Biomedical subjects

Michael P Waalkes

Publications and source records attributed to Michael P Waalkes.

At least 19 recordsLinked to original sources

PubMed2007

Estrogen signaling and disruption of androgen metabolism in acquired androgen-independence during cadmium carcinogenesis in human prostate epithelial cells.

BACKGROUND: Lethal prostate cancers often become androgen-independent due to androgen receptor (AR) overexpression. The role of cadmium in prostate tumor progression was determined. METHODS: Control and cadmium-transformed prostate epithelial cells (CTPE) were compared for steroid-induced proliferation, steroid receptor expression, and androgen metabolism. RESULTS: CTPE cells showed rapid proliferation in complete medium and sustained proliferation in steroid-reduced medium. Androgens stimulated significantly less cell proliferation and AR-related genes expression in CTPE cells. 5alpha-Dihydrotestosterone increased PSA expression more effectively in control cells. Flutamide reduced 5alpha-dihydrotestosterone-stimulated growth less effectively in CTPE cells compared to control. CTPE cells showed decreased p27 expression. Estrogen receptors were overexpressed and estradiol markedly stimulated proliferation in CTPE cells. In CTPE cells 5alpha-aromatase was markedly increased, while 5alpha-reductase was decreased. CONCLUSIONS: Cadmium-induced malignant transformation stimulates androgen independence, unrelated to AR expression or activity. Increased estrogen receptor and 5alpha-aromatase expression suggest estrogen signaling may be critical to this process.

Androgen Antagonists↗

PubMed2006

Fetal onset of aberrant gene expression relevant to pulmonary carcinogenesis in lung adenocarcinoma development induced by in utero arsenic exposure.

Arsenic is a human pulmonary carcinogen. Our work indicates that in utero arsenic exposure in mice can induce or initiate lung cancer in female offspring. To define early molecular changes, pregnant C3H mice were given 85 ppm arsenic in drinking water from days 8 to 18 of gestation and expression of selected genes in the fetal lung or in lung tumors developing in adults was examined. Transplacental arsenic exposure increased estrogen receptor-alpha (ER-alpha) transcript and protein levels in the female fetal lung. An overexpression of various estrogen-regulated genes also occurred, including trefoil factor-3, anterior gradient-2, and the steroid metabolism genes 17-beta-hydroxysteroid dehydrogenase type 5 and aromatase. The insulin growth factor system, which can be influenced by ER and has been implicated in the pulmonary oncogenic process, was activated in fetal lung after gestational arsenic exposure. In utero arsenic exposure also induced overexpression of alpha-fetoprotein, epidermal growth factor receptor, L-myc, and metallothionein-1 in fetal lung, all of which are associated with lung cancer. Lung adenoma and adenocarcinoma from adult female mice exposed to arsenic in utero showed widespread, intense nuclear ER-alpha expression. In contrast, normal adult lung and diethylnitrosamine-induced lung adenocarcinoma showed little evidence of ER-alpha expression. Thus, transplacental arsenic exposure at a carcinogenic dose produced aberrant estrogen-linked pulmonary gene expression. ER-alpha activation was specifically associated with arsenic-induced lung adenocarcinoma and adenoma but not with nitrosamine-induced lung tumors. These data provide evidence that arsenic-induced aberrant ER signaling could disrupt early life stage genetic programing in the lung leading eventually to lung tumor formation much later in adulthood.

Animals↗

PubMed2006

Caspase-independent apoptosis induced in rat liver cells by plancitoxin I, the major lethal factor from the crown-of-thorns starfish Acanthaster planci venom.

