Human death effector domain-associated factor interacts with the viral apoptosis agonist Apoptin and exerts tumor-preferential cell killing.

Apoptin, a protein from chicken anemia virus without an apparent cellular homologue, can induce apoptosis in mammalian cells. Its cytotoxicity is limited to transformed or tumor cells, making Apoptin a highly interesting candidate for cancer therapy. To elucidate Apoptin's mechanism of action, we have searched for binding partners in the human proteome. Here, we report that Apoptin interacts with DEDAF, a protein previously found to associate with death effector domain (DED)-containing pro-apoptotic proteins, and to be involved in regulation of transcription. Like Apoptin, after transient overexpression, DEDAF induced apoptosis in various human tumor cell lines, but not in primary fibroblasts or mesenchymal cells. DEDAF-induced cell death was inhibited by the caspase inhibitor p35. Together with the reported association of DEDAF with a DED-containing DNA-binding protein in the nucleus and the transcription regulatory activity, our findings may provide a clue for the mechanism of Apoptin's actions in mammalian cells.

Common regulation of apoptosis signaling induced by CD95 and the DNA-damaging stimuli etoposide and gamma-radiation downstream from caspase-8 activation.

The death receptor CD95 (APO-1/Fas), the anticancer drug etoposide, and gamma-radiation induce apoptosis in the human T cell line Jurkat. Variant clones selected for resistance to CD95-induced apoptosis proved cross-resistant to etoposide- and radiation-induced apoptosis, suggesting that the apoptosis pathways induced by these distinct stimuli have critical component(s) in common. The pathways do not converge at the level of CD95 ligation or caspase-8 signaling. Whereas caspase-8 function was required for CD95-mediated cytochrome c release, effector caspase activation, and apoptosis, these responses were unaffected in etoposide-treated and irradiated cells when caspase-8 was inhibited by FLIPL. Both effector caspase processing and cytochrome c release were inhibited in the resistant variant cells as well as in Bcl-2 transfectants, suggesting that, in Jurkat cells, the apoptosis signaling pathways activated by CD95, etoposide, and gamma-radiation are under common mitochondrial control. All three stimuli induced ceramide production in wild-type cells, but not in resistant variant cells. Exogenous ceramide bypassed apoptosis resistance in the variant cells, but not in Bcl-2-transfected cells, suggesting that apoptosis signaling induced by CD95, etoposide, and gamma-radiation is subject to common regulation at a level different from that targeted by Bcl-2.

Exposure to fungicides in fruit growing: re-entry time as a predictor for dermal exposure.

As part of a European Concerted Action on Male Reproduction Capability an exposure assessment survey was conducted among seasonal workers in the fruit growing sector in the Netherlands. Dermal exposure to the fungicides captan and tolylfluanid was measured using cotton gloves (12 persons) and skin pads on several body parts (12 persons). In addition, a set of exposure data was used from a study conducted recently among Dutch fruit growers. For harvesting activities, re-entry time appeared to be an important determinant of dermal exposure to captan and tolyfluanid. Explained variance of regression models was moderate to high (range 0.30-0.87). For captan, calculated half-life times from the most recent exposure survey were lower (glove data: 5 days; pad data: 6 days) compared with half-life times based on the previously conducted study (11 days). Possible explanations for the discrepancy are discussed. For tolylfluanid, estimated half-life times during harvesting were 2 and 3 days, based on pad and glove data, respectively. Prediction of captan exposure during other crop activities appeared to be far more difficult (explained variance equal to 0.06), although the estimated half-life time was comparable with that for harvesting. The data suggest that re-entry time gives useful information to group workers in broad exposure categories. Nonetheless, it was concluded that large studies are needed to evaluate the importance of re-entry time in more detail.

Determinants of exposure to captan in fruit growing.

