The chemical composition of smokeless tobacco: a survey of products sold in the United States in 2006 and 2007.

Selected toxicant concentrations and other chemical measures have been determined for 43 U.S. smokeless tobacco products sold in 2006 and 2007. Products evaluated included moist snuff, dry snuff, loose leaf, plug, dissolvable and snus tobacco brands. Reference products available for scientific research purposes and eleven Swedish products were also evaluated and compared to the commercial products studied. Chemical endpoints determined included benzo[a]pyrene (B[a]P), N'-nitrosonornicotine (NNN), N'-nitrosoanatabine (NAT), N'-nitrosoanabasine (NAB), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N-Nitrosodimethylamine (NDMA), nitrite, cadmium, lead, arsenic, nickel, chromium, chloride, water, pH and nicotine. Different toxicant profiles were observed for the products studied, with snus tobacco brands generally containing relatively low concentrations of B[a]P and tobacco specific nitrosamines (TSNAs) compared to other moist snuffs. Smokeless tobacco reference product toxicant profiles were similar to corresponding commercial products, with the exception of the TSNA content of the dry snuff reference material. TSNA concentrations observed for all commercial products were lower than historically reported values, likely reflecting changes in product shelf life, tobacco curing practices and, possibly, product blend formulations during the last 20-30 years. The survey results summarized provide a temporal point of comparison with future data anticipated from FDA "harmful and potentially harmful constituents in tobacco products" reporting.

Identification of an autocrine mechanism for regulating cell-cycle progression in murine keratinocytes.

Primary murine keratinocytes possess a limited doubling potential regardless of plating density or the inclusion of competence factors insulin, epidermal growth factor, and/or fetal bovine serum within the culture medium. In contrast, a murine cell line (CH-72), derived from a 7,12-dimethylbenz[a]-anthraceneinitiated, 12-O-tetra-decanoylphorbol-13-acetate-promoted mouse skin carcinoma, was found to exhibit unlimited proliferative potential; this was demonstrated by the ability of these cells to produce the progression factor required for entry into the DNA-synthesis phase in the absence of competence-factor stimulation. Conditioned medium, collected from murine carcinoma cells, was subsequently shown to increase the level of [3H]thymidine incorporation in competence-factor-deprived CH-72 cultures by more than a factor of 4 within 16 h. Moreover, consistent with its ability of recruit cells cycling within the first gap phase directly into the DNA-synthesis phase, the autocrine progression factor present in conditioned medium decreased the G1:S ratio from the 55:29 observed with growth medium controls to 38:46. Preliminary characterization of the autocrine factor produced by cultured murine carcinoma cells using gel-filtration chromatography revealed a molecular mass of less than 2 kDa, similar in size to the factor previously shown by our laboratory to promote G1-phase progression in cultures of normal human foreskin keratinocytes.

Interaction of phosphatidylinositol-4-monophosphate with a low activity form of DNA polymerase alpha: a potential mechanism for enzyme activation.

1. DNA polymerase alpha isolated from Norman murine myxosarcoma exhibited two isozyme forms, one with low specific activity and low DNA binding affinity (A1), and one with high specific activity and high DNA binding affinity (A2). 2. DNA polymerase alpha A1, but not A2, showed a significant increase in specific activity after treatment with phosphatidylinositol, ATP and phosphatidylinositol kinase, or with phosphatidylinositol-4-monophosphate. 3. Treatment of DNA polymerase alpha A1 with the phospholipase C hydrolysis product of phosphatidylinositol-4-monophosphate, inositol-1,4-bisphosphate, was sufficient to effect the transient increase in activity of polymerase A1 to a form not chromatographically distinguishable from isozyme form A2.

Purification of Norman Murine Sarcoma DNA polymerase alpha forms with different DNA template primer binding affinity and different specific activity.

1. DNA polymerase alpha was isolated from Norman Murine Myxosarcoma cells using ion exchange, immunoaffinity, and DNA affinity chromatography, showing two distinct enzyme forms designated A1 and A2. 2. Chromatographic analysis of polymerase alpha forms A1 and A2 indicate a charge difference and a difference in affinity of binding to DNA between polymerase alpha forms which were equally reactive to anti-DNA polymerase alpha monoclonal IgG. 3. Polymerase A1 specific activity was about 3600 U/mg while A2 specific activity was about 40,000 U/mg.

Monoclonal antibody that blocks phosphoinositide-dependent activation of mouse tumor DNA polymerase alpha.

A monoclonal antibody (MaB) against mouse sarcoma DNA polymerase alpha was isolated from the culture medium of an IgG-secreting hybridoma. The MaB demonstrated reactivity against two murine DNA polymerase alpha preparations and a calf thymus DNA polymerase alpha. Murine sarcoma polymerase was activated in vitro by phosphatidylinositol-4-monophosphate (PIP) showing increased deoxynucleotidyltransferase activity and enhanced binding affinity to activated DNA template. The MaB did not neutralize polymerase activity, but blocked further activation of the enzyme by PIP. Treatment of polymerase with MaB prior to treatment with PIP inhibited both increased enzyme activation and increased binding of the enzyme to DNA template. Treatment of polymerase with MaB subsequent to treatment with PIP did not block enzyme activation or increased DNA template binding. The data suggest that this anti-DNA polymerase alpha IgG is directed against a regulatory subunit of the polymerase rather than the catalytic subunit. The antibody may serve to distinguish between DNA polymerase alpha preparations with distinctly different regulatory subunits.

Phosphatidylinositol-dependent activation of DNA polymerase alpha.

DNA polymerase alpha was activated in vitro by cAMP-independent, phospholipid-dependent, protein kinase catalytic subunit. Of the phospholipids examined, phosphatidylinositol showed the greatest potential for interaction with protein kinase and ATP to activate DNA polymerase alpha in vitro. DNA polymerase alpha was directly activated by phosphorylated phosphatidylinositol in the absence of protein kinase and ATP. Activation of DNA polymerase alpha as a function of phosphorylation was demonstrated using 32P-ATP as the phosphate donor. In vitro treatment of the enzyme with phosphatidylinositol produced Linweaver-Burk plots showing noncompetitive kinetics of enzyme activation, suggesting that activation occurs prior to binding of the enzyme to DNA template/primer. These data indicate that DNA polymerase alpha may be activated in vitro in the presence of protein kinase, ATP, and phosphatidylinositol, and suggest that phosphorylation of the enzyme may constitute an intracellular mechanism of enzyme activation.