Plancitoxin I, the major lethal factor from the crown-of-thorns starfish Acanthaster planci venom, is quite unique not only in exhibiting potent hepatotoxicity but also in sharing high sequence homology with mammalian deoxyribonulease II. In this study, morphological and biochemical changes in rat liver epithelial cells (TRL 1215 cells) treated with the toxin were examined to understand the mechanism by which plancitoxin I displays hepatotoxicity. AlamarBlue assay established that plancitoxin I is cytolethal to TRL 1215 cells. This cytolethalithy was ascribable to apoptotic cell death. Nuclear fragmentation evidenced by either Diff-Quick or Hoechst 33258 staining, DNA fragmentation by TUNEL assay and electrophoretic analysis on agarose gel and phosphatidylserine externalization by flow cytometric analysis of annexin V-FITC stained cells were all characteristics of apoptosis. The observed apoptosis was shown to be independent of the caspase 3 cascade that is generally accepted as the effector of the apoptotic process. Very interestingly, experiments using FITC-labeled plancitoxin I proved that the toxin can enter the nucleus of TRL 1215 cells. Our results suggested that plancitoxin I induces apoptosis of TRL 1215 cells through the following procedure: binding to a specific receptor in the cytoplasmic membrane, entering the cell, entering the nucleus and degrading DNA.

Animals↗

PubMed2006

Hypersusceptibility to cisplatin carcinogenicity in metallothionein-I/II double knockout mice: production of hepatocellular carcinoma at clinically relevant doses.

Metallothionein (MT) is a high-affinity metal binding protein thought to mitigate the toxicity of various metals. Cisplatin is a widely used cancer chemotherapeutic that is a rodent carcinogen and may have carcinogenic potential in humans. MT seems to reduce cisplatin toxicity by binding the metal compound but how MT deficiency might impact the carcinogenic effects of cisplatin is unknown. Thus, groups (n = 25) of male MT-I/II double knockout (MT-null) or MT wild-type (WT) mice were exposed to a single treatment of cisplatin (5 or 10 mg/kg, i.p.), or left untreated (control) and observed over the next 104 weeks. The doses of cisplatin used equate to only a fraction of the total dose used typically in clinical settings. In cisplatin-treated MT-null mice, a dose-related increase in hepatocellular carcinoma (HCC) occurred (control, 0%; 5 mg/kg, 17%; 10 mg/kg, 36%) that was not seen in WT mice. Similarly, liver carcinoma multiplicity (HCC/liver) was increased markedly by cisplatin but only in MT-null mice, indicating the formation of multiple primaries in MT deficient mice. Harderian gland carcinoma incidence was also increased by cisplatin treatment in MT-null mice but not WT mice. Our results indicate that MT-null mice are hypersusceptible to the hepatocarcinogenic effects of cisplatin, and poor MT expression may be a predisposing factor for cisplatin-induced secondary tumors after chemotherapy.

Animals↗

PubMed2006

Further studies on aberrant gene expression associated with arsenic-induced malignant transformation in rat liver TRL1215 cells.

Chronic arsenic exposure of rat liver epithelial TRL1215 cells induced malignant transformation in a concentration-dependent manner. To further define the molecular events of these arsenic-transformed cells (termed CAsE cells), gene expressions associated with arsenic carcinogenesis or influenced by methylation were examined. Real-time RT-PCR showed that at carcinogenic concentrations (500 nM, and to a less extent 250 nM of arsenite), the expressions of alpha-fetoprotein (AFP), Wilm's tumor protein-1 (WT-1), c-jun, c-myc, H-ras, c-met and hepatocyte growth factor, heme oxygenase-1, superoxide dismutase-1, glutathione-S-transferase-pi and metallothionein-1 (MT) were increased between 3 to 12-fold, while expressions of insulin-like growth factor II (IGF-II) and fibroblast growth factor receptor (FGFR1) were essentially abolished. These changes were not significant at the non-carcinogenic concentration (125 nM), except for IGF-II. The positive cell-cycle regulators cyclin D1 and PCNA were overexpressed in CAsE cells, while the negative regulators p21 and p16 were suppressed. Western-blot confirmed increases in AFP, WT-1, cyclin D1 and decreases in p16 and p21 protein in CAsE cells. The CAsE cells over-expressed MT but the demethylating agent 5-aza-deoxycytidine (5-aza-dC, 2.5 microM, 72 h) stimulated further MT expression. 5-Aza-deoxycytidine restored the loss of expression of p21 in CAsE cells to control levels, but did not restore the expression of p16, IGF-II, or FGFR1, indicating the loss of expression of these genes is due to factors other than DNA methylation changes. Overall, an intricate variety of gene expression changes occur in arsenic-induced malignant transformation of liver cells including oncogene activation and alterations in expression of genes critical to growth regulation.

