Recent Submissions

  • In vitro tests to evaluate immunotoxicity: A preliminary study.

    Carfi', M; Gennari, A; Malerba, I; Corsini, E; Pallardy, M; Pieters, R; Loveren, Henk van; Vohr, H W; Hartung, T; Gribaldo, L (2007-01-05)
    The implementation of Registration, Evaluation and Authorisation of new and existing Chemicals (REACH) will increase the number of laboratory animals used, if alternative methods will not be available. In the meantime, REACH promotes the use of in vitro tests and, therefore, a set of appropriated alternative testing methods and assessment strategies are needed. The immune system can be a target for many chemicals including environmental contaminants and drugs with potential adverse effects on human health. The aim of this study was to evaluate the predictivity of a set of in vitro assays to detect immunosuppression. The tests have been performed on human, rat and murine cells. Different endpoints have been assessed: cytotoxicity, cytokine release, myelotoxicity and mitogen responsiveness. For each of these endpoints IC50s values have been calculated. Six chemical substances, representative of the full range of in vivo responses and for which good human and/or animal data are available either from databases or literature, have been selected: two chemicals classified as not immunotoxic (Urethane and Furosemide), and four (tributyltin chloride (TBTC), Verapamil, Cyclosporin A, Benzo(a)pyrene) with different effect on immune system. All the tests confirmed the strong immunotoxic effect of TBTC as well as they confirmed the negative controls. For one chemical (Verapamil) the IC50 is similar through the different tests. The IC50s obtained with the other chemicals depend on the endpoints and on the animal species. The clonogenic test (CFU-GM) and the mitogen responsiveness showed similar IC50s between human and rodent cells except for Cyclosporin A and TBTC. All different tests classified the compounds analyzed in the same way.
  • Tissue specific mutagenic and carcinogenic responses in NER defective mouse models.

    Wijnhoven, Susan W P; Hoogervorst, Esther M; Waard, Harm de; Horst, Gijsbertus T J van der; Steeg, Harry van (2007-01-03)
    Several mouse models with defects in genes encoding components of the nucleotide excision repair (NER) pathway have been developed. In NER two different sub-pathways are known, i.e. transcription-coupled repair (TC-NER) and global-genome repair (GG-NER). A defect in one particular NER protein can lead to a (partial) defect in GG-NER, TC-NER or both. GG-NER defects in mice predispose to cancer, both spontaneous as well as UV-induced. As such these models (Xpa, Xpc and Xpe) recapitulate the human xeroderma pigmentosum (XP) syndrome. Defects in TC-NER in humans are associated with Cockayne syndrome (CS), a disease not linked to tumor development. Mice with TC-NER defects (Csa and Csb) are - except for the skin - not susceptible to develop (carcinogen-induced) tumors. Some NER factors, i.e. XPB, XPD, XPF, XPG and ERCC1 have functions outside NER, like transcription initiation and inter-strand crosslink repair. Deficiencies in these processes in mice lead to very severe phenotypes, like trichothiodystrophy (TTD) or a combination of XP and CS. In most cases these animals have a (very) short life span, display segmental progeria, but do not develop tumors. Here we will overview the available NER-related mouse models and will discuss their phenotypes in terms of (chemical-induced) tissue-specific tumor development, mutagenesis and premature aging features.
  • A 28-day oral dose toxicity study enhanced to detect endocrine effects of hexabromocyclododecane in Wistar rats.