A series of studies investigated occupational exposure to pesticides among fruit growers in The Netherlands during spraying and reentry of orchards between 1990 and 1992 to identify and quantify determinants of exposure. Determinants of exposure are discussed as a starting point for hazard identification and control. Captan was used as a marker for exposure. Cabin use of the tractor was the most prominent determinant of dermal exposure during spraying. For respiratory exposure, factors related to preparation of pesticides were most prominent. A long duration of exposure may reflect a different exposure situation compared with a short duration of exposure. As different determinants of exposure prevailed for each subgroup, consideration should be given to constructing exposure models for each group separately. Dislodgeable foliar residue (DFR) was the most prominent determinant of exposure for both respiratory and dermal exposure during reentry. However, no significant relation between DFR and dermal exposure of forehead and sternal area was found, perhaps because there was no direct contact with foliage here. Therefore, use of a transfer factor based on DFR to estimate total dermal exposure is only a crude estimate. The half-life of captan on crops varied from 10-17 days, so substantial exposure when entering the orchard is very likely, particularly when spraying frequency is high. The main starting points for reduction of exposure are use of a cabin, DFR, and individual time spent on different tasks. Determinants that are constant over time (cabin use) may have an especially great influence on grouping workers, according to long-term exposure in epidemiological studies. As determinants of exposure vary for the different exposure routes and body locations (for dermal exposure), the measure of interest for a specific study design will decide which determinants are most relevant.

Exposure to captan in fruit growing.

This study characterized occupational exposure to pesticides in fruit growing in The Netherlands to assess determinants of exposure. Large-scale exposure surveys were carried out during application of pesticides and during reentry activities. Data on contamination inside the fruit growers' homes were obtained, and total potential exposure for the fruit grower and his family during the growing and harvesting season was estimated. Repeated measurements on the same subject were collected to study components of exposure variability. Relative contribution of the respiratory route and different skin sites to total exposure were assessed. Captan was used as a marker for exposure. Inhalable dust exposure was measured with a personal monitor and potential dermal exposure with skin pads and hand rinsing. Dislodgeable foliar residue was measured by taking leaf punches. For respiratory exposure and potential dermal exposure, differences were observed between several tasks. Workers were categorized according to tasks performed depending on the exposure measure(s) (e.g., hands, forehead, inhalable dust) considered relevant for a specific study purpose. In general, within-worker variability of all exposure measurements was larger than between-worker variability. Variability in dermal exposure on the same body location was small relative to variability between different body locations. Differences in total exposure, including exposure inside the home, between the fruit grower and the son were small. Exposure of the wife was two to three times lower than for the fruit grower and the son. As exposure per unit of time was in the same order of magnitude for different tasks, individual time spent on these tasks is crucial for estimating total potential exposure. Repeated measurements are necessary to estimate individual exposure accurately because of the large within-worker variability.

The anti-cancer drug etoposide can induce caspase-8 processing and apoptosis in the absence of CD95 receptor-ligand interaction.

Caspase-8 (FLICE) can associate with and be activated by CD95 (APO-1/Fas), an apoptosis-inducing member of the Tumour Necrosis Factor receptor family. We find that, in Jurkat T cells, the DNA damaging anti-cancer drug etoposide induces apoptosis and, surprisingly, processing of caspase-8. Therefore, we have investigated whether etoposide involves CD95 receptor activation. We find that etoposide does not induce CD95 ligand expression at the mRNA level. In addition, blocking of CD95 receptor function with a specific antibody does not inhibit etoposide-induced apoptosis. Apparently, in Jurkat cells, etoposide can induce caspase-8 processing and apoptosis in a CD95-independent fashion. Likewise, we find that thymocytes from the CD95-deficient lpr/lpr mouse strain readily undergo apoptosis in response to etoposide. Moreover, since inhibition of the secretory pathway with brefeldin A does not inhibit etoposide-induced apoptosis, we exclude the requirement for a newly synthesized receptor ligand to induce the apoptotic pathway. We conclude that, at least in certain cell types, etoposide does not require CD95 receptor function to induce caspase-8 processing and apoptosis.

