Chronic exposure to arsenite enhances influenza virus infection in cultured cells.

Arsenic is a ubiquitous environmental toxicant that has been associated with human respiratory diseases. In humans, arsenic exposure has been associated with increased risk of respiratory infection. Considering the existing epidemiological evidence and the well-established impact of arsenic on epithelial cell biology, we posited that the effect of arsenic exposure in epithelial cells could enhance viral infection. In this study, we characterized influenza virus A/WSN/33 (H1N1) infection in Madin-Darby Canine Kidney (MDCK) cells chronically exposed to low levels of sodium arsenite (75 ppb). We observed a 27.3-fold increase in viral matrix (M2) protein (24 hours postinfection [p.i.]), a 1.35-fold increase in viral mRNA levels, and a 126% increase in plaque area in arsenite-exposed MDCK cells (48 hours p.i.). Arsenite exposure resulted in 114% increase in virus attachment-positive cells (2 hours p.i.) and 224% increase in α-2,3 sialic acid-positive cells. Interestingly, chronic exposure to arsenite reduced the effect of the antiviral drug, oseltamivir in MDCK cells. We also found that exposure to sodium arsenite resulted in a 4.4-fold increase in viral mRNA levels and significantly increased cytotoxicity in influenza A/Udorn/72 (H3N2) infected BEAS-2B cells. This study suggests that chronic arsenite exposure could result in enhanced influenza infection in epithelial cells, and that this may be mediated through increased sialic acid binding. Finally, the decreased effectiveness of the anti-influenza drug, oseltamivir, in arsenite-exposed cells raises substantial public health concerns if this effect translates to arsenic-exposed, influenza-infected people.

Culture conditions profoundly impact phenotype in BEAS-2B, a human pulmonary epithelial model.

BEAS-2B, an immortalized, human lung epithelial cell line, has been used to model pulmonary epithelial function for over 30 years. The BEAS-2B phenotype can be modulated by culture conditions that include the presence or absence of fetal bovine serum (FBS). The popularity of BEAS-2B as a model of arsenic toxicology, and the common use of BEAS-2B cultured both with and without FBS, led us to investigate the impact of FBS on BEAS-2B in the context of arsenic toxicology. Comparison of genome-wide gene expression in BEAS-2B cultured with or without FBS revealed altered expression in several biological pathways, including those related to carcinogenesis and energy metabolism. Real-time measurements of oxygen consumption and glycolysis in BEAS-2B demonstrated that FBS culture conditions were associated with a 1.4-fold increase in total glycolytic capacity, a 1.9-fold increase in basal respiration, a 2.0-fold increase in oxygen consumed for ATP production and a 2.8-fold increase in maximal respiration, compared with BEAS-2B cultured without FBS. Comparisons of the transcriptome changes in BEAS-2B resulting from FBS exposure to the transcriptome changes resulting from exposure to 1 µM sodium arsenite revealed that mRNA levels of 43% of the arsenite-modulated genes were also modulated by FBS. Cytotoxicity studies revealed that BEAS-2B cells exposed to 5% FBS for 8 weeks were almost 5 times more sensitive to arsenite cytotoxicity than non-FBS-exposed BEAS-2B cells. Phenotype changes induced in BEAS-2B by FBS suggest that culture conditions should be carefully considered when using BEAS-2B as an experimental model of arsenic toxicity.

Arsenite-induced pseudo-hypoxia results in loss of anchorage-dependent growth in BEAS-2B pulmonary epithelial cells.

Epidemiology studies have established a strong link between lung cancer and arsenic exposure. Currently, the role of disturbed cellular energy metabolism in carcinogenesis is a focus of scientific interest. Hypoxia inducible factor-1 alpha (HIF-1A) is a key regulator of energy metabolism, and it has been found to accumulate during arsenite exposure under oxygen-replete conditions. We modeled arsenic-exposed human pulmonary epithelial cells in vitro with BEAS-2B, a non-malignant lung epithelial cell line. Constant exposure to 1 µM arsenite (As) resulted in the early loss of anchorage-dependent growth, measured by soft agar colony formation, beginning at 6 weeks of exposure. This arsenite exposure resulted in HIF-1A accumulation and increased glycolysis, similar to the physiologic response to hypoxia, but in this case under oxygen-replete conditions. This "pseudo-hypoxia" response was necessary for the maximal acquisition of anchorage-independent growth in arsenite-exposed BEAS-2B. The HIF-1A accumulation and induction in glycolysis was sustained throughout a 52 week course of arsenite exposure in BEAS-2B. There was a time-dependent increase in anchorage-independent growth during the exposure to arsenite. When HIF-1A expression was stably suppressed, arsenite-induced glycolysis was abrogated, and the anchorage-independent growth was reduced. These findings establish that arsenite exerts a hypoxia-mimetic effect, which plays an important role in the subsequent gain of malignancy-associated phenotypes.