    Ven, Leo T M van der; Verhoef, Aart; Kuil, Ton van de; Slob, Wout; Leonards, Pim E G; Visser, Theo J; Hamers, Timo; Herlin, Maria; Håkansson, Helen; Olausson, Hanna; Piersma, Aldert H; Vos, Josephus G (2006-12-01)
    A 28-day repeated dose study in rats (OECD407) enhanced for endocrine and immune parameters was performed with hexabromocyclododecane (HBCD). Rats were exposed by daily gavage to HBCD dissolved in corn oil in 8 dose groups with doses ranging between 0 and 200 mg/kg bw per day (mkd). Evaluation consisted of dose-response analysis with calculation of a benchmark dose at the lower 95% one-sided confidence bound (BMDL) at predefined critical effect sizes (CESs) of 10-20%. The most remarkable findings were dose-related effects on the thyroid hormone axis, that is, decreased total thyroxin (TT4, BMDL 55.5 mkd at CES--10%), increased pituitary weight (29 mkd at 10%) and increased immunostaining of TSH in the pituitary, increased thyroid weight (1.6 mkd at 10%), and thyroid follicle cell activation. These effects were restricted to females. Female rats also showed increased absolute liver weights (22.9 mkd at 20%) and induction of T4-glucuronyl transferase (4.1 mkd at 10%), suggesting that aberrant metabolization of T4 triggers feedback activation of the thyroid hormone system. These effects were accompanied by possibly secondary effects, including increased cholesterol (7.4 mkd at 10%), increased tibial bone mineral density (> 49 mkd at 10%), both in females, and decreased splenocyte counts (0.3-6.3 mkd at 20%; only evaluated in males). Overall, female rats appeared to be more sensitive to HBCD than male rats, and an overall BMDL is proposed at 1.6 mkd, based on a 10% increase of the thyroid weight, which was the most sensitive parameter in the sequence of events.
  • In vivo and in vitro Ah-receptor activation by commercial and fractionated pentabromodiphenylether using zebrafish (Danio rerio) and the DR-CALUX assay.

    Kuiper, Rienk V; Murk, A J; Leonards, Pim E G; Grinwis, G C M; Berg, M van den; Vos, Josephus G (2006-10-12)
    The present study addresses the toxicity of a commercial pentabrominated diphenylether (PeBDE) flame retardant mixture, DE-71, in a model aquatic vertebrate. Four weeks' exposure of juvenile zebrafish (Danio rerio) to water-borne DE-71 resulted in dose-dependent induction of CYP1A immunoreactivity, predominantly in the endocardium and the endothelium of larger blood vessels, such as ventral aorta and branchial arteries, as well as the larger hepatic and pancreatic blood vessels. To investigate the impact of possible contaminating PBDD/Fs in the DE-71 product, the study was repeated after DE-71 had been fractionated into a non-planar (cleaned PBDEs) and a planar fraction (PBDD/Fs). Zebrafish were exposed under similar conditions to the planar and cleaned DE-71 fractions, and to uncleaned DE-71. In addition, the above fractions were chemically analyzed and tested in a reporter gene assay (DR-CALUX) for their aromatic hydrocarbon-receptor (AhR) stimulating potencies. A relatively strong CALUX response was detected from the planar DE-71 fraction (19.7ng TCDD equivalent (TEQ)/g DE-71), coinciding with a strong induction of CYP1A immunoreactivity in zebrafish. CYP1A immunoreactivity in zebrafish exposed to uncleaned DE-71 was intense, although the CALUX response was 10-fold less compared to the planar fraction. Only weak CYP1A immunoreactivity was found in fish exposed to cleaned DE-71, and none in control animals; no CALUX response was detected in cleaned DE-71. The present findings indicate that chemical impurities of the commercial PeBDE product account for AhR-mediated effects. Analytical isolation of a planar fraction from the commercial product increased the in vitro (DR-CALUX) signal 10 times. Immunohistochemistry showed a strong tissue specific reaction to DE-71 in vivo at these relatively low TEQ levels regardless of chemical pretreatment of the mix, reflecting the sensitivity of CYP1A induction in juvenile zebrafish to AhR agonists.
  • Modeling responses of Daphnia magna to pesticide pulse exposure under varying food conditions: intrinsic versus apparent sensitivity.

