name for several plants, chiefly of the pulse family, having trifoliate leaves. Best known of the trefoils is clover. factor 3 (TFF TFF The Final Frontier (Star Trek movie)
TFF Tribeca Film Festival
TFF Tears for Fears (band)
TFF The Transnational Foundation for Peace and Future Research
TFF Tangential Flow Filtration 3) and cysteine-rich protein 61 (Cyr61). No apparent changes occurred for CYP2C39, CYP2E1, CYP2J5, CYP3A25, and CYP4A14 (data not shown). Interestingly, the enzymes for lipid metabolism such as lipoprotein lipase (Lpl, 33-fold), cytosolic acyl-CoA thioesterase (Ctel, 5-fold), and proteasome Proteasomes are large protein complexes inside all eukaryotes and archaea, as well as in some bacteria. In eukaryotes, they are located in the nucleus and the cytoplasm. 26S subunit ATPase-3 (Pmsc3, 4-fold) were increased. Conversely, betaine betaine /be·ta·ine/ (be´tah-en) the carboxylic acid derived by oxidation of choline; it acts as a transmethylating metabolic intermediate and is used in the treatment of homocystinuria. homocysteine methyltransferase (BHMT, 60% of control), serum amyloid amyloid /am·y·loid/ (am´i-loid)
1. starchlike; amylaceous.
2. the pathologic, extracellular, waxy, amorphous substance deposited in amyloidosis, being composed of fibrils in bundles or in a meshwork of polypeptide 3 (Saa3, 30% of control), and sulfotransferase-related protein 2 (SULT-X2, 1% of control) were decreased.
For cell--cell interaction genes and genes involved in signal transduction, marked increases occurred in annexin A2 (48-fold), nidogen 1 (50-fold), E-cadherin (11-fold), and [beta]-catenin (7-fold) in arsenic-induced HCC. Moderate increases in the ras homolog hom·o·log
Variant of homologue. gene family U (Rhou, 4-fold), in fibronection (CTGF CTGF connective tissue growth factor
CTGF Cytokine-Transforming Growth Factor
CTGF Clean Tanks, Gas Free , 4-fold), and in prostaglandin-endoperoxide synthase synthase /syn·thase/ (-thas) a term used in the names of some enzymes, particularly lyases, when the synthetic aspect of the reaction is dominant or emphasized.
n. 2 (Ptgs-2, 5.5-fold) were also evident. In contrast, the prolactin receptor (Prlr, 40% of control) and the epidermal growth factor receptor This article is about a cell suface receptor. For estimated measure of kidney function (eGFR), see Glomerular filtration rate.
The epidermal growth factor receptor (Egfr, 40% of control) were decreased as a result of in utero exposure to inorganic arsenic.
Western blot analysis of aberrantly expressed proteins. We also performed Western blot analysis on selected gene products (Figure 3). Western blot analysis largely confirmed the gene expression results and clearly showed the increases in the expression of potential oncogenes AFP, K-ras, and c-Myc in both arsenic-exposed nontumorous livers and arsenic-induced HCC. The cell cycle regulators, such as ER-[alpha], the ER-[alpha] linked cyclin D1, cyclin-dependent kinase 4 (cdk4), and proliferating cell nuclear antigen (PCNA), all followed this pattern of high in tumor tissues, intermediate in tumor-surrounding livers and low in control liver. The HCC biomarkers cytokeratin 8 and plasminogen activator inhibitor 1 (PAI-1) were all significantly increased in arsenic-induced HCC, and also higher in arsenic-exposed tumor-surrounding tissues. Endothelial growth factor receptor (EGFR) was down-regulated in arsenic-induced HCC. The expression of [beta]-actin was consistent among lanes.
[FIGURE 3 OMITTED]
Our most recent transplacental arsenic carcino-genesis study (Waalkes et al. 2004b) confirmed the hepatocarcinogenic potential of arsenic in adult male mice after transplacental exposure (Waalkes et al. 2003), and it also provided an opportunity for a genomewide gene expression analysis. The 22K mouse chip array revealed several novel pathways and gene expression alterations associated with arsenic-induced HCC and in arsenic-exposed nontumorous normal liver samples, which were not detected in previous limited gene expression analysis. Liver is a major target organ for arsenic in rodents and in humans (Liu et al. 2000; 2002; Santra et al. 2000). In other studies, HCC formation has been associated with arsenic exposure in humans (Centeno et al. 2002; Chen et al. 1997; Chin et al. 2004; Zhou et al. 2002). Therefore, the present gene profiling study in mice of a concordant human target tissue is noteworthy because it clearly demonstrates that multiple genetic events occurred during the carcinogenic process in the liver induced by in utero exposure to arsenic. It is reasonable to anticipate a complicated series of genetic interactions in arsenic-induced carcino-genesis, which this study certainly shows. However, the present results also provide a variety of candidate genes to investigate in greater detail, such as with time-course experiments, to define events early after transplacental arsenic exposure. Furthermore, recent evidence indicates that early life chemical exposures may aberrantly "reprogram re·pro·gram
tr.v. re·pro·grammed or re·pro·gramed, re·pro·gram·ming or re·pro·gram·ing, re·pro·grams
To program again.
