Phthalate intake by infants calculated from biomonitoring data.

Urine samples (n=207) of 47 infants between 1- and 5-month of age were quantitated for 12 metabolites of 7 phthalates and compared with samples collected from the mothers of the infants at different time points. Median and 95-percentile were lower for all metabolites in urine samples of infants compared to mothers. For di-2-ethylhexyl phthalate (DEHP) the 95-percentile daily intake was 23.3µg/kg b.w. for mothers and 5.4µg/kg b.w. for infants and for di-isobutyl phthalate (DiBP) 10.1µg/kg b.w. and 8.5µg/kg b.w. Some values exceeded the corresponding tolerable daily intake (TDI) for DiBP for infants and mothers and for DEHP and di-n-butyl phthalate (DnBP) only for mothers. Both, infants and mothers are able to efficiently form phase II metabolites but infants with a slightly lower degree. Therefore, a distinguished risk assessment with respect to the formed toxic metabolites of phthalates would be necessary in combination with a reduction of the most toxic phthalates.

Phthalate and di-(2-ethylhexyl) adipate (DEHA) intake by German infants based on the results of a duplicate diet study and biomonitoring data (INES 2).

Phthalates as well as di-(2-ethylhexyl) adipate (DEHA) are used as plasticizers in diverse applications and are of toxicological concern. The study was conducted with a study population of 25 German subjects aged between 15 and 21 months. Overall, 16 phthalates and DEHA were measured by gas chromatography-mass spectrometry in a total of 171 duplicate diet samples collected over 7 consecutive days, and 20 phthalate metabolites were analyzed in the urine samples collected over 7 consecutive days using a liquid chromatography-tandem mass spectrometry method. The median "high" daily dietary intake based on 95th percentiles was 4.66 µg/kg b.w. for di-2-ethylhexyl phthalate (DEHP), 1.03 µg/kg b.w. for di-isobutyl phthalate (DiBP), and 0.70 µg/kg b.w. for di-n-butyl phthalate (DnBP), and 1.0 µg/kg b.w. for DEHA. The "high" daily total intake from biomonitoring data was 4.9 µg/kg b.w. for DEHP, 2.2 µg/kg b.w. for DnBP, 3.9 µg/kg b.w. for DiBP, and 2.6 µg/kg b.w. for di-isononyl phthalate. The comparison of the two intake estimates indicates that the dominant intake source of DEHP was food ingestion, whereas other sources considerably contributed to the total intake of other phthalates. Using our "high" intake scenario, none of the analyzed phthalates reached the recommended tolerable daily intake levels.

Differential effects on apoptosis induction in hepatocyte lines by stable expression of hepatitis B virus X protein.

AIM: Hepatitis B virus protein X (HBx) has been shown to be weakly oncogenic in vitro. The transforming activities of HBx have been linked with the inhibition of several functions of the tumor suppressor p53. We have studied whether HBx may have different effects on p53 depending on the cell type. METHODS: We used the human hepatoma cell line HepG2 and the immortalized murine hepatocyte line AML12 and analyzed stably transfected clones which expressed physiological amounts of HBx. P53 was induced by UV irradiation. RESULTS: The p53 induction by UV irradiation was unaffected by stable expression of HBx. However, the expression of the cyclin kinase inhibitor p21(waf/cip/sdi) which gets activated by p53 was affected in the HBx transformed cell line AML12-HBx9, but not in HepG2. In AML-HBx9 cells, p21(waf/cip/sdi)-protein expression and p21(waf/cip/sdi) transcription were deregulated. Furthermore, the process of apoptosis was affected in opposite ways in the two cell lines investigated. While stable expression of HBx enhanced apoptosis induced by UV irradiation in HepG2-cells, apoptosis was decreased in HBx transformed AML12-HBx9. P53 repressed transcription from the HBV enhancer I, when expressed from expression vectors or after induction of endogenous p53 by UV irradiation. Repression by endogenous p53 was partially reversible by stably expressed HBx in both cell lines. CONCLUSION: Stable expression of HBx leads to deregulation of apoptosis induced by UV irradiation depending on the cell line used. In an immortalized hepatocyte line HBx acted anti-apoptotic whereas expression in a carcinoma derived hepatocyte line HBx enhanced apoptosis.

Variable ratio of hepatitis C virus RNA to viral core antigen in patient sera.

Quantification of hepatitis C virus (HCV) core antigen and RNA in serum samples leads to a highly variable ratio of both. It is not clear whether this is due to the inaccuracy of RNA quantification or whether both are independent parameters in a certain range. We established a real-time reverse transcription (RT)-PCR for HCV RNA that combines very high sensitivity with a large dynamic range and minimal standard deviations. The assay was calibrated with the first international standard, 96/790, and the international genotype panel for HCV from the National Institute of Biological Standardisation and Control. A linear readout was obtained between 200 and 5 x 10(7) IU/ml. The detection limit was 80 IU/ml, the reproducibility was <0.05 log, and the standard error within one run was <0.01. Comparison of the method with the Roche Monitor competitive RT-PCR revealed its high accuracy. The core protein concentration was determined within a range from 1.5 to 400 pg/ml by using the preliminary trak-C assay from Ortho Clinical Diagnostics. Correlating the HCV RNA levels with core antigen concentrations in 197 serum samples from 23 interferon-treated patients, a average ratio of 7,900 IU of HCV RNA per pg of core antigen was estimated, but the variability of this ratio exceeded largely the variability of the two assays, ranging from 50 to 20,000 IU/pg. Theoretically, HCV should contain ca. 43,000 IU of RNA/pg core. In conclusion, the core antigen assay seems to detect, in addition to complete virions, RNA-free core protein structures, which enhances its sensitivity (98% in this group). The variable ratio of RNA and core protein is not mainly due to standard deviations of quantification but could be an additional parameter for treatment follow-up and state of viral replication.

Conserved transactivating and pro-apoptotic functions of hepadnaviral X protein in ortho- and avihepadnaviruses.

Two established activities of the multifunctional human hepatitis B virus X-protein are its transactivating and pro-apoptotic potential. We analysed whether X-proteins from other orthohepadnaviruses and the newly discovered avihepadnaviral X-proteins have similar functions as HBx. Previously, we have shown that HBx suppresses oncogenic transformation of primary rat embryo fibroblasts (REF) by induction of apoptosis. Using this system, we found that the wildtype X-proteins of woodchuck, ground squirrel, arctic squirrel and woolly monkey hepatitis B virus exhibit similar levels of pro-apoptotic activity as HBx, whereas mutants with carboxyterminal deletions were severely impaired in this activity. A strong correlation between the pro-apoptotic and transactivating abilities of the mammalian X-proteins was found. The newly discovered avihepadnaviral X-like proteins showed similar and Raf-MAPK pathway-dependent transactivating abilities and induced apoptosis in the REF-assay. Our data indicate that the transactivating and pro-apoptotic activities reside in the carboxyterminal half of orthohepadnaviral X and are conserved in avihepadnaviral X-proteins.