    Pieters, Barry J; Jager, Tjalling; Kraak, Michiel H S; Admiraal, Wim (2006-10-01)
    Recent studies showed that limiting food conditions resulted in either increased or decreased sensitivity of Daphnia magna to toxicants. It remained unclear whether these contrasting food-dependent alterations in toxicity resulted from differences in intrinsic sensitivity of the daphnids or from changes in toxicokinetics and resource allocation. It is hypothesized here that, if food level only affects accumulation kinetics and resource allocation, then the intrinsic sensitivity to this toxicant should be the same for all food regimes. This hypothesis was investigated using the DEBtox model, which is based on the theory of Dynamic Energy Budgets. We examined results of two recently conducted life-cycle studies on the combined effects of food level and a pulsed exposure to the pyrethroid insecticide fenvalerate (FV) on D. magna. The model described the effects of the time-varying exposure well, and indicated that when the animals did not die from exposure to FV, full reversibility of toxic effects was possible, allowing a complete recovery. Results revealed furthermore that the data from both studies could be described by the same NECs for survival and assimilation, killing rate and tolerance concentration (132 (49.2-228) x 10(-6) mug/L, 0 (0-1.18 x 10(-5)) mug/L, 74.4 (55.6-96.4) L (mug d)(-1) and 5.39 (2.72-18.5) x 10(-3) mug/L, respectively). It is therefore concluded that food-dependent FV toxicity can be explained by altered toxicokinetics and resource allocation, but not by changes in the intrinsic sensitivity of the daphnids. This study implies that the effect of pesticide application in the field depends on the trophic state of the receiving water body, but also that full recovery of survivors is possible after FV application.
  • A category approach for reproductive effects of phthalates.

    Fabjan, Evelin; Hulzebos, Etje; Mennes, W; Piersma, Aldert H (2006-10-01)
    In regulatory toxicology, the experimental assessment of reproductive toxicity is one of the most costly endpoints to perform. Categorizing chemicals is an approach that can be used to reduce animal tests in risk assessments of chemicals, for example, via REACH (Registration, Evaluation, and Authorization of Chemicals). The category approach was tested for reproductive toxicity with a group of 10 ortho-phthalate esters, with different side chain lengths. Three ortho-phthalates were used for testing the category. Phthalates with side-chain lengths C4 to C6 that are commonly known to cause reproductive effects were included, as well as the recently discovered mechanism that indicates antiandrogenic effects. The differences in physicochemical properties, absorption rates, and metabolism between the phthalates investigated could not fully explain the difference in reproductive toxicity. It was concluded that phthalates with the alkyl side-chain length from C4 to C6 produce similar severe reproductive effects in experimental animals. It is expected that phthalates included in the tight boundaries of the proposed category would all show severe reproductive effects, especially the antiandrogenic effects. Further testing might not be needed for phthalates within these boundaries. If necessary, limited testing could focus on the critical endpoints. Detailed mechanistic information is needed on phthalates to apply the categories for regulatory toxicology.
  • Noise exposure and children's blood pressure and heart rate: the RANCH project.

    Kempen, Elise van; Kamp, I van; Fischer, P; Davies, Hugh W; Houthuijs, D; Stellato, Rebecca K; Clark, Charlotte; Stansfeld, Stephen A (2006-09-01)
    BACKGROUND: Conclusions that can be drawn from earlier studies on noise and children's blood pressure are limited due to inconsistent results, methodological problems, and the focus on school noise exposure. OBJECTIVES: To investigate the effects of aircraft and road traffic noise exposure on children's blood pressure and heart rate. METHODS: Participants were 1283 children (age 9-11 years) attending 62 primary schools around two European airports. Data were pooled and analysed using multilevel modelling. Adjustments were made for a range of socioeconomic and lifestyle factors. RESULTS: After pooling the data, aircraft noise exposure at school was related to a statistically non-significant increase in blood pressure and heart rate. Aircraft noise exposure at home was related to a statistically significant increase in blood pressure. Aircraft noise exposure during the night at home was positively and significantly associated with blood pressure. The findings differed between the Dutch and British samples. Negative associations were found between road traffic noise exposure and blood pressure, which cannot be explained. CONCLUSION: On the basis of this study and previous scientific literature, no unequivocal conclusions can be drawn about the relationship between community noise and children's blood pressure.
  • Dioxin and dioxin-like PCB exposure of non-breastfed Dutch infants.

