Dimethylarsinic acid in drinking water changed the morphology of urinary bladder but not the expression of DNA repair genes of bladder transitional epithelium in F344 rats.

Inorganic arsenic increases urinary bladder transitional cell carcinoma in humans. In F344 rats, dimethylarsinic acid (DMA[V]) increases transitional cell carcinoma. Arsenic-induced inhibition of DNA repair has been reported in cultured cell lines and in lymphocytes of arsenic-exposed humans, but it has not been studied in urinary bladder. Should inhibition of DNA damage repair in transitional epithelium occur, it may contribute to carcinogenesis or cocarcinogenesis. We investigated morphology and expression of DNA repair genes in F344 rat transitional cells following up to 100 ppm DMA(V) in drinking water for four weeks. Mitochondria were very sensitive to DMA(V), and swollen mitochondria appeared to be the main source of vacuoles in the transitional epithelium. Real-time reverse transcriptase polymerase chain reaction (Real-Time RT PCR) showed the mRNA levels of tested DNA repair genes, ataxia telangectasia mutant (ATM), X-ray repair cross-complementing group 1 (XRCC1), excision repair cross-complementing group 3/xeroderma pigmentosum B (ERCC3/XPB), and DNA polymerase beta (Polbeta), were not altered by DMA(V). These data suggested that either DMA(V) does not affect DNA repair in the bladder or DMA(V) affects DNA repair without affecting baseline mRNA levels of repair genes. The possibility remains that DMA(V) may lower damage-induced increases in repair gene expression or cause post-translational modification of repair enzymes.

Transcriptional responses to complex mixtures: a review.

Exposure of people to hazardous compounds is primarily through complex environmental mixtures, those that occur through media such as air, soil, water, food, cigarette smoke, and combustion emissions. Microarray technology offers the ability to query the entire genome after exposure to such an array of compounds, permitting a characterization of the biological effects of such exposures. This review summarizes the published literature on the transcriptional profiles resulting from exposure of cells or organisms to complex environmental mixtures such as cigarette smoke, diesel emissions, urban air, motorcycle exhaust, carbon black, jet fuel, and metal ore and fumes. The majority of the mixtures generally up-regulate gene expression, with heme oxygenase 1 and CYP1A1 being up-regulated by all of the mixtures. Most of the mixtures altered the expression of genes involved in oxidative stress response (OH-1, metallothioneins), immune/inflammation response (IL-1b, protein kinase), xenobiotic metabolism (CYP1A1, CYP1B1), coagulation and fibrinolysis (plasminogen activator/inhibitor), proto-oncogenes (FUS1, JUN), heat-shock response (HSP60, HSP70), DNA repair (PCNA, GADD45), structural unit of condensed DNA (Crf15Orf16, DUSP 15), and extracellular matrix degradation (MMP1, 8, 9, 11, 12). Genes involved in aldehyde metabolism, such as ALDH3, appeared to be uniquely modulated by cigarette smoke. Cigarette smoke-exposed populations have been successfully distinguished from control nonexposed populations based on the expression pattern of a subset of genes, thereby demonstrating the utility of this approach in identifying biomarkers of exposure and susceptibility. The analysis of gene-expression data at the pathway and functional level, along with a systems biology approach, will provide a more comprehensive insight into the biological effects of complex mixtures and will improve risk assessment of the same. We suggest critical components of study design and reporting that will achieve this goal.

Identification of interspecies concordance of mechanisms of arsenic-induced bladder cancer.

