Combined retrospective analysis of 498 rat multi-generation reproductive toxicity studies: on the impact of parameters related to F1 mating and F2 offspring.

The multi-generation reproductive toxicity study (OECD TG 416 and USEPA 870.3800) has been extensively used internationally to assess the adverse effects of substances on reproduction. Recently the necessity of producing a second generation to assess the potential for human health risks has been questioned. The present standardized retrospective analysis of the impact of the second generation on overall study outcome combines earlier analyses and includes 498 rat multi-generation studies representing 438 different tested substances. Detailed assessment of study reports revealed no critical differences in sensitivities between the generations on the basis of a consideration of all endpoints evaluated. This analysis indicates that the second generation mating and offspring will very rarely provide critical information. These findings are consistent with the conclusions of previous retrospective analyses conducted by RIVM, USEPA and PMRA and support adoption of the proposed OECD extended one-generation reproductive toxicity study protocol in regulatory risk assessment testing strategies.

Refinement of risk assessment of dermally and intermittently exposed pesticide workers: a critique.

The regulatory requirements for the registration of pesticides are mainly evolved from concern about dietary exposure and risk, i.e. chronic oral exposure. Pesticide workers, however, are predominantly exposed dermally and intermittently. The present critique provides suggestions for improvement of toxicity studies to refine the risk assessment of pesticide workers. In this respect it is considered of utmost importance that toxicity studies (either toxicokinetic or toxicodynamic) should be tuned towards the anticipated exposure scenario. Apart from suggestions for improvement of dermal toxicokinetic and dynamic studies, recommendations for further research and guidance are given, amongst which the request for information on the robustness of in vitro dermal absorption studies and guidance on how to use these data. With respect to the intermittent exposure of pesticide workers it is recognised that both information on the anticipated exposure scenario as well as knowledge on the effect of intermittent exposure on the toxicity are needed. From a toxicological point of view, the setting of more than one Acceptable Operator Exposure Level (AOEL), covering effects that may arise after different periods of exposure, as well as the development of more robust acute and short term studies are strongly recommended.

Comparative in vitro-in vivo percutaneous absorption of the pesticide propoxur.

In vitro and in vivo skin absorption of the pesticide propoxur (2-isopropoxyphenyl N-methyl carbamate, commercially Baygon(TM) and Unden (TM); log Po/w 1.56, MW 209.2) was investigated. In vivo studies were performed in rats and human volunteers, applying the test compound to the dorsal skin and the volar aspect of the forearm, respectively. In vitro experiments were carried out in static diffusion cells using viable full-thickness skin membranes (rat and human), non-viable epidermal membranes (rat and human) and a perfused-pig-ear model. Percutaneous penetration of propoxur in human volunteers was measured by analysis of its metabolite (2-isopropoxyphenol) in blood and urine; in all other studies radiolabeled propoxur ([ring-U-(14)C]propoxur) was used. In order to allow for direct comparison, experimental conditions were standardized with respect to dose (150 microg propoxur per cm(2)), vehicle (60% aqueous ethanol) and exposure time (4 h). In human volunteers, it was found that approximately 6% of the applied dose was excreted via the urine after 24 h, while the potential absorbed dose (amount applied minus amount washed off) was 23 microg/cm(2). In rats these values were 21% and 88 microg/cm(2), respectively. Data obtained in vitro were almost always higher than those obtained in human volunteers. The most accurate in vitro prediction of the human in vivo percutaneous absorption of propoxur was obtained on the basis of the potential absorbed dose. The absorbed dose and the maximal flux in viable full-thickness skin membranes correlated reasonably well with the human in vivo situation (maximal overestimation by a factor of 3). Epidermal membranes overestimated the human in vivo data up to a factor of 8, but the species-differences observed in vivo were reflected correctly in this model. The data generated in the perfused-pig-ear model were generally intermediate between viable skin membranes and epidermal membranes.

Assessment factors for human health risk assessment: a discussion paper.