    Weijs, P J M; Bakker, Martine I; Korver, K R; Goor Ghanaviztchi, K van; Wijnen, Joop H van (2006-08-01)
    The exposure of humans to PCDD/Fs (polychlorinated dibenzo-p-dioxins and dibenzofurans) and dioxin-like PCBs (dl-PCBs, i.e. polychlorinated non-ortho and mono-ortho biphenyls) occurs predominantly via the intake of food. Young children have a relatively high intake of these substances, due to their high food consumption per kilogram body weight. As the exposure of non-breastfed infants to these compounds has not been assessed before in The Netherlands, we studied the dietary intake of 17 PCDD/Fs and 11 dioxin-like PCBs in 188 Dutch non-breastfed infants between 4 and 13 months. The food intake of the infants was assessed by a 2-d food record. From these data PCDD/F and dioxin-like PCB intake was calculated using PCDD/F and dioxin-like PCB concentrations of food products sampled in 1998/1999 in The Netherlands. The long-term PCDD/F and dioxin-like PCB exposure of the infants was calculated using the statistical exposure model (STEM). For infants of 5 months the chronic exposure to PCDD/F and dioxin-like PCB was 1.1pg WHO-TEQ (toxic equivalents) per kilogram body weight (bw) per day (95th percentile: 1.7pg WHO-TEQ/kg bwxd), which mainly originated from infant formula and vegetables and increased to 2.3pg WHO-TEQ/kg bwxd (95th percentile 3.7pg WHO-TEQ/kg bwxd) for infants just over 1 year old eating the same food as their parents. The percentage of formula-fed infants with an exposure exceeding the TDI of 2pg WHO-TEQ/kg bwxd was 5% at 5 months, 49% at 9 months and 64% at 12 months.
  • An Xpd mouse model for the combined xeroderma pigmentosum/Cockayne syndrome exhibiting both cancer predisposition and segmental progeria.

    Andressoo, Jaan-Olle; Mitchell, James R; Wit, Jan de; Hoogstraten, Deborah; Volker, Marcel; Toussaint, Wendy; Speksnijder, Ewoud; Beems, Rudolf B; Steeg, Harry van; Jans, Judith; Zeeuw, Chris I de; Jaspers, Nicolaas G J; Raams, Anja; Lehmann, Alan R; Vermeulen, Wim; Hoeijmakers, Jan H J; Horst, Gijsbertus T J van der (2006-08-01)
    Inborn defects in nucleotide excision DNA repair (NER) can paradoxically result in elevated cancer incidence (xeroderma pigmentosum [XP]) or segmental progeria without cancer predisposition (Cockayne syndrome [CS] and trichothiodystrophy [TTD]). We report generation of a knockin mouse model for the combined disorder XPCS with a G602D-encoding mutation in the Xpd helicase gene. XPCS mice are the most skin cancer-prone NER model to date, and we postulate an unusual NER dysfunction that is likely responsible for this susceptibility. XPCS mice also displayed symptoms of segmental progeria, including cachexia and progressive loss of germinal epithelium. Like CS fibroblasts, XPCS and TTD fibroblasts from human and mouse showed evidence of defective repair of oxidative DNA lesions that may underlie these segmental progeroid symptoms.
  • Hazard classification of chemicals inducing haemolytic anaemia: An EU regulatory perspective.

