Oncogenic human papillomaviruses activate the tumor-associated lens epithelial-derived growth factor (LEDGF) gene.

The expression of the human papillomavirus (HPV) E6/E7 oncogenes is crucial for HPV-induced malignant cell transformation. The identification of cellular targets attacked by the HPV oncogenes is critical for our understanding of the molecular mechanisms of HPV-associated carcinogenesis and may open novel therapeutic opportunities. Here, we identify the Lens Epithelial-Derived Growth Factor (LEDGF) gene as a novel cellular target gene for the HPV oncogenes. Elevated LEDGF expression has been recently linked to human carcinogenesis and can protect tumor cells towards different forms of cellular stress. We show that intracellular LEDGF mRNA and protein levels in HPV-positive cancer cells are critically dependent on the maintenance of viral oncogene expression. Ectopic E6/E7 expression stimulates LEDGF transcription in primary keratinocytes, at least in part via activation of the LEDGF promoter. Repression of endogenous LEDGF expression by RNA interference results in an increased sensitivity of HPV-positive cancer cells towards genotoxic agents. Immunohistochemical analyses of cervical tissue specimens reveal a highly significant increase of LEDGF protein levels in HPV-positive lesions compared to histologically normal cervical epithelium. Taken together, these results indicate that the E6/E7-dependent maintenance of intracellular LEDGF expression is critical for protecting HPV-positive cancer cells against various forms of cellular stress, including DNA damage. This could support tumor cell survival and contribute to the therapeutic resistance of cervical cancers towards genotoxic treatment strategies in the clinic.

Silencing of human papillomavirus (HPV) E6/E7 oncogene expression affects both the contents and the amounts of extracellular microvesicles released from HPV-positive cancer cells.

The human papillomavirus (HPV) E6/E7 oncogenes play a crucial role in the HPV-induced carcinogenesis. In this study, the authors investigated whether silencing of endogenous HPV E6/E7 expression may influence the contents or amounts of extracellular microvesicles (eMVs) released from HPV-positive cancer cells. It was found that eMVs secreted from HeLa cells are enriched for Survivin protein. RNA interference studies revealed that maintenance of both intracellular and microvesicular Survivin amounts was strongly dependent on continuous E6/E7 expression. This indicates that intracellular HPV activities are translated into visible alterations of protein contents in eMVs. Besides Survivin, eMVs from HeLa cells contain additional members of the inhibitor of apoptosis protein (IAP) family (XIAP, c-IAP1 and Livin). In contrast, no evidence for the presence of the HPV E6 and E7 oncoproteins in eMVs was obtained. Moreover, it was found that silencing of HPV E6/E7 expression led to a significant increase of exosomes-representing eMVs of endocytic origin-released from HeLa cells. This effect was associated with the reinduction of p53, stimulation of the p53 target genes TSAP6 and CHMP4C that can enhance exosome production and induction of senescence. Taken together, these results show that silencing of HPV E6/E7 oncogene expression profoundly affects both the composition and amounts of eMVs secreted by HPV-positive cancer cells. This indicates that HPVs can induce molecular signatures in eMVs that may affect intercellular communication and could be explored for diagnostic purposes.

Quality of illegally and informally produced alcohol in Europe: Results from the AMPHORA project.

BACKGROUND: In the WHO region Europe, the average unrecorded adult per capita alcohol consumption was 2.67 L pure ethanol in 2005, which is 22% of the total consumption of 12.20 L. Despite concerns about potential health harms from the chemical composition of unrecorded alcohol, there are surprisingly few data on the problem in the European Region. This study reports the results from the Alcohol Measures for Public Health Research Alliance (AMPHORA) project, which assessed the quality of unrecorded alcohol in a Europe-wide study. METHODS: Samples of unrecorded alcohol were collected in 16 European countries and chemically analyzed for potentially health-relevant parameters. Thresholds for parameters were defined based on potential health hazards of daily drinking. RESULTS: The average alcoholic strength of unrecorded wine products was 14.9% vol, and 47.8% vol in unrecorded spirits. One half of the samples (n=57) showed acceptable alcohol quality. The other half (n=58) showed one or several deficits with the most prevalent problem being ethyl carbamate contamination (n=29). Other problems included copper (n=20), manganese (n=16) and acetaldehyde (n=12). All other parameters (including methanol, higher alcohols, phthalates) were only seldom problematic (limit exceedance in less than 10 samples). The price of unrecorded alcohol was approximately 45% of the price of recorded alcohol. CONCLUSIONS: The major problem regarding unrecorded alcohol appears to be ethanol itself, as it is often higher in strength and its lower price may further contribute to higher drinking amounts. Compared to the health effects of ethanol, the contamination problems detected may be of minor importance as exposure will only in worst-case scenarios reach tolerable daily intakes of these substances.