The general goal of this discussion paper is to contribute toward the further harmonization of human health risk assessment. It first discusses the development of a formal, harmonized set of assessment factors. The status quo with regard to assessment factors is reviewed, that is, the type of factors to be identified, the range of values assigned, as well as the presence or absence of a scientific basis for these values. Options are presented for a set of default values and probabilistic distributions for assessment factors based on the state of the art. Methods of combining default values or probabilistic distributions of assessment factors are also described. Second, the effect parameter, the no-observed-adverse-effect level (NOAEL), is discussed. This NOAEL as selected from the toxicological database may be a poor substitute for the unknown, true no-adverse-effect level (NAEL). New developments are presented with respect to the estimation of the NAEL. The already widely discussed Benchmark Dose concept can be extended to obtain an uncertainty distribution of the Critical Effect Dose (CED). This CED distribution can be combined with estimated uncertainty distributions for assessment factors. In this way the full distribution of the Human Limit Value will be derived and not only a point estimate, whereas information on dose-response relations is taken into account. Finally, a strategy is proposed for implementation of the new developments into human health risk assessments.

Neutrophil migration across monolayers of cytokine-prestimulated endothelial cells: a role for platelet-activating factor and IL-8.

In a previous study we observed that neutrophils respond with a rapid rise in [Ca2+]i during adherence to cytokine-activated endothelial cells (EC), caused by EC membrane-associated platelet-activating factor (PAF). In the present study, we investigated whether this form of PAF was important in neutrophil adherence and migration across monolayers of rIL-1 beta- or rTNF alpha-prestimulated EC. PAF receptor antagonists prevented neutrophil migration across cytokine-pretreated EC by approximately 60% (P less than 0.005) without interfering with the process of adherence. The antagonists WEB 2086 and L-652,731 had no effect on neutrophil migration across resting EC induced by formylmethionyl-leucyl-phenylalanine (FMLP). A murine anti-IL-8 antiserum was found to also partially inhibit the neutrophil transmigration across cytokine-activated EC. When the anti-IL-8 antiserum was used in combination with a PAF receptor antagonist, neutrophil migration across cytokine-pretreated monolayers of EC was completely prevented. During transmigration, LAM-1 and CD44 on the neutrophils were down-modulated; both WEB 2086 and anti-IL-8 antiserum partially prevented this down-modulation caused by cytokine-prestimulated EC. Our results indicate that human neutrophils are activated and guided by EC-associated PAF and EC-derived IL-8 during the in vitro diapedesis in between cytokine-stimulated EC.

Monocytes enhance endothelial von Willebrand factor release and prostacyclin production with different kinetics and dependency on intercellular contact between these two cell types.

Human umbilical vein endothelial cells cultured on a collagen lattice were used to study the effects of the interaction between human monocytes and endothelial cells on endothelial von Willebrand Factor (vWF) release and prostacyclin (PGI2) production by these cells. The effects of monocytes were compared with those of other leucocytes, conditioned media from monocytes, and agonists such as interleukin 1 (IL-1) and the phorbol ester PMA. Because the cell culture system used allows simultaneous analysis of the lumenal and ablumenal compartment of endothelial cell monolayers, we also studied into which direction these products were released by endothelial cells. Under quiescent conditions the concentration of vWF in the ablumenal compartment was about three-fold higher than that in the lumenal compartment, whereas PGI2 was equally distributed between the two compartments. Direct cell-cell contact between purified monocytes and endothelial cells strongly enhanced both vWF release and PGI2 synthesis, in a dose-dependent and monocyte-specific manner. The monocyte-induced enhancement of PGI2 production, however, was much earlier in onset than that of vWF. Secretory products from monocytes also enhanced endothelial PGI2 synthesis, although to a lesser extent than with monocytes that were in direct contact with endothelial cells. In contrast, the monocyte-induced enhancement of endothelial vWF release was completely dependent on the direct interaction between monocytes and endothelial cells.