    Muller, Andre; Jacobsen, Helene; Healy, Edel; McMickan, Sinead; Istace, Fréderique; Blaude, Marie-Noëlle; Howden, Peter; Fleig, Helmut; Schulte, Agnes (2006-08-01)
    Haemolytic anaemia is often induced following prolonged exposure to chemical substances. Currently, under EU Council Directive 67/548/EEC, substances which induce such effects are classified as dangerous and assigned the risk phrase R48 'Danger of serious damage to health by prolonged exposure.' Whilst the general classification criteria for this endpoint are outlined in Annex VI of this Directive, they do not provide specific information to assess haemolytic anaemia. This review produced by the EU Working Group on Haemolytic Anaemia provides a toxicological assessment of haemolytic anaemia and proposes criteria that can be used in the assessment for classification of substances which induce such effects. An overview of the primary and secondary effects of haemolytic anaemia which can occur in rodent repeated dose toxicity studies is given. A detailed analysis of the toxicological significance of such effects is then performed and correlated with the general classification criteria used for this endpoint. This review intends to give guidance when carrying out an assessment for classification for this endpoint and to allow for better transparency in the decision-making process on when to classify based on the presence of haemolytic anaemia in repeated dose toxicity studies. The extended classification criteria for haemolytic anaemia outlined in this review were accepted by the EU Commission Working Group on the Classification and Labelling of Dangerous Substances in September 2004.
  • Depression and adipose and serum cholesteryl ester polyunsaturated fatty acids in the survivors of the seven countries study population of Crete.

    Mamalakis, G; Jansen, E; Cremers, H; Kiriakakis, M; Tsibinos, G; Kafatos, A (2006-08-01)
    BACKGROUND: Studies have shown that depression relates to biomarkers of both short- and long-term polyunsaturated fatty acid (PUFA) intake. However, it is not known which of these two biomarkers has the closest relationship to depression. OBJECTIVE: To examine the relationship of depression with both adipose tissue and serum cholesteryl ester PUFA and to assess the importance of each of these two biomarkers in relating to depression. DESIGN: Cross-sectional study of healthy elderly men from the island of Crete. SETTING: The Preventive Medicine and Nutrition Clinic, University of Crete, Greece. SUBJECTS: A total of 150 males, aged 80-96 years. The subjects were survivors of the Greek Seven Countries Study group. METHODS: Fatty acids were determined by gas chromatography in adipose tissue and serum cholesteryl esters. Information about depression was obtained through the use of the short form of the Geriatric Depression Scale (GDS-15). RESULTS: Regression analysis showed that depression related positively to age and serum cholesteryl ester arachidonic/docosahexaenoic fatty acid ratio. The only significant unadjusted correlation between depression and serum cholesteryl ester and adipose fatty acids was with adipose alpha-linolenic acid (ALA) (r = -0.31, P < 0.01). Depressed males (GDS-15 > 5) had lower adipose ALA and sum n-3 fatty acids than non-depressed ones. There were no significant differences between depressed and non-depressed males in serum cholesteryl ester fatty acids. When adipose tissue ALA was included as one of the independent measures in the regression model, the observed positive relation between GDS-15 depression and cholesteryl ester arachidonic/docosahexaenoic ratio failed to persist. Instead, there was a negative relationship between GDS-15 depression and adipose tissue ALA. CONCLUSIONS: It appears that the fatty acids of the adipose tissue are better predictors of depression than those of serum cholesteryl esters. This indicates that depression relates more strongly to long-term than to short-term fatty acid intake. The reason for this may be the reported slow rate of deposition of dietary PUFA to the brain.
  • On the limits of toxicant-induced tolerance testing: cotolerance and response variation of antibiotic effects.