Determination of diethyl phthalate and polyhexamethylene guanidine in surrogate alcohol from Russia.

Analytical methods based on spectroscopic techniques were developed and validated for the determination of diethyl phthalate (DEP) and polyhexamethylene guanidine (PHMG), which may occur in unrecorded alcohol. Analysis for PHMG was based on UV-VIS spectrophotometry after derivatization with Eosin Y and (1)H NMR spectroscopy of the DMSO extract. Analysis of DEP was performed with direct UV-VIS and (1)H NMR methods. Multivariate curve resolution and spectra computation methods were used to confirm the presence of PHMG and DEP in the investigated beverages. Of 22 analysed alcohol samples, two contained DEP or PHMG. (1)H NMR analysis also revealed the presence of signals of hawthorn extract in three medicinal alcohols used as surrogate alcohol. The simple and cheap UV-VIS methods can be used for rapid screening of surrogate alcohol samples for impurities, while (1)H NMR is recommended for specific confirmatory analysis if required.

The composition of unrecorded alcohol from eastern Ukraine: is there a toxicological concern beyond ethanol alone?

In 2005, approximately half of all alcohol consumption in Ukraine was unrecorded. This paper investigates the chemical composition of unrecorded and low-cost alcohol, including a toxicological evaluation. A sample of alcohol products (n=78) from both recorded and unrecorded sources was obtained mainly from eastern Ukraine, and chemically analyzed. Analysis entailed alcoholic strength, levels of volatile compounds (methanol, acetaldehyde, higher alcohols), ethyl carbamate, anions, and inorganic elements. The majority of unrecorded alcohol was homemade samohon with alcoholic strength averaging close to 40% vol. A limited number of samples, advertised for medicinal purposes, were identified with high alcoholic strengths (above 60% vol.). Single samples showed contamination with acetaldehyde and ethyl carbamate above the levels of toxicological concern. Metal contamination was frequent, with copper levels above 2mg/l in 33 samples, and zinc above 5mg/l in 10 samples. Overall, however, the composition of unrecorded samples did not raise major public health concerns other those for ethanol. The priority of alcohol policy in Ukraine should be the general reduction of alcohol consumption with a specific focus on that from small-scale home production. Further research is needed on potential mitigative measures and the origin of the metal contamination in particular alcoholic beverages.

Chemical analysis and risk assessment of diethyl phthalate in alcoholic beverages with special regard to unrecorded alcohol.

BACKGROUND: Phthalates are synthetic compounds with a widespread field of applications. For example, they are used as plasticizers in PVC plastics and food packaging, or are added to personal care products. Diethyl phthalate (DEP) may be used to denature alcohol, e.g., for cosmetic purposes. Public health concerns of phthalates include carcinogenic, teratogenic, hepatotoxic and endocrine effects. The aim of this study was to develop and validate a method for determining phthalates in alcohol samples and to provide a risk assessment for consumers of such products. METHODOLOGY/PRINCIPAL FINDINGS: A liquid-liquid extraction procedure was optimized by varying the following parameters: type of extraction solvent (cyclohexane, n-hexane, 1,1,2-trichlorotrifluoroethane), the ratio extraction solvent/sample volume (1 ratio 1 to 50 ratio 1) and the number of extraction repetitions (1-10). The best extraction yield (99.9%) was achieved with the solvent 1,1,2-trichlorotrifluoroethane, an extraction solvent volume/sample volume ratio of 10 ratio 1 and a double extraction. For quantification, gas chromatography/mass spectrometry with deuterated internal standards was used. The investigated samples were alcoholic beverages and unrecorded alcohol products from different countries (n = 257). Two unrecorded alcohol samples from Lithuania contained diethyl phthalate in concentrations of 608 mg/L and 210 mg/L. CONCLUSIONS/SIGNIFICANCE: The consumption of the phthalate-positive unrecorded alcohols would exceed tolerable daily intakes as derived from animal experiments. Both positive samples were labelled as cosmetic alcohol, but had clearly been offered for human consumption. DEP seems to be unsuitable as a denaturing agent as it has no effect on the organoleptic properties of ethanol. In light of our results that DEP might be consumed by humans in unrecorded alcohols, the prohibition of its use as a denaturing agent should be considered.