Critical effect sizes in toxicological risk assessment: a comprehensive and critical evaluation.

A key issue in toxicological risk assessment is determining the effect level below which there is no reason for concern. In the Benchmark approach, this breaking point between adverse and non-adverse is called the critical effect size (CES). This study aimed to investigate the possibilities to determine CESs for toxicological effect parameters commonly used in human risk assessment and includes a literature review and an opinion analysis among European toxicologists. The results indicate that the current knowledge is insufficient to define CESs for all individual parameters. Furthermore, the use of a single universal CES seems no option. It is concluded that it is not yet possible to reach international consensus on CESs for most toxicological parameters. However, every parameter for which consensus on the CES is reached is a step forward, because this can facilitate discussions on the adversity and relevance of certain changes in that parameter, irrespective of the method applied in risk assessment.

Selective immunomodulation by the autoimmunity-inducing xenobiotics streptozotocin and HgCl2.

Exposure to certain drugs and environmental chemicals can provoke the onset of autoimmune disease in susceptible individuals by releasing (self) epitopes for which tolerance has not been established, while simultaneously providing the necessary adjuvant activity. The resulting response type is influenced by the genotype of exposed individuals and relates to susceptibility to the adverse immune effects of the chemicals. Here, we assessed the modulatory role of the chemical compounds themselves. A single injection of streptozotocin (STZ) increased the number of CD8+ cells, macrophages, apoptotic cells, and IFN-gamma-producing T helper and T cytotoxic cells, whereas the number of CD4+ cells and B cells was reduced in the draining lymph node. Coinjection with the reporter antigen TNP-OVA resulted in primary and secondary production of TNP-specific antibodies that were predominantly of IgG2a and IgG2b isotype, whereas STZ did not enhance priming for delayed-type hypersensitivity (DTH) responses to TNP-OVA. Injection of HgCl2 on the other hand, reduced the number of IFN-gamma-producing cells, induced accumulation of B cells and CD4+ and CD8+ T cells, enhanced IgG1 and IgE production to TNP-OVA, and primed for secondary IgG1 and IgE production as well as for DTH reactions. Together these results indicate that a single injection of STZ stimulates type-1 responses, whereas HgCl2 enhanced mixed type-1 and -2 responses in BALB/c mice. These response types match the (auto)immune effects elicited to unknown (auto)antigens following multiple injections of these chemicals.

Immunotoxic effects of TCDD and toxic equivalency factors.

This paper first summarizes the studies indicating that the immunotoxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), notably atrophy of the thymus and suppression of the thymus-dependent immunity, are mediated by binding to a soluble cytosolic protein, the aryl hydrocarbon (Ah) or TCDD receptor, present in the thymus in the epithelial cells. On the basis of a common receptor-mediated mechanism of toxic action, the relative (immuno)toxicity of individual PCDDs and PCDFs can be expressed relative to TCDD (i.e., toxic equivalents). Next, studies on TCDD-induced immunosuppression and impaired host resistance, and lowest observed effects levels of TCDD resulting in immune alterations, are summarized. Immune investigations performed in man are discussed and it is concluded that, for risk assessment purposes, further studies are necessary to determine the sensitivity of the human immune system to TCDD. For this purpose, a recent study is summarized in which the sensitivity of the human thymus to TCDD is investigated in so-called severe combined immunodeficient (SCID) mice in which human thymus grafts were transplanted. This study indicates that the human thymus and the Wistar rat thymus display a similar sensitivity to TCDD.