    Schmitt, Heike; Martinali, Bennie; Beelen, Patrick van; Seinen, Willem (2006-07-01)
    Pollution-induced community tolerance (PICT) as an ecotoxicological test system has been claimed to detect pollutant effects highly specifically and sensitively. However, the specificity might be limited by the occurrence of cotolerance. Another limitation of the application of any ecotoxicological test system lies in variation of the measured responses. We tested the variation and the occurrence of cotolerance experimentally, using antibiotics as toxicants, soil microcosms as microbial communities, and tolerance determination in Biolog plates as PICT detection test. Bacteria have been discussed as being prone to multiple tolerances due to the possible accumulation of multiple resistance genes on mobile genetic elements. However, in our experiments, cotolerance occurred only between antibiotics of the same group (oxytetracycline and tetracycline), as expected from their identical mode of action. Cotolerance between oxytetracycline and tylosin in soil microcosms exposed to oxytetracycline was low, as was cotolerance to oxytetracycline in tylosin-exposed microcosms. We conclude that tolerance development to antibiotics in soils reflects the actual selection pressure rather than a general pattern of multiple resistances. Concerning variation, the PICT effect of tetracycline was well reproducible in two consecutive years. The response variation linked to PICT experiments in controlled microcosms was comparable to that of ecotoxicological test systems of equivalent complexity. In conclusion, our results support an application of the PICT methodology as an effective means to study the soil ecotoxicology of antibiotics.
  • In vitro and in vivo (cyto)toxicity assays using PVC and LDPE as model materials.

    Tienhoven, E A E van; Korbee, D; Schipper, L; Verharen, H W; Jong, W H de (2006-07-01)
    The choice for a biomaterial is partly based on the outcome of (cyto)toxicity assays. The rationales behind the selection of certain parameters, such as cell lines, controls, and animals, were evaluated using a positive and a negative control, and one experimental sample designed to induce intermediate toxicity. Extraction and direct contact assays were performed using human epidermal keratinocytes and mouse fibroblasts and mouse epithelial cells. Cell survival was measured with the tetrazolium salt (MTT) reduction assay. In addition, local implantation studies were performed in mice and rats. The positive control induced a high degree of toxicity in all in vitro tests performed, indicating that the toxicity observed in the direct contact assay was due to in situ extraction of toxic components. In the direct contact assay the negative control tested on the mouse fibroblasts resulted in a significant reduction of cell survival. No decrease in cell survival was found using the experimental sample. Subcutaneous implantation studies in mice showed that the positive control material induced a severe degeneration in mice. However, in rats just minimal alterations were noted. The experimental material induced moderate responses only in mice. Our results indicate that the direct contact assay provides limited additional information on the cytotoxicity of materials if certain limitations are not taken into account. For the in vivo implantation assay mice were superior to rats in detecting local toxic responses.
  • Intoxication following minor stabs from the spines of a porcupine fish.

    Gorcum, Teetske F van; Janse, Max; Leenders, Marianne E C; Vries, Irma de; Meulenbelt, Jan (2006-06-01)
    We report an unusual intoxication by tetrodotoxin (TTX). A curator of an aquarium sustained minor punctures in his finger from the spines of a porcupine fish during an autopsy of a dead porcupine fish. He developed paresthesias, numbness, paresis, dizziness and headache. The death of the fish might have caused some autolysis, leading to increased availability of TTX. In combination with direct contact with the organ fluids, this probably led to TTX exposure via minor wounds.
  • Application of physiologically based pharmacokinetic modeling in setting acute exposure guideline levels for methylene chloride.

    Bos, Peter Martinus Jozef; Zeilmaker, Marco Jacob; Eijkeren, Jan Cornelis Henri van (2006-06-01)
    Acute exposure guideline levels (AEGLs) are derived to protect the human population from adverse health effects in case of single exposure due to an accidental release of chemicals into the atmosphere. AEGLs are set at three different levels of increasing toxicity for exposure durations ranging from 10 min to 8 h. In the AEGL setting for methylene chloride, specific additional topics had to be addressed. This included a change of relevant toxicity endpoint within the 10-min to 8-h exposure time range from central nervous system depression caused by the parent compound to formation of carboxyhemoglobin (COHb) via biotransformation to carbon monoxide. Additionally, the biotransformation of methylene chloride includes both a saturable step as well as genetic polymorphism of the glutathione transferase involved. Physiologically based pharmacokinetic modeling was considered to be the appropriate tool to address all these topics in an adequate way. Two available PBPK models were combined and extended with additional algorithms for the estimation of the maximum COHb levels. The model was validated and verified with data obtained from volunteer studies. It was concluded that all the mentioned topics could be adequately accounted for by the PBPK model. The AEGL values as calculated with the model were substantiated by experimental data with volunteers and are concluded to be practically applicable.
  • Toxicity of tetrabromobisphenol A (TBBPA) in zebrafish (Danio rerio) in a partial life-cycle test.

