Mitigation of the effect of variability in digital PCR assays through use of duplexed reference assays for normalization.

Digital PCR has been promoted as a technique for obtaining absolute measures of the amount of nucleic acid target sequence in a sample, but still lacks standardization in data reporting. The initial method of representing data as copies per microliter produced inconsistent results and made inter-assay comparisons difficult. Normalizing copies to amount of nucleic acid gives more uniform results, but factors influencing the effective concentration of nucleic acid in the final digital PCR assay must be considered.

Gene expression changes in immune response pathways following oral administration of tetrabromobisphenol A (TBBPA) in female Wistar Han rats.

Tetrabromobisphenol A (TBBPA) is a brominated flame retardant used globally at high volumes, primarily in the epoxy resin of circuit boards. It has been detected in the environment and in humans. The National Toxicology Program found that chronic oral TBBPA treatment of 250mg/kg and higher caused an increased incidence of uterine lesions in female Wistar Han rats.

Disruption of estrogen homeostasis as a mechanism for uterine toxicity in Wistar Han rats treated with tetrabromobisphenol A.

Chronic oral treatment of tetrabromobisphenol A (TBBPA) to female Wistar Han rats resulted in increased incidence of cell proliferation at 250mg/kg and tumor formation in the uterus at higher doses. The present study was designed to test the hypothesis that disruption of estrogen homeostasis was a major mode-of-action for the observed effects. Biological changes were assessed in serum, liver, and the proximal (nearest the cervix) and distal (nearest the ovaries) sections of the uterine horn of Wistar Han rats 24h following administration of the last of five daily oral doses of 250mg/kg.

Metal ions in human cancer development.

Metals have been in the environment during the entire evolution of man and the use of metals is key to human civilization. None-the-less, several very toxic species are included in the metallic elements and compounds either widely used by man and/or widely found in the human environment. This includes the five metallic agents considered human carcinogens, namely arsenic and arsenic compounds, beryllium and beryllium compounds, cadmium and cadmium compounds, chromium(VI) compounds, and nickel compounds, all of which are proven carcinogens in laboratory animals as well.

Cancer in experimental animals exposed to arsenic and arsenic compounds.

Inorganic arsenic is a ubiquitous environmental contaminant that has long been considered a human carcinogen. Recent studies raise further concern about the metalloid as a major, naturally occurring carcinogen in the environment. However, during this same period it has proven difficult to provide experimental evidence of the carcinogenicity of inorganic arsenic in laboratory animals and, until recently, there was considered to be a lack of clear evidence for carcinogenicity of any arsenical in animals.

Early life inorganic lead exposure induces testicular teratoma and renal and urinary bladder preneoplasia in adult metallothionein-knockout mice but not in wild type mice.

Inorganic lead compounds are carcinogenic in animals and have carcinogenic potential in humans. In mice, lead (Pb) is a transplacental carcinogen in the kidney. Metallothionein (MT) is a metal-binding protein that can reduce the toxicity of various metals, including Pb, either by direct sequestration or as an antioxidant for metals that generate reactive oxygen species.

Arsenic exposure transforms human epithelial stem/progenitor cells into a cancer stem-like phenotype.

Background: Inorganic arsenic is a ubiquitous environmental carcinogen affecting millions of people worldwide. Evolving theory predicts that normal stem cells (NSCs) are transformed into cancer stem cells (CSCs) that then drive oncogenesis. In humans, arsenic is carcinogenic in the urogenital system (UGS), including the bladder and potentially the prostate, whereas in mice arsenic induces multi-organ UGS cancers, indicating that UGS NSCs may represent targets for carcino-genic initiation.

Potential role of alpha-synuclein and metallothionein in lead-induced inclusion body formation.

Lead (Pb) produces aggresome-like inclusion bodies (IBs) in target cells as a toxic response. Our prior work shows metallothionein (MT) is required for this process. We used MT-I/II double knockout (MT-null) and parental wild-type (WT) cell lines to further explore the formation process of Pb-induced IBs.

V-PROLI/NO, a nitric oxide donor prodrug, protects liver cells from arsenic-induced toxicity.

Inorganic arsenic shows great promise in human cancer chemotherapy, although hepatotoxicity is a major limiting side-effect. O(2)-Vinyl 1-[2-(Carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (V-PROLI/NO) [Correction added after publication 19 December 2008: 1-[2-(Carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (V-PROLI/NO) was corrected to O(2)-Vinyl 1-[2-(Carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (V-PROLI/NO)] is a nitric oxide (NO) donor prodrug that is metabolized by liver cytochromes P450 to release NO. Other NO-releasing agents have been shown to mitigate arsenic toxicity.

Inorganic arsenic and human prostate cancer.

Objective: We critically evaluated the etiologic role of inorganic arsenic in human prostate cancer.

Data Sources: We assessed data from relevant epidemiologic studies concerning environmental inorganic arsenic exposure. Whole animal studies were evaluated as were in vitro model systems of inorganic arsenic carcinogenesis in the prostate.

The nitric oxide prodrug, V-PYRRO/NO, mitigates arsenic-induced liver cell toxicity and apoptosis.

Arsenite is an important cancer chemotherapeutic. The liver is a major target tissue of arsenic toxicity and hepatotoxicity may limit its chemotherapeutic efficacy. O(2)-vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO) is a liver-selective nitric oxide (NO)-producing prodrug metabolized by hepatic P450 enzymes to release NO locally.