No evidence for emergence of autoreactive V beta 6+ T cells in Mls-1a mice following exposure to a thymotoxic dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Tolerance to minor lymphocyte stimulatory-1a (Mls-1a) antigens is associated with clonal deletion of cells carrying the T cell receptor variable region V beta 6. Thymic epithelial cells may contribute to the intrathymic negative selection of potentially autoreactive V beta 6+ cells. Because 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) acts on thymic epithelial cells, we hypothesized that it may interfere with intrathymic selection processes. In the present study, this was addressed by exposing Mls-1a DBA/2 mice to a single thymotoxic dose of TCDD. The emergence of V beta 6+ cells in thymus, spleen, and mesenteric lymph nodes was investigated during the subsequent recovery of TCDD-induced thymic atrophy. In addition, the extrathymic differentiation of T lymphocytes in the liver was studied. TCDD exposure resulted in a severe thymic atrophy, and an increase in hepatic mononuclear cells. However, we were not able to demonstrate any emergence of potentially autoreactive mature V beta 6+ T cells, that differentiated either intrathymically or extrathymically, in TCDD-exposed DBA/2 mice.

Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to the human thymus after implantation in SCID mice.

There are conflicting data with regard to the sensitivity of the human immune system to the toxic action of the highly toxic environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). SCID mice engrafted with human fetal thymus and liver tissue fragments (SCID-hu mice), which sustain normal human T cell differentiation in the thymus graft, were used to directly assess the sensitivity of the human thymus for TCDD. Wistar rats and SCID-hu mice were exposed to 1, 5 or 25 micrograms TCDD/kg body weight. Histopathologic effects were evaluated for rat thymus and transplanted human thymus on Day 4 after exposure. The relative size of the cortex showed a dose-dependent decrease in both the normal rat thymus and grafted human thymus (significant at 25 micrograms/kg). SCID-ra mice (SCID mice with a fetal rat thymus and liver graft) were used as an intermediate model between the normal rat and SCID-hu mice, and were exposed to the same dose levels of TCDD. However, 90% of the SCID-ra mice developed a cutaneous graft-versus-host reaction, associated with lymphodepletion of the rat thymus grafts, and hence a limited number of SCID-ra mice were available for evaluation of TCDD effects. The data obtained in SCID-ra mice were in line with those in normal rat and grafted human thymus. In gas chromatography/mass spectrometry analysis, TCDD tissue concentrations in the normal rat thymus and grafted human thymus were similar. We conclude that the human thymus serves as a target for TCDD, and that the human thymus and the Wistar rat thymus display a comparable sensitivity to the toxic action of TCDD.

The SCID-hu mouse as a tool in immunotoxicological risk assessment: effects of 2-acetyl-4(5)-tetrahydroxybutyl-imidazole (THI) and di-n-butyltin dichloride (DBTC) on the human thymus in SCID-hu mice.

SCID mice engrafted with human fetal thymus and liver tissue fragments (SCID-hu mice) are currently considered as a new tool in human immunotoxicological risk assessment. Testing of various immunotoxicants exerting thymotoxicity via different intrathymic target cell types is necessary for validation of this model. Therefore, SCID-hu mice were exposed to 2-acetyl-4(5)-(1,2,3,4-tetrahydroxybutyl)-imidazole (THI), the immunotoxic component in the food additive, Caramel Colour III, or the organotin compound, di-n-butyltin dichloride (DBTC). Histopathological examination of the human thymus grafts of SCID-hu mice either exposed to THI or to DBTC showed a reduction in the relative size of the thymus cortex, an effect also described in rodents. These results indicate that the human thymus is a target for the immunotoxic action of both THI and DBTC. In addition, they indicate the promising potential of the SCID-hu mouse model as a tool for human immunotoxicological risk assessment.

Lymphodepletion of the thymus cortex in rats after single oral intubation of 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Atrophy of the thymus and thymic lymphodepletion occurs after exposure to sublethal doses of TCDD. Previous studies using high doses of TCDD (150 micrograms/kg) have indicated a preferential lymphodepletion of the thymus cortex on day 4 after a single oral intubation. Here we describe the effects of a single oral intubation of lower dose levels of TCDD (1, 5, and 25 micrograms/kg) on thymic weights and thymocyte subpopulations in Wistar rats on day 4 after exposure. A single administration of 1 micrograms/kg TCDD induced a significant reduction in the number of immature CD4+CD8+ double-positive (DP) thymocytes. At this time point the numbers of mature CD3high medullary thymocytes were not affected at any dose level tested. We conclude that also lower dose levels of TCDD induce thymic atrophy via a preferential lymphodepletion of the thymus cortex.