    Kuiper, R V; Brandhof, E J van den; Leonards, P E G; Ven, L T M van der; Wester, P W; Vos, J G (2006-06-01)
    Toxicological effects of the widely used flame retardant, tetrabromobisphenol A (TBBPA) were assessed in a partial life-cycle test with zebrafish (Danio rerio). Exposure of adult fish during 30 days to water-borne TBBPA in nominal concentrations ranging from 0 (control) to 1.5 muM was followed by exposure of the offspring in early life stages up to 47 days posthatching (dph) to the same concentrations. Adults exposed to 3 and 6 muM showed severe disorientation and lethargy shortly after beginning of exposure and were euthanized. Because semistatic exposure resulted in fluctuating water concentrations, pooled fish samples were chemically analyzed for internal dose assessment. Egg production was decreased in fish exposed to TBBPA concentrations of 0.047 muM and higher, and a critical effect level of 7.2 mug/g lipid with a lower 5% confidence limit of 3.9 mug/g lipid for 50% decreased egg production was calculated. Histology of adult ovaries indicated a relative increase of premature oocytes in two surviving females exposed to 1.5 muM. Hatching of TBBPA-exposed larvae was decreased except in animals exposed to 0.375 muM. In the highest exposure concentration, early posthatching mortality was high (81%) in larvae and the surviving juveniles showed a significant predominance of the female phenotype. Exposure of eggs from control parents up to 6 muM TBBPA resulted in increasing malformation and pericardial fluid accumulation from 1.5 muM; at higher concentrations, all embryos failed to hatch. The presented results indicate decreased reproductive success in zebrafish at environmentally relevant TBBPA concentrations.
  • Significance of ammonium compounds on nicotine exposure to cigarette smokers.

    Willems, E W; Rambali, B; Vleeming, W; Opperhuizen, Antoon; Amsterdam, J G C van (2006-05-01)
    The tobacco industry publicly contends that ammonia compounds are solely used as tobacco additive for purposes of tobacco flavoring, process conditioning and reduction of its subjective harshness and irritation. However, neither objective scientific reports, nor the contents of a large number of internal tobacco company documents support this contention. The present review focuses on the hypothesis that addition of ammonium compounds to tobacco enhances global tobacco use due to smoke alkalization and enhanced free-nicotine nicotine exposure. Obviously, ammonia enhances the alkalinity of tobacco smoke. Consequently, the equilibrium shifts from non-volatile nicotine salts to the volatile free base that is more readily absorbed from the airways. The observed change in the kinetics of nicotine (i.e., shorter t(1/2) and higher c(max)) after ammoniation is, however, predominantly due to the higher concentration of nicotine in the smoke, rather than to an increase in the absorption rate of free-base nicotine in the respiratory tract. Although several findings support the hypothesis, additional studies are required and suggested to provide a proper, objective and independent scientific judgment about the effect of tobacco ammoniation on nicotine bioavailability. Scientific and public awareness of the effects of tobacco-specific ammonia compounds may stimulate global control, legislation and restriction of their use in cigarette manufacture.
  • Phenacetin acts as a weak genotoxic compound preferentially in the kidney of DNA repair deficient Xpa mice.

