Investigating the transfer rate of waterpipe additives to smoke as an integral part of toxicological risk assessments.

Waterpipe, also known as hookah, narghile or narghila, shisha or hubbly bubbly, is a tobacco-smoking device. Waterpipe tobacco is heated and consumed by a process of inhaling tobacco smoke, that bubbles through water before being inhaled. To date, limited studies have examined the transfer of waterpipe additives from tobacco to smoke. This study was designed to investigate the filtration ability of water in the waterpipe's bowl to define exposure to additives in waterpipe smoke, which is an essential requirement to perform toxicological risk assessments of waterpipe additives. Within this study, a standard smoking protocol (ISO 22486) was used to evaluate the transfer of > 40 additives from experimental and commercially available samples. These results are the first to provide such an extensive dataset of information showing transfer rates varying between 6% and 61% depending on the additive. Various physicochemical parameters of the additives including water solubility, partition coefficient, molecular weight, boiling point, and vapor pressure were also evaluated to seek to identify any correlation to transfer rate that may be later used to predict transfer. The amount of additive transfer from waterpipe tobacco to the smoke was found to be moderately correlated to vapor pressure (Pearson correlation coefficient = 0.33) with subsequent multivariate analysis using step-wise selection indicating 39% of the transfer rate variance can be explained collectively by the additive boiling point, molecular weight, vapor pressure and water solubility. These findings underscore the complexity of additive transfer and highlight the necessity of exposure assessment for meaningful waterpipe additive risk assessments.

Urinary zearalenone measured with ELISA as a biomarker of zearalenone exposure in pigs.

The suitability to assess zearalenone (ZEA) exposure in pigs of a commercial ELISA kit for ZEA analysis in urine was tested. A daily dose of 0, 5, 10, 20 and 40 µg synthetic ZEA per kilogram BW was administered via the feed to four gilts per dose group, and after 3 and after 7 days of ZEA intake, urine samples were assayed with the ELISA which has a relative cross-reactivity of 42% with α-zearalenol. The concentration of urinary ZEA equivalents (ZEA plus 42% of α-zearalenol present) did not differ between day 4 and day 8 (P = 0.50) within each dose group. The urinary ZEA equivalent/creatinine ratio was tightly correlated with ZEA intake (r = 0.95). The urinary ZEA equivalent/creatinine values at 0 and 40 µg/kg BW were distinctly different from those of the intermediate dose levels, whereas there was some overlapping of the individual values at the dose levels 5, 10 and 20 µg/kg BW. The urinary ZEA equivalent/creatinine ratio can be used as a biomarker for ZEA exposure in pigs provided that urine samples of several animals receiving the same diet are assayed, either separately or after pooling.

Fertility of sows exposed to zearalenone and deoxynivalenol-a case report.

In a commercial breeding herd of 140 sows, the introduction of a lot of ensiled corn cob mix (CCM) contaminated with 6-11 mg deoxynivalenol and 3-5 mg zearalenone per kg dry matter (DM) into the ration of the pregnant sows and of the gilts immediately decreased its palatability. The contaminated CCM in the ration was reduced from 60% to 40% in the DM after 1 month and was completely eliminated after a further 2.5 months. The reproductive performance data of the herd during the 5 months after the introduction of the contaminated CCM (e.g. 88% non-return rate, 10.2 weaned piglets per litter, no abortion, no pseudopregnancy) were similar to the data obtained for the same period of the previous year. The high mycotoxin exposure thus had no obvious negative effects on fertility.

Fusarium toxins in cereals grown in Switzerland.

770 cereal samples of Swiss origin which were collected in various feed mills and cereal collection centres in the years 2000 - 2002 were assayed for Deoxynivalenol (DON) and zearalenone (ZEA). 137 samples were also assayed for T-2 toxin. The prevalence of DON and ZEA contamination was higher in cereals harvested in the rainy summer 2002 than in the previous years. T-2 toxin levels exceeding 100 µg/kg were found only in three oats samples. High levels ofFusarium toxins do not frequently occur in Swiss cereals.