Animals↗

PubMed2006

Pathophysiological role of the acute inflammatory response during acetaminophen hepatotoxicity.

Neutrophils are recruited into the liver after acetaminophen (AAP) overdose but the pathophysiological relevance of this acute inflammatory response remains unclear. To address this question, we compared the time course of liver injury, hepatic neutrophil accumulation and inflammatory gene mRNA expression for up to 24 h after treatment with 300 mg/kg AAP in C3Heb/FeJ and C57BL/6 mice. Although there was no relevant difference in liver injury (assessed by the increase of plasma alanine aminotransferase activities and the areas of necrosis), the number of neutrophils and the expression of several pro-inflammatory genes (e.g., tumor necrosis factor-alpha, interleukin-1beta and macrophage inflammatory protein-2) was higher in C3Heb/FeJ than in C57BL/6 mice. In contrast, the expression of the anti-inflammatory genes interleukin-10 and heme oxygenase-1 was higher in C57BL/6 mice. Despite substantial hepatic neutrophil accumulation, none of the liver sections from both strains stained positive for hypochlorite-modified proteins, a specific marker for a neutrophil-induced oxidant stress. In addition, treatment with the NADPH oxidase inhibitors diphenyleneiodonium chloride or apocynin or the anti-neutrophil antibody Gr-1 did not protect against AAP hepatotoxicity. Furthermore, although intercellular adhesion molecule-1 (ICAM-1) was previously shown to be important for neutrophil extravasation and tissue injury in several models, ICAM-1-deficient mice were not protected against AAP-mediated liver injury. Together, these data do not support the hypothesis that neutrophils aggravate liver injury induced by AAP overdose.

Acetaminophen↗

PubMed2006

Enhanced urinary bladder and liver carcinogenesis in male CD1 mice exposed to transplacental inorganic arsenic and postnatal diethylstilbestrol or tamoxifen.

Pregnant CD1 mice received 85 ppm arsenite in the drinking water from gestation day 8 to 18, groups (n = 35) of male offspring were subsequently injected on postpartum days 1 through 5 with diethylstilbestrol (DES; 2 microg/pup/day) or tamoxifen (TAM; 10 microg/pup/day), and tumor formation was assessed over 90 weeks. Arsenic alone increased hepatocellular carcinoma (14%), adenoma (23%) and total tumors (31%) compared to control (0, 2 and 2%, respectively). Arsenic alone also increased lung adenocarcinoma, adrenal cortical adenoma and renal cystic tubular hyperplasia compared to control. Compared to arsenic alone, arsenic plus DES increased liver tumor incidence in mice at risk 2.2-fold and increased liver tumor multiplicity (tumors/liver) 1.8-fold. The treatments alone did not impact urinary bladder carcinogenesis, but arsenic plus TAM significantly increased formation of urinary bladder transitional cell tumors (papilloma and carcinoma; 13%) compared to control (0%). Urinary bladder proliferative lesions (combined tumors and hyperplasia) were also increased by arsenic plus TAM (40%) or arsenic plus DES (43%) compared to control (0%) or the treatments alone. Urinary bladder proliferative lesions occurred in the absence of any evidence of uroepithelial cytotoxic lesions. Urinary bladder lesions and hepatocellular carcinoma induced by arsenic plus TAM and/or DES overexpressed estrogen receptor-alpha, indicating that aberrant estrogen signaling may have been a factor in the enhanced carcinogenic response. Thus, in male CD1 mice, gestational arsenic exposure alone induced liver adenoma and carcinoma, lung adenocarcinoma, adrenal adenoma and renal cystic hyperplasia. Furthermore, DES enhanced transplacental arsenic-induced hepatocarcinogenesis. In utero arsenic also initiated urinary bladder tumor formation when followed by postnatal TAM and uroepithelial proliferative lesions when followed by TAM or DES.