Time course of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced thymic atrophy in the Wistar rat.

Exposure to sublethal doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in Wistar rats results in thymic atrophy and reduced thymic cellularity. In a first experiment, rats were once orally intubated with 0, 1, 5, 25, 50, and 150 micrograms TCDD/kg body wt and sacrificed at Day 10. The dose that produced one-half of the maximal thymic involution was estimated at about 10-20 micrograms/kg. The time course (Days 0-21) of thymic atrophy induced by a single oral intubation of 25 micrograms TCDD/kg body wt revealed four phases. In phase 1 (initiation phase, Days 0-2) a decrease in proliferative activity was seen in cortical thymocytes, whereas no changes occurred in thymic cellularity. Phase 2 (lymphodepletion phase, Days 2-8) was characterized by an initial strong depletion of immature CD4+CD8+ double-positive (DP) cells (Day 4), followed by a more gradual decrease in the number of mature thymocytes (Day 6). On Day 8 the lymphodepletion was maximal. In phase 3 (stationary phase, Days 8-13) no changes occurred in thymic cellularity. Although the first signs of recovery were already seen on Day 6, indicated by a recovery in proliferative activity in the thymus cortex, an increase in thymic cellularity was observed first after Day 13 (phase 4, recovery phase, Day 13 onward). Reversibility of thymic atrophy is therefore observed within the estimated half-life of TCDD in the rat thymus (> 16 days). We conclude that TCDD exerts a rapidly reversible effect on an intrathymic cortical target cell population.

The intrathymic target cell for the thymotoxic action of 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Sublethal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces thymic atrophy and thymic lymphodepletion in all species investigated. Lymphodepletion is first seen in the cortex of the thymus, but also occurs in the medulla at later time points. This article surveys in vivo and in vitro studies on TCDD-induced thymus toxicity. It is concluded that the first intrathymic target cell for the action of TCDD is the cortical epithelial cell.

The SCID-ra mouse: rat T-cell differentiation in the severe combined immunodeficient mouse.

The SCID-hu mouse model offers the possibility to study the human thymus in vivo in an isolated xenogeneic environment. Whilst studying the toxicology of the human thymus using the SCID-hu model, a "control" model to test for differences in toxicokinetics due to the different localization of the thymus is desirable when SCID-hu data have to be extrapolated to the normal human situation. For this reason, SCID-ra mice were constructed by implanting rat fetal or postnatal thymus and liver explants under the renal capsule of severe combined immunodeficient (SCID) mice. Implantation of rat fetal thymus in combination with fetal liver resulted in thymus grafts with a histological appearance that was virtually identical to that of normal age-matched rat thymus. T cells of rat origin were found in the circulating blood and lymphoid organs of the recipient mice. After implantation of postnatal thymus and liver, the thymus grafts showed a dysplastic morphology. These grafts were devoid of thymocytes and consisted mainly of thymic microenvironmental components and large numbers of thymic macrophages. Some SCID-ra mice showed signs of graft-versus-host (GVH) reactions in the skin. The incidence of GVH reactions was higher with increasing developmental stage of the donor material used for implantation.

The neuro-endocrine component of the rat thymus: studies on cultured thymic fragments before and after transplantation in congenitally athymic and euthymic rats.