Identification and semiquantitative estimation of antibiotics added to complete feeds, premixes, and concentrates.

Classical microbiological methods for determining antimicrobial compounds in feeds are nonspecific. Thus, there is a need to identify biological activity, and bioautography is used for this purpose. A routine method for detecting the following antimicrobial substances in feeds is described: avilamycin, avoparcin, Zn-bacitracin, erythromycin, flavomycin, furazolidone, lasalocid, monensin, narasin, penicillin, salinomycin, spiramycin, tetracyclines, tylosin, and virginiamycin. Carbadox can be detected by UV light examination of the plates prior to bioautography. Semiquantitative estimations of antibiotic content are compared with quantitative determinations of the above mentioned substances in feeds, except erythromycin, penicillin, and tetracyclines. Detection limits range from 0.1 mg/kg (chlortetracycline) to 20 mg/kg (lasalocid). The method involves agar diffusion of buffered samples, a neutral extraction of polyether antibiotics followed by thin-layer chromatography (TLC), and an acid extraction for other antibiotics followed by TLC. Five test bacteria were used for the main detection by agar diffusion: Micrococcus luteus ATCC 9341, Staphylococcus aureus ATCC 6538P, Corynebacterium xerosis NCTC 9755, Bacillus cereus ATCC 11778, and B. subtilis ATCC 6633. Identification after TLC was achieved by bioautography with the most sensitive microorganism(s). This method allows one laboratory technician to analyze up to 30 feed samples within 2.5 working days, provided that feeds of the same category are analyzed in the same run, and that labels of additives are available. Qualitative and semiquantitative information are valuable when performing a quantitative antibiotic determination and it provides proof that the activity determined is due to the tested substance. This last feature is essential from the perspective of quality assurance of results.

Differences in radiation-induced micronuclei yields of human cells: influence of ras gene expression and protein localization.

Expression of ras has been correlated with increased intrinsic resistance to ionizing radiation. In this study we show that increased EJras expression in human cells is associated with a decrease in the frequency of radiation-induced micronuclei. The experimental system consisted of human osteosarcoma-derived cell lines which quantitatively vary in their EJras expression. There was a dose-dependent relationship between radiation dose and micronuclei formation in all cell lines tested. Human osteosarcoma cells, in which the ras level was undetectable, had the highest frequency of micronuclei production at all radiation doses tested. At 4 Gy the most radioresistant cells exhibited a 41.5 +/- 5% decrease in the production of micronuclei concomitant with high ras expression in comparison with the relatively radiosensitive parental cell line. Cells expressing a low amount of EJras demonstrated a 23 +/- 3% decrease in micronuclei induction compared with parental cells. Treatment of cells with lovastatin, an inhibitor of ras-encoded p21ras post-translational processing via the mevalonate pathway, markedly decreased the yield of micronuclei formation in cells transfected with ras; the drug had no effect on radiation-induced micronuclei formation in parental cells. The use of the in vitro micronuclei assay has provided a convenient way to visualize differences in the genotoxic damage induced by ionizing radiation in cells which express different amount of EJras. The results indicate that elevation of ras expression in human cells can lead to a decrease in the number of radiation-induced micronuclei formed and that this relationship is dependent on membrane association of ras-encoded p21.

Posttranscriptional down-regulation of ras oncogene expression by inhibitors of cellular glutathione.