Blood metallothionein transcript as a biomarker for metal sensitivity: low blood metallothionein transcripts in arsenicosis patients from Guizhou, China.

Background: Because metallothionein (MT) is a metal-binding protein that protects against metal intoxication, it could be a biomarker for individual sensitivity to metal toxicity.

Objective: We assessed the use of bloodborne MT transcript as a reflection of tissue MT levels and examined the potential role of MT in arsenic toxicity in an environmentally exposed human population.

Method: Rodents were treated with zinc or nonmetallic MT inducers for 4 days, and the blood and tissues were collected for MT transcript analysis by real-time reverse transcriptase-polymerase chain reaction and MT protein determination by the cadmium-hemoglobin assay.

Acquisition of apoptotic resistance in cadmium-transformed human prostate epithelial cells: Bcl-2 overexpression blocks the activation of JNK signal transduction pathway.

Background: We have recently shown that cadmium can induce malignant transformation of the human prostate epithelial cell line (RWPE-1) and that these cadmium-transformed prostate epithelial (CTPE) cells acquire apoptotic resistance concurrently with malignant phenotype.

Objective: The present study was designed to define the mechanism of acquired apoptotic resistance in CTPE cells.

Methods: Various molecular events associated with apoptosis were assessed in control and CTPE cells that were obtained after 8 weeks of continuous cadmium exposure.

Mechanisms of acquired androgen independence during arsenic-induced malignant transformation of human prostate epithelial cells.

Background: Prostate cancer progression often occurs with overexpression of growth factors and receptors, many of which engage the Ras/mitogen-activated protein MAP kinase (MAPK) pathway.

Objectives: In this study we used arsenic-transformed human prostate epithelial cells, which also show androgen-independent growth, to study the possibility that chronic activation of Ras/MAPK signaling may contribute to arsenic-induced prostate cancer progression.

Methods: Control and chronic arsenic-transformed prostate epithelial cells (CAsE-PE) were compared for Ras/MAPK signaling capacities using reverse transcription-polymerase chain reaction and Western blot analyses.

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.

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.

Acquisition of androgen independence by human prostate epithelial cells during arsenic-induced malignant transformation.

Lethal phenotypes of human prostate cancer are characterized by progression to androgen independence, although the mechanisms behind this progression remain unclear. Arsenic is a potential human prostate carcinogen that may affect tumor progression. In this study, we used a prostate cancer cell model in which an immortalized, nontumorigenic human prostate epithelial cell line (RWPE-1) had been malignantly transformed by chronic low-level arsenic to help determine whether arsenic affects prostate tumor progression.

Metallothionein-I/II double knockout mice are no more sensitive to the carcinogenic effects of nickel subsulfide than wild-type mice.

Metallothionein (MT) is a high-affinity metal-binding protein thought to mitigate the toxicity of various metals. MT may limit the toxicity of a metal by direct binding or through action as an antioxidant for metals that generate reactive oxygen species. Nickel compounds have carcinogenic potential in humans and animals, possibly by production of oxidative stress.

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.

The nitric oxide prodrug, V-PYRRO/NO, protects against cadmium toxicity and apoptosis at the cellular level.

The liver is an important target tissue of cadmium. The compound O2-vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2 diolate (V-PYRRO/NO) is a liver-selective nitric oxide (NO) prodrug that is metabolized by hepatic P450 enzymes to release NO in hepatocytes. In vivo, V-PYRRO/NO can protect against the toxicity of various hepatotoxicants, including cadmium.

Cadmium-induced malignant transformation in rat liver cells: role of aberrant oncogene expression and minimal role of oxidative stress.

Our study examined the role of oxidative stress and aberrant gene expression in malignant transformation induced by chronic, low-level cadmium exposure in non-tumorigenic rat liver epithelial cell line, TRL 1215. Cells were cultured in 1.0 microM cadmium (as CdCl(2)) for up to 28 weeks and compared to passage-matched control cells.

Metallothionein-I/II double knockout mice are hypersensitive to lead-induced kidney carcinogenesis: role of inclusion body formation.

Lead is an environmental nephrotoxicant and probable human carcinogen. Elucidating factors predisposing populations to lead toxicity is an important public health issue. Recently, we found that metallothionein-I/-II double knockout (metallothionein-null) mice that are unable to produce the major forms of metallothionein do not produce lead inclusion bodies, which are thought to mitigate lead toxicity, and were sensitive to the subchronic toxic effects of lead exposure (10 weeks), showing modestly diminished renal function and nephromegaly compared with wild-type (WT) mice.

Minimal influence of metallothionein over-expression on nickel carcinogenesis in mice.

Metallothionein (MT) is a metal-binding protein associated with tolerance to metals and oxidative stress. Nickel is a metal carcinogen potentially acting through oxidative attack on critical biomolecules. We investigated the role of MT in nickel carcinogenesis using MT-transgenic mice that constitutively over-express MT-I in all tissues tested.

Biokinetics and subchronic toxic effects of oral arsenite, arsenate, monomethylarsonic acid, and dimethylarsinic acid in v-Ha-ras transgenic (Tg.AC) mice.

Previous research demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment increased the number of skin papillomas in v-Ha-ras transgenic (Tg.AC) mice that had received sodium arsenite [(As(III)] in drinking water, indicating that this model is useful for studying the toxic effects of arsenic in vivo. Because the liver is a known target of arsenic, we examined the pathophysiologic and molecular effects of inorganic and organic arsenical exposure on Tg.