    Luijten, Mirjam; Speksnijder, Ewoud N; Alphen, Niels van; Westerman, Anja; Heisterkamp, Siem H; Benthem, Jan van; Kreijl, Coen F van; Beems, Rudolf B; Steeg, Harrym van (2006-04-11)
    Chronic use of phenacetin-containing analgesics has been associated with the development of renal cancer. To establish genotoxicity as a possible cause for the carcinogenic effect of phenacetin, we exposed wild type and DNA repair deficient Xpa-/- and Xpa-/-/Trp53+/- mice (further referred as Xpa and Xpa/p53 mice, respectively), carrying a reporter lacZ gene, to 0.75% (w/w) phenacetin mixed in feed. Xpa mice completely lack the nucleotide excision repair pathway, and as such they are sensitive to some classes of genotoxic compounds. Phenacetin exposure induced a significant increase of lacZ mutations in the kidney of both Xpa and Xpa/p53 mice. A minor response was found in liver, whereas no lacZ mutation induction was observed in the spleen of these animals. Interestingly, the observed phenacetin-induced mutant frequencies were higher in male than those found in female mice. This gender difference is probably due to a difference in metabolic rate. Phenacetin-induced mutations mainly consisted of point mutations rather than deletions. The mutational spectra in the kidney of treated WT and Xpa mice were quite similar. Taken together, these results demonstrate that the human carcinogen phenacetin acts as a weak genotoxic agent in an in vivo mouse model system.
  • Predicted effects of toxicant mixtures are confirmed by changes in fish species assemblages in Ohio, USA, rivers.

    Posthuma, Leo; Zwart, Dick de (2006-04-01)
    The purposes of this study were to investigate whether exposure to toxicant mixtures is associated with fish assemblage characteristics in the field and to describe the relationships between predicted chronic and acute mixture risks and observed impacts. Fish abundance and abiotic monitoring data from Ohio, USA, surface waters were compiled and analyzed. Variability of biotic and abiotic parameters was large. Exposure assessment, risk assessment with species-sensitivity distributions, and mixture toxicity rules were used to calculate a relative risk predictor: The multisubstance potentially affected fraction of species (msPAF). Predicted acute and chronic risks ranged from low values to more than 10 and 50% of species potentially affected, respectively. Pearson correlations between predicted risk and observed assemblage characteristics were nonsignificant for total abundance, number of species, Shannon-Weaver index, and evenness. Moderately significant correlations were found between predicted risk and abundance for 23% of individual species. Both abundance increases and decreases were observed. Generalized linear model (GLM) regressions revealed significant nonlinear associations between predicted risk and the abundance for 50% (metals and ammonia) and 55% (household product ingredients) of the species. Local ecological impact was expressed as the fraction of species expected but not observed, both with and without attribution of impact to mixture exposure. The association between predicted impacted fraction and the fraction of species expected but not observed was not significant. Predicted acute and chronic impacted fractions were associated significantly with the observed fraction of species likely lost by the action of toxicant mixtures under field conditions, with wide confidence bounds. These findings confirm the view that higher mixture impacts are expected in the field at higher msPAF.
  • Screening suspected counterfeit Viagra and imitations of Viagra with near-infrared spectroscopy.

    Vredenbregt, M J; Blok-Tip, L; Hoogerbrugge, Ronald; Barends, D M; Kaste, D de (2006-03-03)
    We describe a near-infrared spectroscopy (NIRS) method for fast-screening Viagra tablets, counterfeit Viagra tablets, and imitations of Viagra. The method can (1) check the homogeneity of a batch; (2) distinguish counterfeits and imitations from authentic Viagra; (3) screen for the presence of sildenafil citrate, the pharmacologically active substance in Viagra, irrespectively of the excipients present; (4) and detect whether similar samples have been previously analysed. We applied the method to 103 samples with a diversity of appearance, chemical composition, and origin. Other analytical methods confirmed the positive screening results for sildenafil citrate and the presence of other pharmacological active substances. The NIRS screening indicated the absence of sildenafil citrate in the presence of another pharmacological substance for only 2 samples, where the reference methods showed the presence of sildenafil citrate in addition to that of clomifene citrate. Otherwise, the method gave no false positive or negative results. The NIRS screening method is very fast and reliable for detecting counterfeits and imitations, and it correctly predicts the presence or absence of sildenafil citrate in 98% of the samples.

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