Arsenic exposure induces the Warburg effect in cultured human cells.

Understanding how arsenic exacts its diverse, global disease burden is hampered by a limited understanding of the particular biological pathways that are disrupted by arsenic and underlie pathogenesis. A reductionist view would predict that a small number of basic pathways are generally perturbed by arsenic, and manifest as diverse diseases. Following an initial observation that arsenite-exposed cells in culture acidify their media more rapidly than control cells, the report here shows that low level exposure to arsenite (75ppb) is sufficient to induce aerobic glycolysis (the Warburg effect) as a generalized phenomenon in cultured human primary cells and cell lines. Expanded studies in one such cell line, the non-malignant pulmonary epithelial line, BEAS-2B, established that the arsenite-induced Warburg effect was associated with increased accumulation of intracellular and extracellular lactate, an increased rate of extracellular acidification, and inhibition by the non-metabolized glucose analog, 2-deoxy-D-glucose. Associated with the induction of aerobic glycolysis was a pathway-wide induction of glycolysis gene expression, as well as protein accumulation of an established glycolysis master-regulator, hypoxia-inducible factor 1A. Arsenite-induced alteration of energy production in human cells represents the type of fundamental perturbation that could extend to many tissue targets and diseases.

How can biologically-based modeling of arsenic kinetics and dynamics inform the risk assessment process? - A workshop review.

Quantitative biologically-based models describing key events in the continuum from arsenic exposure to the development of adverse health effects provide a framework to integrate information obtained across diverse research areas. For example, genetic polymorphisms in arsenic metabolizing enzymes can lead to differences in target tissue dosimetry for key metabolites causative in toxic and carcinogenic response. This type of variation can be quantitatively incorporated into pharmacokinetic (PK) models and used together with population-based modeling approaches to evaluate the impact of genetic variation in methylation capacity on dose of key metabolites to target tissue. The PK model is an essential bridge to the pharmacodynamic (PD) models. A particular benefit of PD modeling for arsenic is that alternative models can be constructed for multiple proposed modes of action for arsenicals. Genomics data will prove useful for identifying the key pathways involved in particular responses and aid in determining other types of data needed for quantitative modeling. These models, when linked with PK models, can be used to better understand and explain dose- and time-response behaviors. This in turn assists in prioritizing modes of action with respect to their risk assessment relevance and future research. This type of integrated modeling approach can form the basis for a highly informative mode-of-action directed risk assessment for inorganic arsenic (iAs). This paper will address both practical and theoretical aspects of integrating PK and PD data in a modeling framework, including practical barriers to its application.

Epigenetic inactivation of the HOXA gene cluster in breast cancer.

Using an integrated approach of epigenomic scanning and gene expression profiling, we found aberrant methylation and epigenetic silencing of a small neighborhood of contiguous genes-the HOXA gene cluster in human breast cancer. The observed transcriptional repression was localized to approximately 100 kb of the HOXA gene cluster and did not extend to genes located upstream or downstream of the cluster. Bisulfite sequencing, chromatin immunoprecipitation, and quantitative reverse transcription-PCR analysis confirmed that the loss of expression of the HOXA gene cluster in human breast cancer is closely linked to aberrant DNA methylation and loss of permissive histone modifications in the region. Pharmacologic manipulations showed the importance of these aberrant epigenetic changes in gene silencing and support the hypothesis that aberrant DNA methylation is dominant to histone hypoacetylation. Overall, these data suggest that inactivation of the HOXA gene cluster in breast cancer may represent a new type of genomic lesion-epigenetic microdeletion. We predict that epigenetic microdeletions are common in human cancer and that they functionally resemble genetic microdeletions but are defined by epigenetic inactivation and transcriptional silencing of a relatively small set of contiguous genes along a chromosome, and that this type of genomic lesion is metastable and reversible in a classic epigenetic fashion.

Cross-sectional analysis of oncogenic HPV viral load and cervical intraepithelial neoplasia.