Exposure to arsenic causes cancer by inducing a variety of responses that affect the expression of genes associated with numerous biological pathways leading to altered cell growth and proliferation, signaling, apoptosis and oxidative stress response. Affymetrix GeneChip arrays were used to detect gene expression changes following dimethylarsinic acid (DMA) exposure to human bladder cells (UROtsa) or rat bladder cells (MYP3) and rat bladder epithelium in vivo at comparable doses. Using different experimental models coupled with transcriptional profiling allowed investigation of the correlation of mechanisms of DMA-induced toxicity between in vitro and in vivo treatment and across species. Our observations suggest that DMA-induced gene expression in UROtsa cells is distinct from that observed in the MYP3 cells. Principal component analysis shows a more distinct separation by treatment and dose in MYP3 cells as compared to UROtsa cells. However, at the level of pathways and biological networks, DMA affects both common and unique processes in the bladder transitional cells of human and rats. Twelve pathways were found common between human in vitro, rat in vitro and rat in vivo systems. These included signaling pathways involved in adhesion, cellular growth and differentiation. Fifty-five genes found to be commonly expressed between rat in vivo and rat in vitro systems were involved in diverse functions such as cell cycle regulation, lipid metabolism and protein degradation. Many of the genes, processes and pathways have previously been associated with arsenic-induced toxicity. Our finding reiterates and also identifies new biological processes that might provide more information regarding the mechanisms of DMA-induced toxicity. The results of our analysis further suggest that gene expression profiles can address pertinent issues of relevance to risk assessment, namely interspecies extrapolation of mechanistic information as well as comparison of in vitro to in vivo response.

Camptothecin analogs with enhanced activity against human breast cancer cells. II. Impact of the tumor pH gradient.

Human breast tumors often exist in an acidic and hypoxic microenvironment, which can promote resistance to radiation and chemotherapies. A tumor-selective pH gradient arises in these tumors which favors uptake and retention of drugs like camptothecin that are weak acids. We evaluated the effect of alkyl substitutions at the 7 position in seven CPTs with varying groups at the 10 position on modulation by acidic extracellular pH in three human breast cancer cell lines. Growth inhibition was assessed by propidium iodide staining of nucleic acids in human breast cancer cells cultured at either extracellular pH 6.8 or 7.4 that were (1) hormone-sensitive (MCF-7/wt), (2) hormone insensitive (MDA-MB-231), or (3) alkylator-resistant (MCF-7/4-hc). Over 10-fold pH modulation was observed in 7-halomethyl analogs of methylenedioxy-CPT and in 7-alkyl analogs of 10-amino-CPT. Of 39 analogs tested, the overall pattern of activity across breast tumor cell lines was similar with some notable exceptions. For example, 7-propyl-10-amino-CPT was modulated 16- to 20-fold by acidic extracellular pH in the MCF-7 cell lines, but only 6-fold in MDA-MB-231 cells. One mechanism that can contribute to pH modulation is enhanced cellular drug uptake and retention. In MCF-7/wt cells, uptake of 10-amino-CPT increased 4-fold, while retention increased over 10-fold at acidic extracellular pH. In addition, gene expression analysis of MCF-7/wt cells indicated that expression of a number of genes changed under acidic culture conditions, including down-regulation of the CPT efflux protein pump breast cancer resistance protein (BCRP). Interestingly, expression of topoisomerase I, the molecular target of CPT, was not affected by acidic growth conditions. These results highlight the importance of maintaining key features of tumor physiology in cell culture models used to study cancer biology and to discover and develop new anticancer drugs. While several substitutions at the 7 and 10 positions enhance potency, 7-halomethyl and 10-amino CPT analogs show selective activity at the acidic pH common to the microenvironment of most solid tumors.

Gene expression profiling of responses to dimethylarsinic acid in female F344 rat urothelium.

Gene expression profiling has been shown to be useful for identifying underlying mechanisms of toxicity, determining patterns of biological response, and elucidating candidate markers of exposure and response. Inorganic arsenic (iAs) is a human carcinogen and epidemiologic evidence implicates it in the development of urinary bladder cancer. Dimethylarsinic acid (DMA), the major excreted metabolite of iAs in humans, is a known rat bladder carcinogen. To examine the changes associated with DMA exposure, microarray analysis of the urothelium was performed in female F344 rats exposed to non-toxic and toxic doses of DMA in their drinking water for 28 days. A novel method for isolating predominantly urothelial cells was developed. Gene expression profiling of the urothelium using a custom 2-dye spotted array revealed that DMA treatment modulated the expression of transcripts of genes that regulate apoptosis, cell cycle regulation and the oxidative stress response. Expression of genes mapping to pathways involved in cancer control processes were also altered after DMA exposure. Morphological data suggested a dose dependent increase in cellular toxicity. Significant changes in differential gene expression were present after all treatments event at doses where standard toxicological responses were not detectable. The greatest perturbation in gene expression was present in rats after treatment with 40 ppm DMA. Doses which produced no histologic or ultrastructural evidence of toxicity (non-toxic) could be differentiated from toxic doses based on the expression of a subset of genes, which control cell signaling and the stress response. These reported changes in gene expression show similarities between the mechanisms of action of DMA in vivo and those previously described for iAs in vitro. These data illustrate the utility of transcriptional profiling and its potential in predicting key mechanistic pathways involved in toxicity and as a time efficient tool to inform the mode of action analysis in risk assessment.