re " gene expression patterns, thus resulting in altered growth responses and cancer later in life, as with diethylstilbestrol diethylstilbestrol: see DES. and uterine cancer (Cook et al. 2005). Consistent with this concept, the altered gene expression patterns seen in adults after in utero arsenic exposure could have resulted from the early life reprogramming Reprogramming refers to erasure and remodeling of epigenetic marks, such as DNA methylation, during mammalian development. After fertilization some cells of the newly formed embryo migrate to the germinal ridge and will eventually become the germ cells during a critical stage in development.
Clustering analysis clearly shows that the gene changes in HCC and normal liver samples taken from mice exposed to arsenic in utero are for the most part directionally consistent but with quantitatively greater changes in the arsenic-induced tumors. The gene expression alterations associated with arsenic hepato-carcinogenesis have an important impact on as many as 50 biological networks shown by Ingenuity Pathways Analysis to be significantly altered or interrupted. The observation that a large number of graded gene expression changes occurred both in nontumorous and in tumorous liver samples from adults exposed to arsenic in utero is important because it implies that complex changes initiated during gestation appear to have persisted into adulthood. Some of those alterations are very likely relevant to the carcinogenic process. The complex nature of this arsenic-induced response argues against mutational activation of a few key pathways and perhaps points toward other mechanisms for persistent gene expression change. For example, the central network role for c-Myc seen in the present study is consistent with c-myc activation in arsenic-transformed rat liver cells (Chen et al. 2001b), in hamster embryo cells (Takahashi et al. 2002), and in murine murine /mu·rine/ (mur´en) pertaining to, derived from, or characteristic of mice or rats.
adj. fibroblasts Fibroblasts
A type of cell found in connective tissue; produces collagen.
Mentioned in: Skin Grafting (Trouba et al. 2000). This central network role also agrees with that suggested by results from arsenic-induced skin tumors (Germolec et al. 1998) and from HCC induced by in utero arsenic exposure (Liu et al. 2004). Overexpression of c-myc may be caused by arsenic-induced DNA DNA: see nucleic acid.
or deoxyribonucleic acid
One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes. hypomethylation of the c-myc promoter region (Takahashi et al. 2002). The activation of c-myc oncogene can in turn result in many other gene expression changes. For example, the increased expression of the N-myc downstream regulated gene 1 (NDRG1) is associated with HCC formation. The increased expression of stearoyl-CoA desaturase-1 (SCD-1) plays a key role in lipogenic lipogenic /lip·o·gen·ic/ (-jen´ik) forming, producing, or caused by fat.
producing, forming or caused by fat. gene expression and in metabolic syndrome, which coincides with marked elevations of lipoprotein lipase (Lpl, 33-fold) and cytosolic acyl-CoA thioesterase-1 (Ctel, 5-fold) in arsenic-induced HCC. This increased expression could also be important for arsenic-induced hepatic steatosis steatosis /ste·a·to·sis/ (ste?ah-to´sis) fatty change.
See fatty degeneration.
fatty degeneration. See also muscular steatosis. (Chen et al. 2004). Interestingly, in this network, the increase in C-myc is associated with a decrease in metallothionein-1 expression (MT-1), which was confirmed by RT-PCR analysis. MT is an adaptive protein in response to arsenic exposure (Liu et al. 2000). The decrease in MT could be a marker for liver tumor progression because MT is poorly expressed in both mouse and human HCC (Waalkes et al. 1996). Determining the changes that are specific to arsenic and the changes that are part of the potentially autonomous process of liver oncogenesis oncogenesis /on·co·gen·e·sis/ (-jen´e-sis) tumorigenesis; the production or causation of tumors.oncogenet´ic
The formation and development of tumors. will be a major challenge, but the present work opens various avenues for further study.
In addition to c-myc overexpression, the activation of various oncogenes such as k-ras and c-met was also evident following transplacental arsenic exposure, as seen with arsenic-transformed rat liver cells (Chen et al. 2001a). Overexpression of k-ras is also associated with arsenic-induced malignant transformation in human prostate epithelial cells (Benbrahim-Tallaa et al. 2005). Ha-ras mutation and overexpression, as in genetically altered Tg.AC mice, is associated with arsenic-induced co-promotion of skin tumors (Germolec et al. 1998). In a fashion similar to c-myc, DNA hypomethylation could be responsible for ras oncogene overexpression, and chronic arsenic exposure in vivo appears to induce a loss of methylation methylation,
n a phase-II detoxification pathway in the liver; methyl groups combine with toxins to rid the body of various substances.