Animals↗

PubMed2006

Arsenicals in maternal and fetal mouse tissues after gestational exposure to arsenite.

Exposure of pregnant C3H/HeNCR mice to 42.5- or 85-ppm of arsenic as sodium arsenite in drinking water between days 8 and 18 of gestation markedly increases tumor incidence in their offspring. In the work reported here, distribution of inorganic arsenic and its metabolites, methyl arsenic and dimethyl arsenic, were determined in maternal and fetal tissues collected on gestational day 18 of these exposure regimens. Tissues were collected from three females and from associated fetuses exposed to each dosage level. Concentrations of total speciated arsenic (sum of inorganic, methyl, and dimethyl arsenic) were higher in maternal tissues than in placenta and fetal tissues; total speciated arsenic concentration in placenta exceeded those in fetal tissues. Significant dosage-dependent (42.5 ppm versus 85 ppm of arsenite in drinking water) differences were found in total speciated arsenic concentrations in maternal lung (p<0.01) and liver (p<0.001). Total speciated arsenic concentrations did not differ significantly between dosage levels for maternal blood or for fetal lung, liver, and blood, or for placenta. Percentages of inorganic, methyl, or dimethyl arsenic in maternal or fetal tissues were not dosage-dependent. Over the range of total speciated arsenic concentrations in most maternal and fetal tissues, dimethyl arsenic was the most abundant arsenical. However, in maternal liver at the highest total speciated arsenic concentration, inorganic arsenic was the most abundant arsenical, suggesting that a high tissue burden of arsenic affected formation or retention of methylated species in this organ. Tissue concentration-dependent processes could affect kinetics of transfer of inorganic arsenic or its metabolites from mother to fetus.

Animals↗

PubMed2006

Molecular mechanism of distorted iron regulation in the blood-CSF barrier and regional blood-brain barrier following in vivo subchronic manganese exposure.

Previous studies in this laboratory indicated that manganese (Mn) exposure in vitro increases the expression of transferrin receptor (TfR) by enhancing the binding of iron regulatory proteins (IRPs) to iron responsive element-containing RNA. The current study further tested the hypothesis that in vivo exposure to Mn increased TfR expression at both blood-brain barrier (BBB) and blood-cerebrospinal fluid (CSF) barrier (BCB), which contributes to altered iron (Fe) homeostasis in the CSF. Groups of rats (10-11 each) received oral gavages at doses of 5 mg Mn/kg or 15 mg Mn/kg as MnCl(2) once daily for 30 days. Blood, CSF, and choroid plexus were collected and brain capillary fractions were separated from the regional parenchyma. Metal analyses showed that oral Mn exposure decreased concentrations of Fe in serum (-66%) but increased Fe in the CSF (+167%). Gel shift assay showed that Mn caused a dose-dependent increase of binding of IRP1 to iron responsive element-containing RNA in BCB in the choroid plexus (+70%), in regional BBB of capillaries of striatum (+39%), hippocampus (+56%), frontal cortex (+49%), and in brain parenchyma of striatum (+67%), hippocampus (+39%) and cerebellum (+28%). Real-time RT-PCR demonstrated that Mn exposure significantly increased the expression of TfR mRNA in choroid plexus and striatum with concomitant reduction in the expression of ferritin (Ft) mRNA. Collectively, these data indicate that in vivo Mn exposure results in Fe redistribution in body fluids through regulating the expression of TfR and ferritin at BCB and selected regional BBB. The disrupted Fe transport by brain barriers may underlie the distorted Fe homeostasis in the CSF.

Animals↗

PubMed2006

Urogenital carcinogenesis in female CD1 mice induced by in utero arsenic exposure is exacerbated by postnatal diethylstilbestrol treatment.