Neutrophil and monocyte adherence to and migration across monolayers of cytokine-activated endothelial cells: the contribution of CD18, ELAM-1, and VLA-4.

Pretreatment of endothelial cells with cytokines enhances the adherence of leukocytes, a process that is mediated by surface proteins expressed on both cell types. A three-dimensional model system for the simultaneous determination of leukocyte adherence and migration was used to study the contribution of CD11/CD18, endothelial leukocyte-adhesion molecule-1 (ELAM-1) and VLA-4 in neutrophil and monocyte adherence to and migration through cytokine-activated endothelial cells. Pretreatment of endothelial cells for 4 hours with recombinant interleukin-1 beta (rIL-1 beta) was found to enhance neutrophil adherence and migration to a much greater extent than monocyte adherence and migration. Neutrophil adherence was almost completely prevented by the combined use of monoclonal antibodies (MoAbs) against ELAM-1 and CD18. Although ELAM-1 has been designated an endothelial cell-specific cytokine-inducible receptor for neutrophils, we observed that ENA2, an anti-ELAM-1 MoAb, significantly reduced monocyte adherence about 30%. MoAbs against VLA-4, the ligand of the cytokine-inducible receptor VCAM-1, did not affect monocyte adherence. However, the combined use of the MoAbs against CD18, ELAM-1, and VLA-4 had a very strong and additive inhibitory effect on rIL-1 beta-induced monocyte adherence. The anti-CD18 MoAb reduced both rIL-1 beta-induced neutrophil and monocyte migration far below the level of the unstimulated controls, whereas neither the anti-ELAM-1 nor the anti-VLA-4 MoAb significantly affected the process of migration. Our results indicate that neutrophils and monocytes initially adhere to cytokine-activated endothelial cells by CD18-independent and (to a lesser extent) by CD18-dependent mechanisms and subsequently change gears to a completely CD18-dependent migratory mechanism.

Role of endothelial leukocyte adhesion molecule-1 and platelet-activating factor in neutrophil adherence to IL-1-prestimulated endothelial cells. Endothelial leukocyte adhesion molecule-1-mediated CD18 activation.

Adherence of neutrophils to endothelium is a key event in the sequence of inflammatory leukocyte responses. Double-color FACS analysis was used to determine the extent and kinetics of neutrophil adherence to rIL-1 beta-pretreated endothelial cells (EC). Neutrophils bound very avidly when the EC were prestimulated for 4 to 6 h with rIL-1 beta. Anti-ELAM-1 F(ab)2 fragments inhibited this adherence for more than 80%. On the other hand, anti-CD18 F(ab)2 fragments also inhibited the neutrophil adherence (40 to 50%). Combined use of anti-ELAM-1 and anti-CD18 F(ab)2 fragments completely prevented adherence. Neutrophils became activated as soon as they made contact with the rIL-1 beta-pretreated EC. First, neutrophils depleted of intracellular ATP showed a clearly decreased adherence completely dependent on ELAM-1-mediated binding, i.e., without additional effects of CD18 adhesion proteins. Thus, CD18 is activated during neutrophil adherence and then participates in the binding process. Secondly, the neutrophils responded with a transient rise in [Ca2+]i upon binding to rIL-1 beta-pretreated EC, which was demonstrated to be caused by endothelial cell-associated platelet-activating factor (PAF). However, the extent of neutrophil adherence to rIL-1 beta-pretreated EC was not affected by the use of the PAF-receptor antagonist WEB 2086, or removal of the EC-bound PAF. The only effect was a complete dependency of the neutrophil adherence on ELAM-1-mediated binding, although anti-CD18 mAb still induced 40 to 50% inhibition under these conditions. We therefore conclude that ELAM-1-mediated binding is the major mechanism for CD18 activation during neutrophil adherence to rIL-1 beta-pretreated EC.

Monocytes enhance the bidirectional release of type I plasminogen activator inhibitor by endothelial cells.