Experts' subjective assessment of pesticide exposure in fruit growing.

OBJECTIVES: Exposure to pesticides in fruit growing was estimated by pesticide experts, occupational hygienists, and fruit growing experts to determine whether valid subjective assessments can be made by experts. The study objectives were (i) validation of exposure assessment by experts using different sources of information, (ii) assessment of interrater agreement, (iii) measurement of agreement between experts' assessments and actual quantitative exposure data. METHODS: Three groups with different expertise made four ratings. Three of the ratings were made in three phases in which exposure information was provided. RESULTS: The intraclass correlation was high for each subgroup of experts when tasks in fruit growing were relatively ranked by increasing exposure level. In general, the interrater agreement on factors influencing the internal dose decreased when more information on exposure was provided. Experts correctly considered dermal exposure as the prominent contributor to internal dose. Results were comparable for the three pesticides under study. The ranking of 15 specific sprayings with a fungicide clearly showed differences between raters according to their expertise. The pesticide experts and occupational hygienists were able to rank daily exposure levels during pesticide spraying in a meaningful way. CONCLUSIONS: Experts seem to recognize the most important determinants of external exposure and therefore should be able pay a role in evaluating the effectiveness of control measures taken to reduce external exposure and to determine exposure groups in epidemiologic studies. The expert panel should not be too small, and consensus or average estimates should be used because differences within expert groups can be considerable.

Urinary excretion of tetrahydrophtalimide in fruit growers with dermal exposure to captan.

The relation between dermal and respiratory exposure and uptake into the body of captan, measured as 24 hr cumulative tetrahydrophtalimide (THPI) dose, was studied among 14 male fruit growers applying pesticides in orchards in the Netherlands. No contribution of respiratory exposure was observed on THPI in the urine. Dermal exposure, measured with skin pads, showed a clear relation with THPI in urine when exposure was estimated from exposure on skin pads of ankles and neck. No relation was found for total dermal exposure, calculated from measured exposure on skin pads of representative skin areas according to models described in the literature. Determinants of exposure such as use of a cabin on the tractor, use of gloves during mixing and loading, and use of rubber boots also explained THPI in urine very well. This finding corroborated the findings on measured dermal exposure. Results indicate that more attention should be paid to skin areas which are suspected to be most permeable for a chemical under study. It was concluded that dermal exposure data can be linked better to biological monitoring based on empirical findings as gathered in a pilot study on exposure of specific body areas than on estimations of total skin dose.

Variations in transcription-repair coupling in mouse cells.

Formation and repair of UV-induced cyclobutane pyrimidine dimers (CPDs) was examined in three different genes in mouse L cells: 1) a stably integrated insert (called LTL), consisting of a herpes simplex virus thymidine kinase gene (tk) fused to a hormone inducible promotor (LTR); 2) the constitutively expressed proto-oncogene c-abl; and 3) the inactive immunoglobulin J chain gene. Transcription of the tk gene is induced > 50-fold by dexamethasone. There is a nonuniform distribution of CPDs in LTL DNA irradiated in vitro, being 4-fold higher in the LTR than in the tk gene, indicating the LTR may be damaged preferentially in irradiated cells. Repair of CPDs occurs efficiently in both strands of LTL and is unaffected by hormone induction of tk gene transcription. Transcription of tk mRNA is very sensitive to UV damage and follows single hit kinetics with UV dose. Furthermore, tk mRNA expression rapidly recovers during repair incubation. Transcription-coupled repair occurs in these cells, however, since only the transcribed strand of c-abl is efficiently repaired of CPDs; the non-transcribed strand as well as both strands of the J chain gene are inefficiently repaired. Thus, repair in the LTL construct may reflect a lack of transcription-coupled repair in either the LTR promotor or the LTL insertion region of chromatin.