(meth´ at several cytosine cytosine (sī`tōsēn'), organic base of the pyrimidine family. It was isolated from the nucleic acid of calf thymus tissue in 1894. sites within the promoter region of the hepatic Ha-ras gene in mice (Okoji et al. 2002). The Ras-mediated signal transduction molecule Rhou (ras homolog gene family U) was also increased 3- to 4-fold in arsenic-exposed tissues in the present study. The HCC biomarkers [alpha]-fetoprotein, plasminogen activator inhibitor 1 (PAI-1), cytokeratin 8, cytokeratin 18, and cytokeratin 19, as well as the neoplastic progression protein-3, were all significantly increased, consistent with HCC pathobiology pathobiology /patho·bi·ol·o·gy/ (-bi-ol´ah-je) pathology.
The study or practice of pathology with greater emphasis on the biological than on the medical aspects. and previous findings with arsenic (Liu et al. 2004). Conversely, the tumor suppressor genes BRCA1 and BRCA2 were decreased. BRCA1 protein interacts directly with ER-[alpha], thereby resulting in inhibition of estradiol-stimulated ER-[alpha] transcriptional activity (Ma et al. 2005). Thus, down-regulation of BRCA1 and BRCA2 could play a role in the overexpression of ER-[alpha] and ER-[alpha]-linked gene expression observed in this model of arsenic carcinogenesis (Waalkes et al. 2004a).
The tumors (in the liver, ovary, adrenal, lung) and proliferative lesions (in the uterus and oviduct oviduct: see fallopian tube. ) induced by in utero arsenic exposure can all similarly be induced by estrogenic carcinogens Carcinogens
Substances in the environment that cause cancer, presumably by inducing mutations, with prolonged exposure.
Mentioned in: Colon Cancer, Rectal Cancer (Waalkes et al. 2003; 2004b). Indeed, prior results demonstrated the over-expression of ER-[alpha] with a nuclear localization Customizing software and documentation for a particular country. It includes the translation of menus and messages into the native spoken language as well as changes in the user interface to accommodate different alphabets and culture. See internationalization and l10n. (active form) in adult male liver after in utero arsenic exposure (Waalkes et al. 2004a; Chen et al. 2004), a result confirmed by Western blot analysis in the present study. This finding leads to the hypothesis that arsenic could somehow produce estrogenic-like effects, possibly directly or indirectly through stimulation of ER-[alpha], thus resulting in tumor formation (Waalkes et al. 2004a). Overexpression of ER-[alpha] could be caused by arsenic-induced DNA hypomethylation of the promoter region of the ER-[alpha] gene (Chen et al. 2004; Waalkes et al., 2004a). Such overexpression is associated with the feminization pattern of liver metabolic enzymes in male liver bearing arsenic-induced HCC (Liu et al. 2004; Waalkes et al. 2004a). In the present study, marked elevation of the female predominant CYP2A4, CYP2B9, and CYP2D9 and significant decreases in male dominant CYP7B1, CYP2F2, and CYP41 were evident following transplacental arsenic exposure in male offspring, consistent with this hypothesis. The contribution of ER-[alpha] signaling pathways to arsenic-induced HCC could be multifactorial multifactorial /mul·ti·fac·to·ri·al/ (mul?te-fak-tor´e-al)
1. of or pertaining to, or arising through the action of many factors.
2. , including the increased trefoil factor-3 (TFF3), cychn D1, and the CCN CCN Cloud Condensation Nuclei
CCN Church Communication Network
CCN Conseil Canadien des Normes (Standards Council of Canada)
CCN Critical Care Nurse
CCN Certified Clinical Nutritionist
CCN Community Care Network
CCN Cyclin family members cysteine-rich 61 (Cyr61) and connective tissue growth factor (CTGF). Overexpression of TFF3 is proposed to be a critical process in hepatocarcinogenesis (Okada et al. 2005), and overexpression of Cyr61 and CTGF is associated with recurrence and metastasis metastasis /me·tas·ta·sis/ (me-tas´tah-sis) pl. metas´tases
1. transfer of disease from one organ or part of the body to another not directly connected with it, due either to transfer of pathogenic microorganisms or to of HCC (Zeng et al. 2004).