Transplacental inorganic arsenic carcinogenicity, together with postnatal exposure to diethylstilbestrol or tamoxifen, was studied. Pregnant CD1 mice received 85 ppm arsenic in the drinking water from gestation days 8 to 18 and were allowed to give birth. Groups (n = 35) of female offspring were injected s.c. on postpartum days 1 through 5 with diethylstilbestrol (2 microg/pup/d) or tamoxifen (10 microg/pup/d) and observed for 90 weeks. Arsenic alone induced some urogenital system tumors, including mostly benign tumors of the ovary and uterus, and adrenal adenoma. Diethylstilbestrol alone induced some tumors (primarily cervical) but when given after in utero arsenic, it greatly enhanced urogenital tumor incidence, multiplicity, and progression. For instance, compared with the incidence of urogenital malignancies in the control (0%), arsenic alone (9%), and diethylstilbestrol alone (21%) groups, arsenic plus diethylstilbestrol acted synergistically, inducing a 48% incidence of malignant urogenital tumors. Of the urogenital tumors induced by arsenic plus diethylstilbestrol, 80% were malignant, and 55% were multiple site. Arsenic plus diethylstilbestrol increased ovarian, uterine, and vaginal tumors, and urinary bladder proliferative lesions, including three transitional cell carcinomas. Tamoxifen alone did not increase urogenital tumors or affect arsenic-induced neoplasia but did increase arsenic-induced uroepithelial proliferative lesions. Uterine and bladder carcinoma induced by arsenic plus diethylstilbestrol greatly overexpressed estrogen receptor-alpha (ER-alpha) and pS2, an estrogen-regulated gene. In neonatal uteri, prenatal arsenic increased ER-alpha expression and enhanced estrogen-related gene expression induced by postnatal diethylstilbestrol. Thus, arsenic acts with estrogens to enhance production of female mouse urogenital cancers.

Animals↗

PubMed2006

Chronic exposure to methylated arsenicals stimulates arsenic excretion pathways and induces arsenic tolerance in rat liver cells.

Although inorganic arsenicals are toxic and carcinogenic in humans, inorganic arsenite has recently emerged as a highly effective chemotherapeutic agent for acute promyelocytic leukemia (APL). Inorganic arsenicals are enzymatically methylated to monomethylarsonic acid (MMAs(V)), dimethylarsinic acid (DMAs(V)), and trimethylarsine oxide (TMAs(V)O) in mammals. We examined the effects of chronic exposure to methylated arsenicals on arsenic tolerance by using rat normal liver TRL 1215 cells. TRL 1215 cells were exposed for 20 weeks to MMAs(V), DMAs(V), or TMAs(V)O at levels that produced submicromolar cellular concentrations of arsenic. On chronic exposure to these methylated arsenicals, the cells acquired tolerance to acute arsenic cytolethality. Cellular arsenic uptake was reduced in these cells compared to passage-matched control cells. The long-term arsenic exposure increased glutathione S-transferase (GST) activity and cellular glutathione (GSH) levels. Glutathione S-transferase, multidrug resistance-associated proteins (Mrps; efflux transporters encoded by Mrp genes), and P-glycoprotein [P-gp; efflux transporter encoded by multidrug resistance gene (MDR)] had also increased in these cells at the transcript and protein levels. The depletion of cellular GSH and the inhibition of Mrps and P-gp functions increased cellular arsenic uptake and reduced arsenic tolerance in these cells. These results indicate that chronic exposure to methylated arsenicals induces a generalized arsenic tolerance that is caused by increased arsenic excretion. Because accumulation of methylated arsenicals may occur in patients with chronic arsenic poisoning and arsenic-treated APL patients, this study may provide important information regarding chronic arsenic poisoning and the latent risk of developing multidrug resistance in APL therapy using inorganic arsenite.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2006

Acquisition of apoptotic resistance in cadmium-induced malignant transformation: specific perturbation of JNK signal transduction pathway and associated metallothionein overexpression.