Alterations in intracellular glutathione (GSH) content are known to affect intrinsic responses to ionizing radiation. More recently, it became apparent that radiation responses may depend also on the expression of specific oncogenes, including ras. These findings, suggesting a possible link between GSH and ras, led us to examine the effect of various GSH modulators on ras expression. Treatment of c-Ha-ras-transformed NIH 3T3 cells with L-buthionine S'R'-sulfoximine, dimethylfumarate, or N',N'-1,3-bis(trans-4-hydroxycyclohexyl)-N'-nitrosourea resulted in dose- and time-dependent reduction in ras mRNA steady-state levels followed by a decrease in ras-encoded p21 protein production. The effect on ras correlated with the extent of GSH decline, was common to different members of the ras family, and was independent of the mode of oncogene activation or cell phenotype. Indeed, similar drug effects were observed with murine cells in which overexpression of the c-Ha-ras proto-oncogene was due to transcriptional activation (PR4, nontumorigenic) or gene amplification (NIH 136, tumorigenic) and with malignant cells expressing a mutated Ha-ras (RS504). Moreover, N-ras, EJras, and Ki-ras in human tumor cells were similarly affected. Molecular analysis revealed a significant decrease in ras mRNA half-life in cells subjected to GSH inhibition, an effect that required de novo protein synthesis, but there was no change in the rate of gene transcription. These results indicate that pharmacological manipulation of cellular GSH content can down-regulate ras expression at the posttranscriptional level by destabilizing ras transcripts. The potential clinical implications are discussed.

Increased radiation resistance in transformed and nontransformed cells with elevated ras proto-oncogene expression.

The cellular Ha-ras oncogene, activated by missense mutations, has been implicated in intrinsic resistance to ionizing radiation. This study shows that the overexpression of the unmutated gene (proto-oncogene) may also be involved in how the cells respond to radiation. The experimental system consisted of mouse NIH 3T3-derived cell lines which carry multiple copies of a transcriptionally activated human c-Ha-ras proto-oncogene. Both tumorigenic (RS485) and revertant nontumorigenic subclones (PR4 and 4C3) which have high levels of ras expression exhibited a marked increase in radioresistance as measured by D0 compared to control NIH 3T3 cells. Other nontransformed cells with elevated levels of ras (phenotypically revertant line 4C8-A10) also had a significantly increased resistance to radiation, further indicating an association between ras and radioresistance. The increased radioresistance of the RS485 and phenotypic revertants could not be explained by a differential expression of the myc or metallothionein I genes or by variations in cell cycle. The correlation between increased ras proto-oncogene expression and radioresistance suggests that the ras encoded p21, a plasma membrane protein, may participate in the cellular responses to ionizing radiation.

Gene conversion and reciprocal exchange in a Ty-mediated translocation in yeast.

A haploid yeast mutant carrying a reciprocal translocation was analyzed. Cloning and comparison of sequences involved in the translocation event in wildtype and mutant revealed that the crossover between nonhomologous chromosomes has occurred within Ty sequences. By DNA sequence analysis it could be demonstrated that the reciprocal recombination event is accompanied by a short segment of non-reciprocal exchange (gene conversion) in the immediate vicinity of the crossover. Analysis of the translocation mutant and revertant isolates also indicated that the regulatory effect of Ty elements on adjacent genes can be modified by discrete changes within a Ty element.

Delta sequences in the 5' non-coding region of yeast tRNA genes.

Two so far undetected tRNA genes were found close to delta (delta) sequences at the sup4 locus on chromosome X in the genome of Saccharomyces cerevisiae. The two genes were identified from their abundant transcription products in frog oocytes. Hybridisation experiments allowed the mapping of the transcripts in cloned DNA and DNA sequence analysis revealed the presence of one AGGtRNA and one GACtRNA gene. tRNA genes with sequences similar or identical to GACtRNA exist in 14-16 copies per haploid yeast genome, whereas only one copy was detected for AGGtRNA. In vivo labelling of total yeast tRNA with P followed by hybridisation revealed that the unique AGGtRNA gene is transcribed in S. cerevisiae. delta sequences are present 120 bp upstream from the first coding nucleotide in the case of AGGtRNA, 80 bp in the case of GACtRNA and 405 bp in the case of the known UACtRNA (sup4) gene. delta sequences, as part of Ty elements or alone, were also found by other investigators at similar distances upstream of the mRNA start in mutant alleles of protein-coding yeast genes. Although protein-coding genes are transcribed by RNA polymerase II and tRNA genes by RNA polymerase III, the 5' non-coding region of both types of genes could conceivably have a peculiar DNA or chromatin structure used as preferred landing sites by transposable elements.