Dust exposure indices and lung function changes in longshoremen and dock workers.

A group of Dutch harbor workers involved in loading and unloading bulk products from sea vessels such as coal, cokes, and some other products like alumina, borax, phosphate ore, and vermiculite was studied. Exposures were characterized by personal and environmental monitoring. This information was subsequently used to estimate several dust exposure indices and to study relationships with lung function variables and respiratory symptoms. Average respirable dust exposure levels ranged from 0.3-4.0 mg/m3. Workers involved in unloading products from sea vessels were exposed to the highest dust levels. Supervisors and workers with tasks in the dock had an intermediate to low exposure. Office workers had the lowest exposure to respirable dust. Inhalable dust levels were considerably higher and average exposures ranged from 0.3-80 mg/m3. The ranking of occupational titles by inhalable dust exposure was almost identical to the rank order of respirable dust levels. Workers with higher current and cumulative dust exposures tended to have a lower lung function, and only shortness of breath had a statistically significant relationship with current and cumulative inhalable dust exposure. In general, relationships between lung function and inhalable dust levels tended to be somewhat stronger in terms of statistical significance, because inhalable dust is an estimate of dust deposition in the upper airways and lung function is a measurable parameter of airway obstruction in that region. However, the differences with respirable dust were minimal, and variability in dust exposure levels was extremely large for this population. It was concluded that harbor workers involved in unloading ships containing coal and various kinds of ore can be exposed to high dust levels. Relationships between dust exposure and lung function illustrate that these exposures are a respiratory hazard. Our finding that inhalable dust levels have a somewhat stronger relationship with lung function level than respirable dust levels deserves further attention.

Time to pregnancy and occupational exposure to pesticides in fruit growers in The Netherlands.

OBJECTIVES: Although pesticides are regularly used in agriculture, relatively little is known about possible adverse health effects, especially reproductive effects, due to occupational exposure. This explorative study investigates the relation between exposure of the fruit grower to pesticides and fecundability (probability of pregnancy) in a population of fruit growers. METHODS: The analysis is based on self reported data and includes 91 pregnancies during 1978-1990 of 43 couples. Cox' proportional hazards model was used to analyse time to pregnancy after correction for gravidity and consultation with a physician for fertility problems. RESULTS AND CONCLUSIONS: Application of pesticides solely by the owner was associated with a long time to pregnancy, resulting in a fecundability ratio of 0.46 (95% confidence interval (95% CI) 0.28-0.77). Similarly a low spraying velocity (< or = 1.5 hectares/h) resulted in a fecundability ratio of 0.47 (95% CI 0.29-0.76) and is associated with the use of older spraying techniques and tractors without a cabin. These factors were assumed to cause high exposure, which was confirmed by exposure measurements in the field. The effect of high exposure was mainly apparent if the couple had intended to become pregnant in the period from March-November (fecundability ratio 0.42, 95% CI 0.20-0.92). This is the period in which pesticides are applied. Out of the spraying season the effect of a high exposure was absent (fecundability ratio 0.82, 95% CI 0.33-2.02). In the high exposure group 28% of the pregnancies had been preceded by consulting a physician because of fertility problems, compared with 8% in the low exposure group. These findings indicate that an adverse effect of exposure to pesticides on fecundability is likely.

Neither enhanced removal of cyclobutane pyrimidine dimers nor strand-specific repair is found after transcription induction of the beta 3-tubulin gene in a Drosophila embryonic cell line Kc.