Prior work has shown that chronic cadmium exposed rat liver epithelial cells (CCE-LE) become malignantly transformed after protracted low level cadmium exposure. Acquisition of apoptotic resistance is common in oncogenesis and the present work explores this possibility in CCE-LE cells. CCE-LE cells were resistant to apoptosis induced by etoposide or an acute high concentration of cadmium as assessed by flow cytometry with annexin/FITC. Three key mitogen-activated protein kinases (MAPKs), namely ERK1/2, JNK1/2, and p38, were phosphorylated in CCE-LE cells after acute cadmium exposure. However, the levels of phosphorylated JNK1/2 were markedly decreased in CCE-LE cells compared to control. JNK kinase activity was also suppressed in CCE-LE cells exposed to cadmium. Epidermal growth factor (EGF), used as a positive control for stimulating JNK phosphorylation, was much less effective in CCE-LE cells than control cells. Ro318220 (Ro), a strong activator of JNK, increased phosphorylated JNK1/2 to levels similar to the cadmium-treated control cells and also enhanced apoptosis in response to cadmium in CCE-LE cells. Metallothionein (MT), which is thought to potentially inhibit apoptosis, was strongly overexpressed in CCE-LE cells. Further, in MT knockout (MT-/-) fibroblasts, JNK1/2 phosphorylation was markedly increased after cadmium exposure compared with similarly treated wild-type (MT+/+) cells. These results indicate cadmium-transformed cells acquired apoptotic resistance, which may be linked to the specific suppression of the JNK pathway and is associated with MT overexpression, which, in turn, may impact this signal transduction pathway. The acquisition of apoptotic resistance may play an important role in cadmium carcinogenesis by contributing to both tumor initiation and malignant progression.

Animals↗

PubMed2006

24-norUrsodeoxycholic acid is superior to ursodeoxycholic acid in the treatment of sclerosing cholangitis in Mdr2 (Abcb4) knockout mice.

BACKGROUND & AIMS: Current therapy for primary sclerosing cholangitis is of limited efficacy. Multidrug resistance gene 2 knockout mice (Mdr2(-/-)) represent a well-characterized model for sclerosing cholangitis. Experiments were performed to test in such mice the therapeutic effects of 24-norUrsodeoxycholic acid, a C(23) homologue of ursodeoxycholic acid with 1 fewer methylene group in its side chain. METHODS: Mdr2(-/-) mice were fed a diet containing 24-norUrsodeoxycholic acid (0.5% wt/wt) or ursodeoxycholic acid (0.5% wt/wt) as a clinical comparator for 4 weeks; controls received standard chow. Effects on serum liver tests, liver histology, markers of inflammation and fibrosis, and bile acid transport and metabolism were compared. 24-norUrsodeoxycholic acid metabolism was studied in serum, liver, bile, and urine. RESULTS: 24-norUrsodeoxycholic acid markedly improved liver tests and liver histology and significantly reduced hydroxyproline content and the number of infiltrating neutrophils and proliferating hepatocytes and cholangiocytes. 24-norUrsodeoxycholic acid underwent extensive phase I/II metabolism (hydroxylation, sulfation, and glucuronidation), thereby increasing the hydrophilicity of biliary bile acid secretion. There was a coordinated induction of bile acid detoxifying enzymes (Cyp2b10, Cyp3a11, and Sult2a1) and efflux pumps (Mrp3 and Mrp4). Ursodeoxycholic acid, in contrast, increased alanine transaminase and alkaline phosphatase levels, had no significant effects on hydroxyproline content, and induced biliary transporters and detoxification enzymes to a much smaller extent than 24-norUrsodeoxycholic acid. CONCLUSIONS: 24-norUrsodeoxycholic acid ameliorates sclerosing cholangitis in Mdr2(-/-) mice. Its therapeutic mechanisms involve (1) increasing the hydrophilicity of biliary bile acids, (2) stimulating bile flow with flushing of injured bile ducts, and (3) inducing detoxification and elimination routes for bile acids.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2006

Differential effects between maotai and ethanol on hepatic gene expression in mice: Possible role of metallothionein and heme oxygenase-1 induction by maotai.

