Micronucleus induction in mice exposed to diazoaminobenzene or its metabolites,benzene and aniline: implications for diazoaminobenzene carcinogenicity

Diazoaminobenzene (DAAB), a manufacturing intermediate metabolized primarily to the known carcinogens benzene and aniline, has been identified as an impurity in a number of dyes and coloring agents that are components of cosmetics, food products, and pharmaceuticals. Several structural analogs of DAAB are carcinogenic as well. DAAB was selected for metabolism and toxicity studies by the National Toxicology Program (NTP) based on the potential for human exposure, positive Salmonella data, and lack of adequate toxicological data. In the toxicology studies in mice, DAAB exhibited properties similar to benzene and aniline. Because both these metabolites induce micronuclei (MN) in rodent bone marrow erythrocytes, DAAB was tested for induction of micronuclei in male B6C3F(1) mice. DAAB was administered twice by corn oil gavage at 24 h intervals, at doses of 25, 50, and 100 mg/kg per day. In addition, comparative micronucleus tests were conducted with benzene, aniline, and a mixture of benzene plus aniline; doses were based on the respective molar equivalents of each metabolite to DAAB. It was hypothesized that any observed increase in micronuclei seen in DAAB-treated mice would be due primarily to the effects of the benzene metabolite, as benzene is a more potent inducer of chromosomal damage than aniline. Results of this study showed that DAAB and benzene were effective inducers of micronuclei, with stronger responses noted for DAAB at higher doses. Positive results were also obtained with the mixture of benzene and aniline, although the magnitude of the response was lower than for DAAB. Aniline gave a weak positive response at doses exceeding its molar equivalent to 100 mg/kg DAAB. Overall, the data indicated that DAAB is a potent inducer of micronuclei in mice, and its activity appears to be closely related to the activity of benzene, one of its primary metabolites. The results are consistent with a prediction of carcinogenicity for DAAB.     

Mouse ribonuclease III. cDNA structure, expression analysis, and chromosomal location

Background  

Members of the ribonuclease III superfamily of double-stranded(ds)-RNA-specific endoribonucleases participate in diverse RNA maturation and decay pathways in eukaryotic and prokaryotic cells. A human RNase III orthologue has been implicated in ribosomal RNA maturation. To better understand the structure and mechanism of mammalian RNase III and its involvement in RNA metabolism we determined the cDNA structure, chromosomal location, and expression patterns of mouse RNase III.     

Chronic intrauterine pulmonary hypertension compromises fetal pulmonary artery smooth muscle cell O2 sensing

To test the hypothesis that chronic intrauterine pulmonary hypertension (PHTN) compromises pulmonary artery (PA) smooth muscle cell (SMC) O2 sensing, fluorescence microscopy was used to study the effect of an acute increase in Po2 on the cytosolic Ca2+ concentration ([Ca2+]i) of chronically hypoxic subconfluent monolayers of PA SMC in primary culture. PA SMCs were derived from fetal lambs with PHTN due to intrauterine ligation of the ductus arteriosus. Acute normoxia decreased [Ca2+]i in control but not PHTN PA SMC. In control PA SMC, [Ca2+]i increased after Ca2+-sensitive (KCa) and voltage-sensitive (Kv) K+ channel blockade and decreased after diltiazem treatment. In PHTN PA SMC, KCa blockade had no effect, whereas Kv blockade and diltiazem increased [Ca2+]i. Inhibition of sarcoplasmic reticulum Ca2+ ATPase activity caused a greater increase in [Ca2+]i in controls compared with PHTN PA SMC. Conversely, ryanodine caused a greater increase of [Ca2+]i in PHTN compared with control PA SMC. KCa channel mRNA is decreased and Kv channel mRNA is unchanged in PHTN PA SMC compared with controls. We conclude that PHTN compromises PA SMC O2 sensing, alters intracellular Ca2+ homeostasis, and changes the predominant ion channel that determines basal [Ca2+]i from KCa to Kv.     

Choline phospholipid metabolism: A target in cancer cells?

The experience of treating cancer over the past several decades overwhelmingly demonstrates that the disease continues to evade the vast array of drugs and treatment modalities available in the twenty-first century. This is not surprising in view of the complexity of this disease, and the multiplicities of pathways available to the cancer cell to enable its survival. Although the progression of cancer arrives at a common end point of cachexia, organ failure, and death, common pathways are rare in cancer. Identifying and targeting common pathways that would act across these levels of multiplicity is essential for the successful treatment of this disease. Over the past decade, one common characteristic consistently revealed by magnetic resonance spectroscopic studies is the elevation of phosphocholine and total choline-containing compounds in cancer cells and solid tumors. This elevation has been observed in almost every single cancer type studied with NMR spectroscopy and can be used as an endogenous biomarker of cancer. In this article, we have summarized some of the observations on the choline phospholipid metabolism of cancer cells and tumors, and make a case for targeting the aberrant choline phospholipid metabolism of cancer cells.     

