Nitric oxide donor, V-PROLI/NO, provides protection against arsenical induced toxicity in rat liver cells: requirement for Cyp1a1.

Arsenic is a cancer chemotherapeutic but hepatotoxicity can be a limiting side effect. 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 and metabolized by liver cytochromes P450 (CYP450) to release NO. The effects of V-PROLI/NO pretreatment on the toxicity of arsenic (as NaAsO(2)) were studied in a rat liver cell line (TRL 1215). The cells acted upon the prodrug to release NO, as assessed by nitrite levels, in a time-dependent fashion to maximal levels of 8-fold above basal levels. Pretreatment with V-PROLI/NO markedly reduced arsenic cytolethality which was directly related to the level of NO produced by V-PROLI/NO treatment. Cyp1a1 expression was directly related to the level of NO production and to reduced arsenic cytotoxicity. V-PROLI/NO pretreatment markedly reduced arsenic-induced apoptosis and suppressed phosphorylation of JNK1/2. V-PROLI/NO pretreatment facilitated additional increases in arsenic-induced metallothionein, a metal-binding protein important in arsenic tolerance. Thus, V-PROLI/NO protects against arsenic toxicity in rat liver cells, reducing cytolethality, apoptosis and dysregulation of MAPKs, through generation of NO formed after metabolism by liver cell enzymes, possibly including Cyp1a1. CYP450 required for NO production from V-PROLI/NO treatment in the rat and human appears to differ as we have previously studied the ability of V-PROLI/NO to prevent arsenic toxicity in human liver cells where it reduced toxicity apparently through a CYP2E1-mediated metabolic mechanism. None-the-less, it appears that both rat and human liver cells act upon V-PROLI/NO via a CYP450-related mechanism to produce NO and subsequently reduce arsenic toxicity.

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. Thus, the effects of V-PROLI/NO pretreatment on the toxicity of inorganic arsenic (as NaAsO(2)) were studied in vitro in a human liver (HepG2) cell line. HepG2 cells acted upon the prodrug to release NO, as assessed by nitrite levels, in a dose- and time-dependent fashion to maximal levels of 57-fold above control levels. In cells pretreated with V-PROLI/NO (200 microM, 24 h) then exposed to arsenic for an additional 24 h, arsenic was much less toxic (LC(50) = 151.9 +/- 5.9 microM) than in control cells (LC(50) = 90.5 +/- 6.5 microM) and the reduced cytolethality was directly related to the level of NO produced. V-PROLI/NO also increased CYP2E1 transcriptional expression in a dose-dependent manner and CYP2E1 expression was directly related to the level of NO produced and the reduction in arsenic cytotoxicity. V-PROLI/NO pretreatment markedly reduced arsenic-induced apoptosis as measured by DNA fragmentation. Pretreatment with V-PROLI/NO suppressed phosphorylation of JNK1/2 after arsenic exposure. Arsenic increased metallothionein, a metal-binding protein important in arsenic tolerance, and V-PROLI/NO pretreatment caused additional increases in metallothionein levels. Thus, the prodrug, V-PROLI/NO, protects against arsenic toxicity in cultured human liver cells, reducing cytolethality, apoptosis and dysregulation of mitogen-activated protein kinases, through generation of NO formed after metabolism by liver cell enzymes, possibly including CYP2E1.

Cadmium malignantly transforms normal human breast epithelial cells into a basal-like phenotype.