Estrogen-induced progression through the [G.sub.1] phase of the cell cycle is preceded by increased expression of the [G.sub.1]-phase regulatory proteins c-Myc and Cyclin D1 (Prall et al. 1998). Cyclin D1 overexpression is important for HCC formation and is considered a hepatic oncogene (Deane et al. 2001). Overexpression of cyclin D1 has been reported in arsenic-transformed cells (Chen et al. 2001b), during arsenic-induced skin co-carcinogenicity in mice (Rossman et al. 2001), in dimethylarsinic acid--induced bladder proliferative lesions in rats (Wei et al. 2002), in chronic arsenate-exposed rats (Cui et al. 2004), and in the livers of mice bearing HCC induced by in utero exposure to arsenic (Liu et al. 2004). Cyclin D1 overexpression, together with other positive cell cycle regulators such as cyclin E, cdk4, cdkn2a, cdkn2b, and PCNA was evident in arsenic-exposed tissues in the present study. Thus, cell cycle dysregulation, as manifested in cyclin D1 overexpression, appears to be a consistent event in arsenic-induced oncogenesis. In comparison, expression of the negative cell cycle regulator p16 was decreased, consistent with prior observations with arsenic (Chen et al. 2001a; Liu et al. 2004). In addition to these cell cycle regulators, annexin A2 was also dramatically increased (48-fold) in arsenic-induced HCC. Annexins belong to a family of the calcium-dependent phospholipid phospholipid (fŏs'fōlĭp`ĭd), lipid that in its simplest form is composed of glycerol bonded to two fatty acids and a phosphate group. binding proteins and are substrates of receptor tyrosine kinases. Annexin A2 overexpression is common in various carcinomas (Emoto et al. 2001), and annexin A2 can interact with c-Myc in increasing cell proliferation (Micldeburgh et al. 2005). Dysregulation of the IGF (Internet Governance Forum) An international organization of governments and U.N. agencies that was founded to discuss Internet issues such as security and spam. It was created at the United Nations Summit in 2005 after the U.S. axis, including IGF-1 and IGF-2, and IGF binding proteins, was evident in our prior work (Liu et al. 2004) and in our present studies. A decrease in IGF-1 and an increase in IGFBP1 in arsenic-exposed livers are similar to findings with the nongenotoxic model carcinogens such as oxazepam oxazepam /ox·az·e·pam/ (ok-saz´e-pam) a benzodiazepine tranquilizer, used as an antianxiety agent and as an adjunct in the treatment of acute alcohol withdrawal symptoms.
n. and Wyeth-14,643 (Iida et al. 2003). Dysregulation of the IGF axis may also well contribute to uncontrolled cell proliferation and hepato-carcinogenesis (Scharfand Braulke 2003).
The expression of oxidative stress-related genes is associated with arsenic toxicity and cell proliferation (Liu et al. 2001; Pi et al. 2003). For example, overexpression of early growth protein 1 (EGR1) is associated with arsenic-induced proliferation in urinary bladder cells (Simeonova et al. 2000) and in arsenic-induced HCC (Liu et al. 2004). The activation of transcription factor Nrf2 by arsenic and the Nrf2-mediated overexpression of antioxidants Antioxidants
Substances that reduce the damage of the highly reactive free radicals that are the byproducts of the cells.
Mentioned in: Aging, Nutritional Supplements
n. such as Cu,Zn superoxide dismutase (SODI Sodi (sō`dī), in the Bible, father of Gaddiel, a spy sent into Canaan. ) is evident in arsenic-exposed cells (Pi et al. 2003). Expression of SOD1 and the Cu-binding protein ceruloplasmin was increased in arsenic-exposed liver and in arsenic-induced liver tumors in the present work. The increases in these acute phase proteins may have clinical relevance because increased ceruloplasmin (Bencko et al. 1988) and SOD1 (Lu et al. 2001) have been reported in arsenic-exposed humans. Heine oxygenase-1, a biomarker for acute arsenic-induced oxidative stress (Liu et al. 2001a; NRC 1999), was not significantly altered in the present study. In comparison, GST-alpha, GST-mu, GST-pi, and GST-theta expressions were all increased following in utero arsenic exposure, similar to previous observations in chronic arsenic exposure settings (Liu et al. 2000; Lu et al. 2001; Xie et al. 2004). Increased GSTs likely play a role in conjugation conjugation, in genetics
conjugation, in genetics: see recombination.
conjugation, in grammar
conjugation: see inflection. and detoxication detoxification, detoxication
1. reduction of the toxic properties of a substance.
2. treatment designed to assist in recovery from the toxic effects of a drug.
metabolic detoxification of lipid peroxides produced by arsenic, but they also play a role for cellular efflux efflux Medtalk That which flows outward of arsenic via MRP (Material Requirements Planning) An information system that determines what assemblies must be built and what materials must be procured in order to build a unit of equipment by a certain date. pumps (Leslie et al. 2004; Liu et el. 2001b). The increased demand for GSH GSH reduced glutathione.
reduced glutathione. utilization may activate the use of homocysteine Homocysteine Definition
Homocysteine is a naturally occurring amino acid found in blood plasma. High levels of homocysteine in the blood are believed to increase the chance of heart disease, stroke, Alzheimer's disease, and osteoporosis. for GSH synthesis, rather than for S-adenosylmethionine (SAM), and, consequently, disrupt cellular methyl homeostasis homeostasis
Any self-regulating process by which a biological or mechanical system maintains stability while adjusting to changing conditions. Systems in dynamic equilibrium reach a balance in which internal change continuously compensates for external change in a feedback (Lu et al. 2002). In this regard, expression of betaine-homocysteine methyltransferase was markedly decreased, and it is a key enzyme in SAM recycling. This disruption of SAM production may contribute to a cellular environment conducive to errors in DNA methylation, which can be a contributing factor to aberrant gene expression.