SEP-class genes in Populus tremuloides and their likely role in reproductive survival of poplar trees.

One of the most important processes to the survival of a species is its ability to reproduce. In plants, SEPALLATA-class MADS-box genes have been found to control the development of the inner whorls of flowers. However, while much is known about floral development in herbaceous plants, similar systems in woody trees remain poorly understood. Populus tremuloides (trembling aspen) is a widespread North American tree having important economic value, and its floral development differs from that of well-studied species in that the flowers have only two whorls and are truly unisexual. Sequence based analyses indicate that PTM3 (Populus tremuloides MADS-box 3), and a duplicate gene PTM4, are related to the SEPALLATA1-and 2-class of MADS-box genes. Another gene, PTM6, is related to SEP3, and each of these genes has a counterpart in the poplar genomic database along with additional members of the A, B, C, D, and E-classes of MADS-box genes. PTM3/4 and 6 are expressed in all stages of male and female aspen floral development. However, PTM3/4 is also expressed in the terminal buds, young leaves, and young stems. In situ RNA localization identified PTM3/4 and 6 transcripts predominantly in the inner, sexual whorl, within developing ovules of female flowers and anther primordia of male flowers. Tree researchers often use heterologous systems to help study tree floral development due to the long juvenile periods found in most trees. We found that the participation of PTM3/4 in floral development is supported by transgenic experiments in both P. tremuloides and heterologous systems such as tobacco and Arabidopsis. However, phenotypic artifacts were observed in the heterologous systems. Together the results suggest a role for poplar SEP-class genes in reproductive viability.

The transcriptional profile of the kidney in Tsc2 heterozygous mutant Long Evans (Eker) rats compared to wild-type.

Hereditary renal cell carcinoma (RCC) in Eker rats results from an inherited insertional mutation in the Tsc2 tumor suppressor gene and provides a valuable experimental model to characterize the function of the Tsc2 gene product, tuberin in vivo. The Tsc2 mutation predisposes the Eker rat to develop renal tumors at an early age. The exact mechanism of Tsc2 mediated tumor suppression is not known, however, there is evidence that it is most likely mediated by changes in cell cycle regulation via the PI3K/Akt pathway. The present study was designed to identify if gene expression was different in Tsc2 heterozygous mutant rat kidney compared to wild-type and if any of those differences are associated with tumorigenesis. cDNA microarray analysis of the untreated Tsc2 (+/-) mutant Long Evans (Eker) rat was compared to the Tsc2 (+/+) wild-type Long Evans rat to search for patterns that might be indicative of the intrinsic role of Tsc2. Of 4395 genes queried, 3.2% were significantly altered in kidneys from heterozygous mutant rats, of which 110 (76%) were up-regulated and 34 (24%) were down-regulated relative to the wild-type. The genes with altered expression belonged to the functional categories of cell cycle regulation, cell proliferation, cell adhesion and endocytosis. Many of these genes appear to be directly or indirectly regulated by the PI3K/Akt pathway. In addition to the PI3K/Akt pathway, other signaling pathways were also differentially expressed in Tsc2 mutant Eker rat kidneys compared to wild-type rats. The gene expression profiles of the Tsc2 heterozygous mutant and wild-type animals highlights new pathways for investigation that may be associated with the tumorigenic activity of tuberin loss and correlate with the enhanced susceptibility of the Tsc2 mutant animal's tendency to develop renal cell carcinoma.