Alcohol is a risk factor for liver fibrosis and hepatocellular carcinoma. On the other hand, light alcoholic beverage consumption is believed to be beneficial because of the effects of both alcohol and nonalcoholic components of the beverage. Maotai is a commonly consumed beverage in China containing 53% alcohol. Epidemiological and experimental studies show that Maotai is less toxic to the liver than ethanol alone. To examine the differential effects of Maotai and ethanol, a low dose of Maotai or an equal amount of ethanol (53%, v/v in water, 5 ml/kg) were given to male mice daily for 1 week, and hepatic RNA was extracted for microarray analysis. Approximately 10% of genes on the liver-selective custom array (588 genes) were altered following Maotai or ethanol administration, but Maotai treated livers had fewer alterations compared with ethanol alone. Real-time reverse transcription-polymerase chain reaction confirmed and extended microarray results on selected genes. An induction of metallothionein and heme oxygenase-1 occurred with Maotai, which could not be explained by alcohol consumption alone, whereas the attenuation of ethanol responsive genes such as quinone dehydrogenase, DNA-ligase 1, IGFBP1, and IL-1beta suggests less liver injury occurred with Maotai. The expression of genes related to liver fibrosis, such as cytokeratin-18, was slightly increased by the high dose of ethanol, but was unchanged in the Maotai group. In summary, gene expression analysis indicates that Maotai induces a different response than ethanol alone. The dramatic induction of metallothionein and heme oxygenase-1 with Maotai could be important adaptive responses to reduce alcoholic liver injury.

Animals↗

PubMed2006

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 produces hepatocellular carcinoma (HCC) in adult male mice. To explore further the molecular mechanisms of transplacental arsenic hepatocarcinogenesis, we conducted a second arsenic transplacental carcinogenesis study and used a genomewide microarray to profile arsenic-induced aberrant gene expression more extensively. Briefly, pregnant C3H mice were given drinking water 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 of selected genes/proteins showed > 90% concordance. Arsenic-altered gene expression included activation of oncogenes and HCC biomarkers, and increased expression of cell proliferation-related genes, stress proteins, and insulin-like growth factors and genes involved in cell-cell communications. Liver feminization 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.

Animals↗

PubMed2005

Transplacental arsenic plus postnatal 12-O-teradecanoyl phorbol-13-acetate exposures associated with hepatocarcinogenesis induce similar aberrant gene expression patterns in male and female mouse liver.

Our prior work shows that in utero arsenic exposure alone is a complete transplacental carcinogen, producing hepatocellular carcinoma in adult male offspring but not in females. In a follow-up study to potentially promote arsenic-initiated tumors, mice were exposed to arsenic (85 ppm) from gestation day 8 to 18 and then exposed to 12-O-teradecanoyl phorbol-13-acetate (TPA), a well-known tumor promoter after weaning. The dermal application of TPA (2 mug/0.1 ml acetone, twice/week for 21 weeks) after transplacental arsenic did not further increase arsenic-induced liver tumor formation in adult males but significantly increased liver tumor formation in adult females. Thus, for comparison, liver tumors and normal liver samples taken from adult male and female mice at necropsy were analyzed for aberrant gene/protein expression by microarray, real-time RT-PCR and Western blot analysis. Arsenic/TPA treatment resulted in increased expression of alpha-fetoprotein, k-ras, c-myc, estrogen receptor-alpha, cyclin D1, cdk2na, plasminogen activator inhibitor-1, cytokeratin-8, cytokeratin-18, glutathione S-transferases and insulin-like growth factor binding proteins in liver and liver tumors from both male and female mice. Arsenic/TPA also decreased the expression of BRCA1, betaine-homocysteine methyltransferase, CYP7B1, CYP2F2 and insulin-like growth factor-1 in normal and cancerous livers. Alterations in these gene products were associated with arsenic/TPA-induced liver tumors, regardless of sex. Thus, transplacental arsenic plus postnatal TPA exposure induced similar aberrant gene expression patterns in male and female mouse liver, which are persistent and potentially important to the mechanism of arsenic initiation of hepatocarcinogenesis.

Administration, Topical↗

PubMed2005

Acquired cadmium resistance in metallothionein-I/II(-/-) knockout cells: role of the T-type calcium channel Cacnalpha1G in cadmium uptake.