Both [beta]-catenin and E-cadherins are important cell--cell communication molecules. The overexpression of [beta]-catenin is linked to cyclin D1 overexpression in HCC (Iida et al., 2003). Furthermore, [beta]-catenin can also affect tumor progression by stimulating proliferation, and overexpression is associated with a poor prognosis for patients with HCC. E-cadherin is a cell--cell adhesion molecule that plays a key role in the development and maintenance of cell polarity. Dysregulation of E-cadherin is also associated with HCC (Calvisi et al. 2004). Nidogen-1 acts as a bridge between the extra-cellular matrix molecules laminin-1 and collagen IV, and it participates in the assembly of the basement membranes. In the present study, nidogen 1 (50-fold), [beta]-catenin (7-fold), and E-cadherin (11-fold) were all dramatically increased in transplacental arsenic-induced HCC and in arsenic-exposed liver tissues. Conversely, the prolactin receptor and EGFR were decreased in arsenic-induced HCC, thereby suggesting that the dysregulation of cell-cell communications and signal transduction pathways could be another important aspect of arsenic hepatocarcinogenesis, as has been proposed for liver cancer in general.
In summary, we used a genomewide analysis to dissect dissect /dis·sect/ (di-sekt´) (di-sekt´)
1. to cut apart, or separate.
2. to expose structures of a cadaver for anatomical study.
v. further the toxicogenomics changes of transplacental arsenic hepatocarcinogenesis in the mouse. These findings clearly demonstrate that arsenic carcinogenesis in the liver involves a complex interplay between multiple genetic events, including stimulation of oncogene expression, liver feminization, cell cycle dysregulation, and disruption of cell--cell communication. This study indicates that early life exposure to arsenic during a critical stage in development may have resulted in aberrant genetic reprogramming that leads to events associated with hepatocarcinogenesis later in life. Finally, the present results highlight the importance of protecting pregnant women from excessive arsenic exposure.
Received 22 July 2005; accepted 6 October 2005.
Anderson LM, Diwan Noun 1. diwan - a Muslim council of state
privy council - an advisory council to a ruler (especially to the British Crown)
2. diwan - a collection of Persian or Arabic poems (usually by one author)
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Damaging or destructive to the liver.
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Of or occurring after birth, especially in the period immediately after birth. phorbol phorbol /phor·bol/ (for´bol) a polycyclic alcohol occurring in croton oil; it is the parent compound of the phorbol esters.
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Jie Liu, (1) Yaxiong Xie, (1) Danica M.K. Ducharme, (2) Jun Shen Shen, in the Bible, place, perhaps close to Bethel, near which Samuel set up the stone Ebenezer. , (1) Bhalchandra A. Diwan, (3) B. Alex Merrick, (2) Sherry F. Grissom, (2) Charles J. Tucker, (2) Richard S. Paules, (2) Raymond Tennant, (2) and Michael P. Waalkes (1)
(1) Inorganic Carcinogenesis Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute at the National Institute of Environmental Health Sciences The National Institute of Environmental Health Sciences (NIEHS) is one of 27 Institutes and Centers of the National Institutes of Health (NIH),which is a component of the Department of Health and Human Services (DHHS). The Director of the NIEHS is Dr. David A. Schwartz. , and (2) National Center For Toxicogenomics, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services Noun 1. Department of Health and Human Services - the United States federal department that administers all federal programs dealing with health and welfare; created in 1979
Health and Human Services, HHS , Research Triangle Park Research Triangle Park, research, business, medical, and educational complex situated in central North Carolina. It has an area of 6,900 acres (2,795 hectares) and is 8 × 2 mi (13 × 3 km) in size. Named for the triangle formed by Duke Univ. , North Carolina, USA; (3) Basic Research Program, Science Applications International Corporation, National Cancer Institute, National Institutes of Health, Department of Health and Human Services, Frederick, Maryland, USA
Address correspondence to M.P. Waalkes, Inorganic Carcinogenesis Section, NCI See Liberate. at NIEHS NIEHS National Institute of Environmental Health Sciences (NIH, DHHS) , Mail Drop F0-09, Research Triangle Park, NC 27709 USA. Telephone: (919) 541-2328. Fax: (919) 541-3970. E-mail: waalkes@ niehs.nih.gov
The authors thank W. Qu, J-F. Coppin, and L. Keefer for their critical review of this manuscript.