Nucleotide excision repair (NER) of ultraviolet (UV) light induced cyclobutane pyrimidine dimers (CPDs) was assayed in a Drosophila melanogaster Kc subline that responds to treatment with the steroid hormone 20-hydroxyecdysone (20-OH-E; beta-ecdysone, ecdysterone). In this cell line the hormone induces transcription of the beta 3-tubulin gene which is not expressed under standard culture conditions. Cells were exposed to either 10 or 15 J/m2 UV (predominantly 254-nm) and removal of CPDs from several genes, including beta 3-tubulin, and total cellular DNA was assayed. We show that upon induction of transcription of the beta 3-tubulin gene, its repair is not enhanced. In non-treated as well as 20-OH-E treated cells, repair kinetics in beta 3-tubulin resemble those in the active genes Gart and Notch, the inactive locus white and total cellular DNA. Moreover, in the presence as well as in the absence of transcription, the separate strands of the beta 3-tubulin gene are repaired with the same rate and to the same extent: about 90% after 24 h. It can be concluded from these observations that transcription is not a prerequisite for the efficient repair of CPDs in the Drosophila embryonic Kc cell line.

Repair of UV-induced (6-4)photoproducts measured in individual genes in the Drosophila embryonic Kc cell line.

The nucleotide excision repair (NER; dark-repair) of (6-4)photoproducts ((6-4)PPs) was assayed in cells from a permanent Drosophila melanogaster embryonic cell line, Kc, after exposure to 20 or 40 J/m2 ultraviolet (UV) light. Induction rates in the transcriptionally active genes Gart and Notch as well as in the inactive white locus is similar. They are formed with a frequency of about one-third of that of cyclobutane pyrimidine dimers (CPDs). In all three genes, (6-4)PPs are repaired with the same rate and to the same extent: 31% of the (6-4)PPs are removed in 4 hours post-irradiation and after 16 hours repair is nearly complete. In none of the three genes strand-specific repair was found. Exposure of cells that were irradiated with 40 J/m2 UV to photoreactivating light for 1 hour prior to dark-repair incubation, resulted in enhanced repair of (6-4)PPs.

Absence of strand-specific repair of cyclobutane pyrimidine dimers in active genes in Drosophila melanogaster Kc cells.

Strand-specific excision repair of UV-induced cyclobutane pyrimidine dimers was investigated in three genes: Gart, Notch and white in the permanent Kc cell line derived from wild-type Drosophila melanogaster embryonic cells. In this cell line Gart and Notch are transcriptionally active, whereas white is not expressed. Cells were irradiated with 10 or 15 J/m2 ultraviolet (UV) light (predominantly 254 nm). In all three genes, cyclobutane pyrimidine dimers were removed from the non-transcribed strand at the same rate and to the same extent as from the transcribed strand, indicating the absence of strand-specific repair in permanent Drosophila embryonic cell lines.

Repair of UV-induced pyrimidine dimers in the individual genes Gart, Notch and white from Drosophila melanogaster cell lines.

The excision repair of UV-induced pyrimidine dimers was investigated in three genes: Gart, Notch and white in a permanent Drosophila cell line Kc, derived from wild type Drosophila melanogaster embryonic cells. In this cell line Gart and Notch are actively transcribed, whereas white is not expressed. In all three genes UV-induced pyrimidine dimers were removed with the same rate and to the same extent: 60% removal within 16 hours, up to 80-100% in 24 hours after irradiation with 10 or 15 J/m2 UV. These kinetics are similar to the time course of dimer removal measured in the genome overall. No difference in repair of the inactive white locus compared to the active Gart and Notch genes was found. Similar results were obtained using a different wild type cell line, SL2, although repair appeared to be somewhat slower in this cell line. The results are discussed with respect to the data found for gene specific repair in other eukaryotic systems.

Unusual migration of a straight Amsterdam-type endoprosthesis for bile duct stones.

We describe an 84-yr-old woman in whom a straight Amsterdam-type biliary endoprosthesis was inserted because of endoscopically unremovable large common duct stones. After 18 months, the prosthesis migrated through the duodenum and the intrapancreatic portion of the bile duct, with its distal tip close to the vena cava and the aorta. In view of this high-risk complication, the authors avoid placing straight biliary endoprotheses for common bile duct stones in patients who might be lost to follow-up.