Metallothioneins (MTs) are cytoplasmic proteins that sequester certain divalent cations and are considered a primary cellular defense against the toxic transition metal cadmium (Cd(2+)). MT-I/II(-/-) knockout [MT(-/-)] cells are available and serve as an excellent tool to study non-MT-related mechanisms in metal tolerance. In the current study, Cd(2+)-resistant MT(-/-) (CdR) and CdR revertant (CdR-rev) cell lines were developed and characterized to investigate non-MT-mediated cellular protection mechanisms. Resistance to Cd(2+) was approximately 70-fold higher in CdR than the parental MT(-/-) cell line (IC(50) = 20 versus 0.3 microM, respectively) and was stable in the absence of Cd(2+) for 35 days. Accumulation of Cd(2+) by the CdR cell line was reduced by approximately 95% compared with parental cells, primarily because of a decreased Cd(2+) uptake. Cd(2+) uptake by the MT(-/-) parental cell line was independent of sodium, energy, and electrogenic potential. Uptake was saturable (K(m) = 65 nM; V(max) = 4.9 pmol/mg/min) and pH-dependent (maximal at pH 6.5-7). Potent inhibitors of Cd(2+) uptake included Zn(2+) (IC(50) = 7 microM), Mn(2+) (IC(50) = 0.4 microM), and the T-type Ca(2+) channel antagonist mibefradil (IC(50) = 5 microM), whereas other metals (including Fe(2+)) and L-type Ca(2+) channel antagonists had little effect. Immunoblot and real-time reverse transcription-polymerase chain reaction analysis indicated that the Cacnalpha(1G) T-type Ca(2+) channel was expressed at a reduced level in CdR compared with the parental MT(-/-) cell line, suggesting it is important for Cd(2+) uptake. The CdR1-rev cell line was found to have a Cd(2+) uptake and sensitivity level in between that of the CdR1 and MT(-/-) cell lines. Consistent with this was an intermediate expression of Cacnalpha(1G) in the CdR-rev cell line. These data suggest that decreased expression of Cacnalpha(1G) protects cells from Cd(2+) exposure by limiting Cd(2+) uptake.

Animals↗

PubMed2005

Low level, long-term inorganic arsenite exposure causes generalized resistance to apoptosis in cultured human keratinocytes: potential role in skin co-carcinogenesis.

Inorganic arsenic is a human carcinogen that targets the skin. Carcinogenesis is a multistep process in which acquired apoptotic resistance is a common event and prior work in non-skin cells shows acquired resistance to apoptosis occurs with chronic arsenite exposure. In the present study, when HaCaT cells, an immortalized, non-tumorigenic human keratinocyte cell line, were continuously exposed to low-level inorganic arsenite (as sodium arsenite; 100 nM) for 28 weeks, the cells acquired a generalized resistance to apoptosis. This included resistance to apoptosis induced by acute high concentrations of arsenite, ultraviolet A (UVA) irradiation, and several chemotherapeutic compounds (cisplatin, etoposide and doxorubicin). These arsenite-tolerant (As-TL) cells showed similar levels of UVA-induced reactive oxygen species (ROS) and oxidative DNA damage when compared to passage match control cells. Because cellular apoptosis is dependent on the balance between proapoptotic and survival pathways, the roles of protein kinase B (PKB), a key antiapoptotic molecule, in this acquired apoptotic resistance were investigated. Stimulation of apoptosis markedly decreased nuclear phosphorylated PKB (P-PKB) levels in control cells, but As-TL cells showed greatly increased stability of nuclear P-PKB. Pretreatment of the As-TL cells with LY294002 or Wortmannin, which specifically inhibit PKB phosphorylation, completely blocked apoptotic resistance in As-TL cells, indicating acquired apoptotic resistance is associated with increased stability of nuclear P-PKB. Because arsenic and UV irradiation are co-carcinogenic in mouse skin, resistance to UV-induced apoptosis in As-TL cells may allow UV-damaged cells to escape normal cell population controls and initiate the carcinogenic cascade. The observation that As-TL cells show no lessening of UV-induced genotoxicity supports this possibility.

Apoptosis↗