Human umbilical vein endothelial cells cultured on a collagen lattice were used to study the effects of the interaction between human monocytes and endothelial cells on the production of type 1 plasminogen activator inhibitor (PAI-1) by endothelial cells. The effects of adherence and transendothelial migration of monocytes on endothelial PAI-1 release were compared with those of other leukocytes, conditioned media from monocytes, and interleukin-1 beta (IL-1 beta). Because the cell culture system used allows simultaneous analysis of the lumenal and the subendothelial compartment of endothelial cell monolayers, we also studied into which direction PAI-1 is released by endothelial cells. Under quiescent conditions, the net amount of PAI-1 accumulated at the lumenal side was twofold higher than that accumulated at the subendothelial side (about 2.0 micrograms PAI-1/10(6) cells and 1.1 microgram PAI-1/10(6) cells, respectively, in 24 hours), as analyzed by a quantitative immunoradiometric assay (IRMA). Direct cell-cell contact between highly purified monocytes and endothelial cells strongly enhanced the PAI-1 release by endothelial cells in a dose-dependent way, whereas lymphocytes and neutrophils did not affect endothelial PAI-1 production. The monocyte-mediated increase was first detected after 12 hours of incubation and lasted for at least 48 hours. In the presence of two monocytes per endothelial cell, the increases of PAI-1 at the lumenal side and at the subendothelial side were 87% and 32% in 24 hours, respectively. The effect of IL-1 beta on PAI-1 release by endothelial cells closely resembled that observed for monocytes. Monocyte-conditioned medium contained heat-labile product(s) which also, although to a much lesser extent than intact monocytes, enhanced endothelial PAI-1 release. Similarly, monocytes cultured on top endothelial cell separated by a microporous filter enhanced the release of PAI-1 to a lesser extent. Thus, these findings indicate that monocytes enhance endothelial PAI-1 release by mechanisms that are, at least in part, dependent on cell-cell contact.

A three-dimensional model system to study the interactions between human leukocytes and endothelial cells.

Leukocyte adhesion to endothelial cells and migration into the subendothelial matrix was studied with a three-dimensional model system, consisting of human endothelial cells cultured on a loose collagen matrix. We developed a new method to separate the endothelial cell monolayer and adhering leukocytes, from the subendothelial matrix, allowing simultaneous analysis of leukocyte adhesion and transendothelial migration. Monocytes adhered more avidly to untreated endothelial cells than did neutrophils (2.5 +/- 0.3 vs. 1.0 +/- 0.2 leukocytes per endothelial cell). Only a small fraction (10%-20%) of these leukocytes migrated into the subendothelium. Pretreatment of endothelial cells with interleukin 1 (IL 1) enhanced adhesion (20%), but not migration of monocytes. In contrast, neutrophil adhesion was markedly and in a time-dependent manner increased by IL 1 treatment (i.e. 200% after 6 h and 110% after 24 h of IL 1 treatment). Moreover, IL 1 pretreatment enhanced neutrophil migration twofold. Activation of leukocytes with formyl-methionyl-leucyl-phenylalanine (fMLP) enhanced both monocyte and neutrophil adhesion, but did not affect leukocyte migration. Under all conditions, monocyte adhesion was only partly (30%-40%) inhibited by monoclonal antibodies (mAb) against the common beta subunit of the leukocyte-cell adhesion molecules (LeuCAM: CD18) and 25%-30% by mAb against the alpha subunit of LFA-1 (CD11a). In contrast, mAb against the alpha subunits of Mac-1 (CD11b) and p150.95 (CD11c) were hardly effective. fMLP-mediated neutrophil adhesion was reduced to below baseline levels by anti-LeuCAM (CD18) mAb, whereas the LeuCAM contribution in IL 1-mediated neutrophil adhesion was less pronounced and varied in time. IL 1-mediated neutrophil migration, however, was completely blocked by anti-LeuCAM mAb. fMLP-mediated neutrophil adhesion was inhibited by mAb against the alpha subunits of Mac, while mAb against the alpha subunits of LFA-1 and Mac-1 both reduced IL 1-mediated adherence. In summary, we describe a novel leukocyte adhesion/migration method and demonstrate that the contribution of the LeuCAM complex in leukocyte-endothelium interaction varies depending on cell type and stimulus used.