The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services.
Research was supported in part by the Intramural intramural /in·tra·mu·ral/ (-mu´r'l) within the wall of an organ.
Occurring or situated within the walls of a cavity or organ. Research Program of the NIH, National Cancer Institute, Center for Cancer Research, the National Center for Toxicogenomics, and the National Cancer Institute under contract NO1-CO-12400.
The authors declare they have no competing financial interests.
Table 1. Real-time RT-PCR and microarray analysis of liver samples from adult male C3H mice exposed to arsenic in utero. Accession Control Gene categories (a) no. (a) (PCR) Oncogenes and HCC-related genes alpha-fetoprotein V00743 1.0 [+ or -] 0.2 c-myc X01023 1.0 [+ or -] 0.2 c-met Y00671 1.0 [+ or -] 0.2 K-ras U49448 1.0 [+ or -] 0.2 PAI-1, plasminogen M33960 1.0 [+ or -] 0.3 activator inhibitor-1 Cytokeratin-8 X12789 1.0 [+ or -] 0.2 Cytokeratin-18 Ml_1686 1.0 [+ or -] 0.3 Cytokeratin-19 NM_008471 1.0 [+ or -] 0.4 BRCA1 U31625 1.0 [+ or -] 0.3 BRCA2 U65594 1.0 [+ or -] 0.1 Npn3, neoplastic Z31362 1.0 [+ or -] 0.4 progression protein-3 Cell cycle regulators and IGFs Cychn D1 M64403 1.0 [+ or -] 0.2 Cyclin E X75888 1.0 [+ or -] 0.1 Cdk2na NM_009877 1.0 [+ or -] 0.2 Cdk2nb AF_059567 1.0 [+ or -] 0.3 Cdk4 L01640 1.0 [+ or -] 0.3 PCNA X53068 1.0 [+ or -] 0.1 P16 U79632 1.0 [+ or -] 0.3 IGF-1 X04480 1.0 [+ or -] 0.1 IGF-2 M14951 1,0 [+ or -] 0.2 IGFBP1 X81579 1.0 [+ or -] 0.3 IGFBP3 X81581 1.0 [+ or -] 0.4 IGFBP5 X81583 1.0 [+ or -] 0.2 Stress-related genes GST-alpha4 AK008490 1.0 [+ or -] 0.4 GST-mu J03953 1.0 [+ or -] 0.4 GST-theta X98055 1.0 [+ or -] 0.5 GST-pi NM_013541 1.0 [+ or -] 0.4 EGR1,early response M20157 1.0 [+ or -] 0.5 protein-1 SOD-1 M01143 1.0 [+ or -] 0.4 Ceruloplasmin U49430 1.0 [+ or -] 0.1 HO-1, heme oxygenase-1 M33203 1.0 [+ or -] 0.1 MT-1 BC027262 1.0 [+ or -] 0.2 Genes for metabolic enzymes CYP2A4 J03549 1.0 [+ or -] 0.1 CYP2F2 M77497 1.0 [+ or -] 0.2 CYP2B9 M21855 1.0 [+ or -] 0.4 CYP2D9 M27168 1.0 [+ or -] 0.4 CYP3A41 NM_017396 1.0 [+ or -] 0.5 CYP7B1 U36993 1.0 [+ or -] 0.3 Akr1c18, aldo-keto NM_134066 1.0 [+ or -] 0.5 reductase 1cl8 HSD1707 NM_010476 1.0 [+ or -] 1.4 TFF3, trefoil factor 3 NM_011575 1.0 [+ or -] 0.1 Cyr61, cysteine-rich NM_010516 1.0 [+ or -] 1.0 protein 61 Lp1, lipoprotein lipase NM_008509 1.0 [+ or -] 0.1 Cte1, cytosolic acyl-CoA NM_012006 1.0 [+ or -] 0.2 thioestaset Pmsc3 proteasome 26S D49686 1.0 [+ or -] 1.1 subunit, ATPase3 BHMT, homocysteine AF033381 1.0 [+ or -] 0.1 methyltransferase Saa3, serum amyloid 3 NM_011315 1.0 [+ or -] 0.3 SULT-X2 AF26075 1.0 [+ or -] 0.1 Cell communication and signal transduction Annexin A2 M14044 1.0 [+ or -] 0.3 Nid1, nidogen 1 NM_010917 1 0 [+ or -] 0.2 [beta]-catenin