Brain lipid binding protein in axon-Schwann cell interactions and peripheral nerve tumorigenesis

Loss of axonal contact characterizes Schwann cells in benign and malignant peripheral nerve sheath tumors (MPNST) from neurofibromatosis type 1 (NF1) patients. Tumor Schwann cells demonstrate NF1 mutations, elevated Ras activity, and aberrant epidermal growth factor receptor (EGFR) expression. Using cDNA microarrays, we found that brain lipid binding protein (BLBP) is elevated in an EGFR-positive subpopulation of Nf1 mutant mouse Schwann cells (Nf1(-/-) TXF) that grows away from axons; BLBP expression was not affected by farnesyltransferase inhibitor, an inhibitor of H-Ras. BLBP was also detected in EGFR-positive cell lines derived from Nf1:p53 double mutant mice and human MPNST. BLBP expression was induced in normal Schwann cells following transfection with EGFR but not H-Ras12V. Furthermore, EGFR-mediated BLBP expression was not inhibited by dominant-negative H-Ras, indicating that BLBP expression is downstream of Ras-independent EGFR signaling. BLBP-blocking antibodies enabled process outgrowth from Nf1(-/-) TXF cells and restored interaction with axons, without affecting cell proliferation or migration. Following injury, BLBP expression was induced in normal sciatic nerves when nonmyelinating Schwann cells remodeled their processes. These data suggest that BLBP, stimulated by Ras-independent pathways, regulates Schwann cell-axon interactions in normal peripheral nerve and peripheral nerve tumors.     

A novel method of imaging lysosomes in living human mammary epithelial cells

Cancer cells invade by secreting degradative enzymes which, under normal conditions, are sequestered in lysosomal vesicles. The ability to noninvasively label lysosomes and track lysosomal trafficking would be extremely useful to understand the mechanisms by which degradative enzymes are secreted in the presence of pathophysiological environments, such as hypoxia and acidic extracellular pH, which are frequently encountered in solid tumors. In this study, a novel method of introducing a fluorescent label into lysosomes of human mammary epithelial cells (HMECs) was evaluated. Highly glycosylated lysosomal membrane proteins were labeled with a newly synthesized compound, 5-dimethylamino-naphthalene-1-sulfonic acid 5-amino-3,4,6-trihydroxy-tetrahydro-pyran-2-ylmethyl ester (6-O-dansyl-GlcNH2). The ability to optically image lysosomes using this new probe was validated by determining the colocalization of the fluorescence from the dansyl group with immunofluorescent staining of two well-established lysosomal marker proteins, LAMP-1 and LAMP-2. The location of the dansyl group in lysosomes was also verified by using an anti-dansyl antibody in Western blots of lysosomes isolated using isopycnic density gradient centrifugation. This novel method of labeling lysosomes biosynthetically was used to image lysosomes in living HMECs perfused in a microscopy-compatible cell perfusion system.     

Zn(2+) site engineering at the oligomeric interface of the dopamine transporter

The hetN gene plays an important role in heterocyst differentiation and pattern formation. An immunoblotting study showed that the hetN gene in Anabaena sp. PCC 7120 was expressed in vegetative cells grown with combined nitrogen. After a switch to a medium without combined nitrogen, hetN expression first declined and was then followed by a rapid increase in its product, HetN, which was only present in mature heterocysts. HetN is located on both thylakoid membranes and plasma membranes as determined by immunoblotting using purified membranes. Overexpression of hetN completely prevented hetR up-regulation under nitrogen-deprivation conditions, suggesting that its role in pattern control may depend on its inhibition of hetR expression.     

Neuronal activity and its links with the perception of multi-stable figures

In order to isolate the neuronal activity that relates to the making of perceptual decisions, we have made use of a perceptually ambiguous motion stimulus. This stimulus lies on the boundary between two perceptual categories that correspond to clockwise and counter-clockwise rotation of a three-dimensional figure. It consists of a two-dimensional pattern of moving dots that are capable of generating these two, distinct, three-dimensional percepts. We have studied the responses of neurons in cortical area V5/MT whilst macaque monkeys report judgements about the perceptual configuration of this stimulus. We extract a quantitative statistic called 'choice probability' that expresses the covariation of neuronal activity and perceptual choice. An analysis of choice probabilities shows that the pool of neurons involved in the perceptual decisions is a tightly constrained subset of the population of sensory neurons relevant to the perceptual task.