BACKGROUND: Breast cancer has recently been linked to cadmium exposure. Although not uniformly supported, it is hypothesized that cadmium acts as a metalloestrogenic carcinogen via the estrogen receptor (ER). Thus, we studied the effects of chronic exposure to cadmium on the normal human breast epithelial cell line MCF-10A, which is ER-negative but can convert to ER-positive during malignant transformation. METHODS: Cells were continuously exposed to low-level cadmium (2.5 µM) and checked in vitro and by xenograft study for signs of malignant transformation. Transformant cells were molecularly characterized by protein and transcript analysis of key genes in breast cancer. RESULTS: Over 40 weeks of cadmium exposure, cells showed increasing secretion of matrix metalloproteinase-9, loss of contact inhibition, increased colony formation, and increasing invasion, all typical for cancer cells. Inoculation of cadmium-treated cells into mice produced invasive, metastatic anaplastic carcinoma with myoepithelial components. These cadmium-transformed breast epithelial (CTBE) cells displayed characteristics of basal-like breast carcinoma, including ER-alpha negativity and HER2 (human epidermal growth factor receptor 2) negativity, reduced expression of BRCA1 (breast cancer susceptibility gene 1), and increased CK5 (cytokeratin 5) and p63 expression. CK5 and p63, both breast stem cell markers, were prominently overexpressed in CTBE cell mounds, indicative of persistent proliferation. CTBE cells showed global DNA hypomethylation and c-myc and k-ras overexpression, typical in aggressive breast cancers. CTBE cell xenograft tumors were also ER-alpha negative. CONCLUSIONS: Cadmium malignantly transforms normal human breast epithelial cells-through a mechanism not requiring ER-alpha-into a basal-like cancer phenotype. Direct cadmium induction of a malignant phenotype in human breast epithelial cells strongly fortifies a potential role in breast cancer.

Tumor suppressor gene inactivation during cadmium-induced malignant transformation of human prostate cells correlates with overexpression of de novo DNA methyltransferase.

BACKGROUND: Aberrant DNA methylation is common in carcinogenesis. The typical pattern appears to involve reduced expression of maintenance DNA methyltransferase, DNMT1, inducing genomic hypomethylation, whereas increased expression of de novo DNMT3a or 3b causes gene-specific hypermethylation. OBJECTIVES: During cadmium-induced malignant transformation, an unusual pattern of genomic hypermethylation occurred that we studied to provide insight into the roles of specific DNMTs in oncogenesis. METHODS: Gene expression and DNA methylation were assessed in control and chronic cadmium-transformed prostate epithelial cells (CTPE) using reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis, methylation-specific PCR, and methyl acceptance assay. RESULTS: During the 10-weeks of cadmium exposure that induced malignant transformation, progressive increases in generalized DNMT enzymatic activity occurred that were associated with over-expression of DNMT3b without changes in DNMT1 expression. Increased DNMT3b expression preceded increased DNMT enzymatic activity. Procainamide, a specific DNMT1 inhibitor, reversed cadmium-induced genomic DNA hypermethylation. Reduced expression of the tumor suppressor genes, RASSF1A and p16, began about the time DNMT3b overexpression first occurred and progressively decreased thereafter. RASSF1A and p16 promoter regions were heavily methylated in CTPE cells, indicating silencing by hypermethylation, while the DNA demethylating agent, 5-aza-2'-deoxycytidine, reversed this silencing. DNMT1 inhibition only modestly increased RASSF1A and p16 expression in CTPE cells and did not completely reverse silencing. CONCLUSIONS: These data indicate that DNMT3b overexpression can result in generalized DNA hypermethylation and gene silencing but that DNMT1 is required to maintain these effects. The pattern of genomic DNA hypermethylation together with up-regulation of DNMT3b may provide a unique set of biomarkers to specifically identify cadmium-induced human prostate cancers.

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. Blood and buccal cell samples were collected from arsenicosis patients and healthy subjects residing in Guizhou, China, and total RNA was isolated for MT transcript analysis. RESULTS: There was a positive correlation between blood MT-1 and MT-2 transcripts and corresponding hepatic or renal MT transcript levels in rats and mice. Furthermore, there was a positive correlation between blood MT-1 and MT-2 transcript and tissue MT protein levels in these animals. A positive correlation also occurred between human blood MT and buccal cell MT transcript levels. MT-1A and MT-2A were the major isoform transcripts in human blood and buccal cells, and significantly lower MT levels were seen in arsenicosis patients compared with healthy subjects. CONCLUSIONS: Blood MT transcript appears to be a useful biomarker of tissue MT levels. Arsenicosis patients in Guizhou show significantly lower MT transcript levels in blood, which may have predisposed this population to arsenic intoxication.