Distinct adhesive properties of granulocytes and monocytes to endothelial cells under static and stirred conditions.

Adherence of neutrophils and monocytes to endothelium is an important event in the sequence of leukocyte responses to inflammatory disease. Double-color flow microfluorimetry analysis was used to determine neutrophil and monocyte adherence to suspended endothelial cells under stirred conditions. The static adherence to endothelial cell monolayers was simultaneously determined. In both assays, neutrophils behaved in a similar way. Basal adherence of neutrophils was very low. Activation by PMA or by the chemoattractants platelet-activating factor and FMLP induced rapid binding, primarily mediated by CR3. Nonactivated neutrophils showed CD18-dependent (lymphocyte function-associated Ag-1 and CR3) and CD18-independent adherence to endothelial cells when the endothelial cells were prestimulated with rIL-1 beta. In contrast to neutrophils, nonactivated monocytes adhered avidly to resting endothelial cells. This adherence was partly CD18 dependent and partly CD18 independent. Whereas monocyte adherence under stirred conditions strongly increased upon activation by PMA, a significant decrease in adherence was found under static conditions, which was prevented by cytochalasin B. This decrease was limited to a distinct CD18-independent binding mechanism, and absent under stirred conditions. We conclude that monocytes adhere with (at least) three binding mechanisms to endothelial cells, a CD18-dependent and two CD18-independent mechanisms.

Inhibitory effects of the phorbolester TPA and cigarette smoke condensate on the mutagenicity of benzo[a]pyrene in a co-cultivation system.

The transport of reactive intermediates was studied in a co-cultivation system of primary chick embryo hepatocytes and V79 Chinese hamster cells. Two test systems with different genetic endpoints--sister-chromatid exchange (SCE) and gene mutation at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus--were used. Benzo[a]pyrene (B[a]P) was positive in both test systems. When the V79 cells were co-cultivated with the hepatocytes at a distance of 1 mm, only a slight increase in the number of SCEs was observed after exposure to benzo[a]pyrene. When the two cell types were in direct contact, addition of the phorbolester TPA or cigarette smoke condensate inhibited the mutagenic effects of B[a]P in both assays by 50%. No influence of TPA on the number of SCEs induced by B[a]P was observed in a preincubation assay using Aroclor-1254-induced rat liver homogenate. The results indicate that metabolic co-operation may play a role in the transport of reactive intermediates in this co-cultivation system. The mutagenic potential of compounds may be underestimated in systems using intact cells for metabolic activation if the compounds or their metabolites are capable of inhibiting metabolic co-operation.

Genotoxicity testing of cigarette-smoke condensate in the SCE and HGPRT assays with V79 Chinese hamster cells.

The genotoxicity of cigarette-smoke condensate (CSC) was investigated using V79 Chinese hamster fibroblasts in the sister-chromatid exchange (SCE) and HGPRT (hypoxanthine-guanine-phosphoribosyl-transferase) tests. CSC was negative in the SCE test both with and without metabolic activation (co-cultivation with chick-embryo primary hepatocytes). In the HGPRT test no direct mutagenicity of CSC was observed but after metabolic activation there was a considerable increase in the number of mutants. Comparison of different metabolizing systems showed that non-induced chick hepatocytes, liver homogenate from non-induced chick embryos and liver homogenate from rats pretreated with Aroclor 1254 generated similar numbers of mutants in cells treated with CSC. In addition the capacity of CSC to inhibit metabolic co-operation between V79 cells was studied. A dose-related increase in the inhibition of metabolic co-operation was observed.