NM_007614 1.0 [+ or -] 0.3 E-cadherin NM_009864 1.0 [+ or -] 0.3 Ptgs-2, prostaglandin- NM_011198 1.0 [+ or -] 0.4 endoperoxidesynthase 2 Rhou, ras homolog gene NM_133955 1.0 [+ or -] 0.2 family U CTGF, fibronectin NM_022266 1.0 [+ or -] 0.3 Prlr, prolactin receptor NM_11169 1.0 [+ or -] 0.4 Eafr, enidermal orowth NM_07912 1.0 [+ or -] 0.2 factor receptor As-Normal Gene categories (a) PCR Array-fold Oncogenes and HCC-related genes alpha-fetoprotein 2.3 [+ or -] 0.5 NA c-myc 2.2 [+ or -] 0.5 1.67 * c-met 2.4 [+ or -] 0.6 1.03 K-ras 1.4 [+ or -] 0.4 1.41 PAI-1, plasminogen 2.7 [+ or -] 0.9 NA activator inhibitor-1 Cytokeratin-8 1.6 [+ or -] 0.2 NA Cytokeratin-18 1.3 [+ or -] 0.3 1.04 Cytokeratin-19 12.1 [+ or -] 4.7 * 1.53 * BRCA1 0.7 [+ or -] 0.1 1.08 BRCA2 0.8 [+ or -] 0.1 NA Npn3, neoplastic 1.0 [+ or -] 0.3 1.05 progression protein-3 Cell cycle regulators and IGFs Cychn D1 4.8 [+ or -] 1.0 * 1.62 * Cyclin E 4.4 [+ or -] 1.5 * 1.19 * Cdk2na 7.1 [+ or -] 1.9 * 1.01 Cdk2nb 2.3 [+ or -] 0.6 NA Cdk4 2.5 [+ or -] 0.5 1.53 * PCNA 2.5 [+ or -] 0.8 1.73 * P16 0.6 [+ or -] 0.2 0.85 IGF-1 0.7 [+ or -] 0.1 0.65 * IGF-2 3.3 [+ or -] 2.1 3.77 * IGFBP1 2.7 [+ or -] 0.5 * 1.15 IGFBP3 2.9 [+ or -] 0.8 * 0.88 IGFBP5 4.6 [+ or -] 1.5 * 0.94 Stress-related genes GST-alpha4 2.5 [+ or -] 0.6 * 1.94 * GST-mu 3.5 [+ or -] 0.8 * 1.54 * GST-theta 3.5 [+ or -] 0.8 * 1.14 GST-pi 2.5 [+ or -] 0.6 * 2.23 EGR1,early response 3.5 [+ or -] 0.8 * 1.24 * protein-1 SOD-1 2.5 [+ or -] 0.6 * NA Ceruloplasmin 4.1 [+ or -] 1.0 * 1.02 HO-1, heme oxygenase-1 1.0 [+ or -] 0.2 1.51 * MT-1 0.6 [+ or -] 0.3 0.50 * Genes for metabolic enzymes CYP2A4 3.1 [+ or -] 0.9 * 1.05 CYP2F2 1.0 [+ or -] 0.2 1.12 CYP2B9 1.9 [+ or -] 0.6 0.85 CYP2D9 5.3 [+ or -] 1.0 * 1.15 * CYP3A41 0.4 [+ or -] 0.2 0.55 * CYP7B1 0.7 [+ or -] 0.3 0.82 * Akr1c18, aldo-keto 2 5 [+ or -] 0.8 1.39 reductase 1cl8 HSD1707 3.1 [+ or -] 0.4 * 0.92 TFF3, trefoil factor 3 3.2 [+ or -] 1.0 * 5.82 * Cyr61, cysteine-rich 1.7 [+ or -] 0.3 0.85 protein 61 Lp1, lipoprotein lipase 12.1 [+ or -] 5.1 * 4.61 Cte1, cytosolic acyl-CoA 2.7 [+ or -] 0.8 1.29 * thioestaset Pmsc3 proteasome 26S 2.6 [+ or -] 0.4 1.62 * subunit, ATPase3 BHMT, homocysteine 0.7 [+ or -] 0.1 0.84 * methyltransferase Saa3, serum amyloid 3 2.2 [+ or -] 0.7 0.23 * SULT-X2 0.1 [+ or -] 0.0 * 0.21 * Cell communication and signal transduction Annexin A2 9.0 [+ or -] 2.9 * 2.30 * Nid1, nidogen 1 3.1 [+ or -] 1.1 2.60 * [beta]-catenin 4.2 [+ or -] 0.8 * 2.01 * E-cadherin 4.4 [+ or -] 1.5 * 1.24 * Ptgs-2, prostaglandin- 2.6 [+ or -] 1.0 1.12 endoperoxidesynthase 2 Rhou, ras homolog gene 3.1 [+ or -] 0.8 * NA family U CTGF, fibronectin 2.7 [+ or -] 0.6 2.50 * Prlr, prolactin receptor 0.4 [+ or -] 0.2 0.37 * Eafr, enidermal orowth 1.1 [+ or -] 0.2 0.71 * factor receptor As-HCC Gene categories (a) PCR Array-fold Oncogenes and HCC-related genes alpha-fetoprotein 19.1 [+ or -] 5.4 * NA c-myc 3.1 [+ or -] 0.5 * 2.36 * c-met 3.4 [+ or -] 0.8 * 1.26 * K-ras 2.5 [+ or -] 0.6 * 1.54 * PAI-1, plasminogen 9.2 [+ or -] 2.9 * NA activator inhibitor-1 Cytokeratin-8 6.0 [+ or -] 1.3 * NA Cytokeratin-18 2.9 [+ or -] 0.4 * 2.34 * Cytokeratin-19 11.1 [+ or -] 5.4 * 1.24 BRCA1 0.4 [+ or -] 0.1 * 0.81 * BRCA2 0.7 [+ or -] 0.1 * NA Npn3, neoplastic 3.1 [+ or -] 0.8 * 2.41 * progression protein-3 Cell cycle regulators and IGFs Cychn D1 5.1 [+ or -] 0.9 * 1.51 * Cyclin E 5.2 [+ or -] 1.3 * 1.31 * Cdk2na 13.9 [+ or -] 3.8 * 1.20 Cdk2nb 6.3 [+ or -] 1.8 * NA Cdk4 4.4 [+ or -] 0.2 * 1.76 * PCNA 2.6 [+ or -] 0.5 * 1.76 * P16 0.6 [+ or -] 0.1 * 0.83 * IGF-1 0.6 [+ or -] 0.1 * 0.53 IGF-2 4.4 [+ or -] 2.0 * 4.21 * IGFBP1 9.1 [+ or -] 3.2 * 4.18 * IGFBP3 2.9 [+ or -] 0.8 * 0.88 IGFBP5 2.4 [+ or -] 0.9 * 0.91 Stress-related genes GST-alpha4 2.5 [+ or -] 0.6 * 2.16 * GST-mu 4.8 [+ or -] 1.1 * 1.26 * GST-theta 3.1 [+ or -] 0.9 * 1.31 GST-pi 2.5 [+ or -] 0.6 * 1.11 EGR1,early response 3.1 [+ or -] 0.9 * 5.20 * protein-1 SOD-1 2.5 [+ or -] 0.6 * NA Ceruloplasmin 4.3 [+ or -] 1.5 * 1.14 * HO-1, heme oxygenase-1 0.9 [+ or -] 0.2 1.23 * MT-1 0.5 [+ or -] 0.1 * 0.59 * Genes for metabolic enzymes CYP2A4 25.3 [+ or -] 8.7 * 2.51 * CYP2F2 0.5 [+ or -] 0.2 * 0.68 * CYP2B9 2.6 [+ or -] 1.1 * 0.43 CYP2D9 4.7 [+ or -] 1.2 * 1.06 CYP3A41 0.4 [+ or -] 0.1 * 0.57 * CYP7B1 0.5 [+ or -] 0.1 * 0.39 * Akr1c18, aldo-keto 61.7 [+ or -] 27.8 * 15.3 * reductase 1cl8 HSD1707 2.8 [+ or -] 0.4 0.99 TFF3, trefoil factor 3 6.3 [+ or -] 1.8 * 10.9 * Cyr61, cysteine-rich 4.9 [+ or -] 1.7 * 1.17 protein 61 Lp1, lipoprotein lipase 33.2 [+ or -] 13.1 * 9.21 * Cte1, cytosolic acyl-CoA 4.8 [+ or -] 1.8 * 2.18 ** thioestaset Pmsc3 proteasome 26S 4.1 [+ or -] 1.1 * 1.61 subunit, ATPase3 BHMT, homocysteine 0.6 [+ or -] 0.1 * 0.63 * methyltransferase Saa3, serum amyloid 3 0.3 [+ or -] 0.1 * 0.05 * SULT-X2 0.0 [+ or -] 0.0 * 0.16 * Cell communication and signal transduction Annexin A2 48.5 [+ or -] 16.9 * 8.64 * Nid1, nidogen 1 56.2 [+ or -] 9.8 * 10.5 * [beta]-catenin 6.9 [+ or -] 2.4 * 2.03 * E-cadherin 11.2 [+ or -] 4.5 * 4.34 * Ptgs-2, prostaglandin- 5.6 [+ or -] 2.5 * 1.18 endoperoxidesynthase 2 Rhou, ras homolog gene 3.9 [+ or -] 1.0 * NA family U CTGF, fibronectin 3.7 [+ or -] 0.8 * 1.90 * Prlr, prolactin receptor 0.4 [+ or -] 0.1 * 0.23 * Eafr, enidermal orowth 0.5 [+ or -] 0.1 * 0.63 * factor receptor NA, same gene access clone is not available. Data are mean [+ or -] SEM of 5-7 individual animals. * Gene names, symbols, and accession numbers are from GenBank (http://www.ncbi-nih.gov/GenBank/). * Significantly different from controls p < 0.05.