In human papillomavirus (HPV)-associated carcinogenesis, HPV infection characteristics such as viral load may play an important role in lesion development. The purpose of this study was to determine the association between quantitative assessment of oncogenic HPV viral load, and abnormal cytology among women residing along the United States-Mexico border. A cross-sectional study of 2,319 women was conducted between 1997 and 1998. Viral load of oncogenic HPV types (16, 18, 31, 39, 45, 51, 52, and 58) was measured among 173 HPV (+) women using quantitative real-time PCR. Overall, HPV 16, 31, 52 and 58 showed the highest viral load. Single type infection had higher viral loads compared to multiple type infections. HPV viral load declined significantly (p = 0.04) with age. No significant association was observed with other known HPV risk factors such as oral contraceptive use, parity, sexual and STD history. Viral load was independently associated with degree of cervical lesions. An adjusted odds ratio (AOR) of 4.7 for the association between increasing total viral load and Atypical Squamous Cells of Undetermined Significance (ASCUS)/Atypical Glandular Cells of Undetermined Significance (AGUS) was observed (p for trend <0.01). Increased risk of low-grade SIL was observed with higher viral load compared with HPV negative women (AOR = 47.7 for total viral load; AOR = 37.1 for HPV viral load not including HPV16, and AOR = 25.9 for HPV16 viral load). Likewise, increased risk of high-grade SIL with higher viral loads was observed (AOR = 58.4 for high total viral load compared with HPV negative women, AOR = 58.1 for HPV viral load not including HPV16, and AOR = 69.8 for HPV16 high viral load). Results from this study suggest a dose-response relationship between increasing oncogenic HPV viral load and risk of LSIL and HSIL.

Epigenetic regulation of the cell type-specific gene 14-3-3sigma.

Epigenetic control participates in processes crucial in mammalian development, such as X-chromosome inactivation, gene imprinting, and cell type-specific gene expression. We provide evidence that the p53-inducible gene 14-3-3sigma is a new example of a gene important to human cancer, where epigenetic mechanisms participate in the control of normal cell type-specific expression, as well as aberrant gene silencing in cancer cells. Like a previously identified cell type-specific gene maspin, 14-3-3sigma is a p53-inducible gene; however, it participates in G2/M arrest in response to DNA-damaging agents. 14-3-3Sigma expression is restricted to certain epithelial cell types, including breast and prostate, whereas expression is absent in nonepithelial tissues such as fibroblasts and lymphocytes. In this report, we show that in normal cells expressing 14-3-3sigma, the 14-3-3sigma CpG island is unmethylated; associated with acetylated histones, unmethylated histone H3 lysine 9; and an accessible chromatin structure. By contrast, normal cells that do not express 14-3-3sigma have a methylated 14-3-3sigma CpG island with hypoacetylated histones, methylated histone H3 lysine 9, and an inaccessible chromatin structure. These findings extend the spectrum of cell type-specific genes controlled, partly, by normal epigenetic mechanisms, and suggest that this subset of genes may represent important targets of epigenetic dysregulation in human cancer.

Performance assessment of eight high-throughput PCR assays for viral load quantitation of oncogenic HPV types.

Infection with mucosotropic human papillomavirus (HPV) is the necessary cause of cervical intraepithelial neoplasia. Several epidemiological studies suggest that HPV viral load can be a risk factor of cervical dysplasia. The purpose of the present study was to evaluate a methodology to determine HPV viral load of eight oncogenic HPV types (16, 18, 31, 39, 45, 51, 52, and 58). The quantitation assay is based on a high-throughput real-time PCR. The E6-E7 region of HPV types 16, 18, 45, and 51 were used to amplify specific DNA sequences and cloned into a plasmid vector. The constructs for HPV types 16, 18, 45, and 51, and the whole genome for HPV types 31, 39, 52, and 58 were quantitated using a limiting dilution analysis and used to create standard curves. Type-specific HPV clones were used to determine specificity, linearity, and intra- and inter-assay variation. The sensitivity of our assay covered a dynamic range of up to seven orders of magnitude with a coefficient of intra-assay variation less than 6% and the inter-assay variation less than 20%. No cross-reactivity was observed on any of the type-specific standard curves when phylogenetically close HPV types were used as templates. Our real-time PCR methodologies are highly reproducible, sensitive, and specific over a sevenfold magnitude dynamic range.