An immunohistochemical study was done for the presence of tyrosine hydroxylase (noradrenergic innervation), neuron-specific protein PGP9.5, and anterior pituitary hormones (beta-subunit of follicle-stimulating hormone, growth hormone, beta-subunit of luteinizing hormone, prolactin, and beta-subunit of thyroid-stimulating hormone) in cultured thymic fragments before and after transplantation in congenitally athymic and euthymic rats. The cultured thymic fragments consisted of epithelial cells and were depleted of lymphocytes. After implantation in syngeneic and allogeneic athymic recipients and in syngeneic euthymic recipients, a recovery of the original architecture was found within 6 weeks; rejection occurred within 3 weeks for allogeneic transplantation in euthymic rats. During culture nerve-like profiles almost disappeared from the tissue, and reappeared simultaneously with the influx of host-derived cells and the restoration of the original thymic architecture. A high immunoreactivity for hormones and PGP9.5 was found in epithelial cells after culture and in the first phase after transplantation. These epithelial cells may represent precursor-epithelial cells, based on their unusual ultrastructure and combined expression of markers that in the normal thymus occur only on subcapsular/medullary epithelium or on cortex epithelium. These data indicate a potential role of the neuroendocrine function of the thymus during restoration of the thymus architecture starting from precursor-like epithelial cells.

Transplantation of cultured thymic fragments in congenitally athymic and euthymic rats. Culture with deoxyguanosine or cyclosporin A does not influence the histologic characteristics and outcome after transplantation in syngeneic and allogeneic combinations.

Cultured thymic fragments (CTF) from WAG/CPB (RT1u) and DA/01a (RT1a) rats were prepared in the presence or absence of 2'deoxyguanosine or cyclosporin A, and subsequently transplanted under the kidney capsule of congenitally athymic and euthymic WAG/CPB recipients. The rationale of the culture supplements was that these may affect the disappearance of medullary dendritic cells, with subsequent induction of allotolerance. However, the immunohistology of the CTF showed more RT1 class II-positive cells than keratin-positive cells, indicative of the maintenance of dendritic cells. Grafts in athymic animals showed the recovery of the original thymic architecture within 6 weeks after transplantation. The influx of host-derived lymphocytes was accompanied by an influx of dendritic cells in the medulla-like area and macrophages in the cortex. A similar recovery was observed for syngeneic CTF in euthymic recipients. In addition lymphocytic infiltration was seen in the connective tissue surrounding the epithelial areas. Allogeneic grafts in euthymic animals were rejected within 3 weeks after transplantation. This outcome of the transplanted CTF under different conditions was not affected by the supplementation of the thymic culture before transplantation with 2'deoxyguanosine or cyclosporin A. We conclude that there is no tolerance induction after transplantation in euthymic allogeneic rats of CTF prepared in the presence of 2'deoxyguanosine. This conclusion is in contrast to data in the mouse, which may be explained by the maintenance of dendritic cells during culture. A chimaeric state of donor-derived epithelium and host-derived dendritic cells is obtained by transplantation of allografts in athymic rats.

V beta gene family usage in spontaneous lymphomas of AKR mice: evidence for defective clonal deletion.

T-cell receptor (TCR) beta-chain usage and expression of the CD3, CD4, and CD8 differentiation antigens were analyzed in 14 spontaneous AKR lymphomas. Lymphoma cells massively infiltrated and/or proliferated in the organs analyzed (thymus, spleen, and mesenteric lymph nodes), giving rise to a loss of organ structure. One lymphoma occurred only in the thymus, and failed to express CD3, CD4, and CD8. All other lymphomas expressed the CD3/TCR complex. With respect to CD4 and CD8 expression, the lymphomas were either double-negative (DN), double-positive (DP), or single-positive (SP). The frequency of DP (CD4+8+) lymphomas was low compared to the frequency of DP thymocytes in a normal AKR thymus. A substantial heterogeneity was seen in the intensity of CD4 and CD8 expression among various lymphomas, which was independent of the level of CD3 expression. Considering TCR V beta gene family usage, 2 out of 14 lymphomas expressed V beta 6. Normally, V beta 6+ thymocytes are deleted from the thymocyte pool at the immature DP stage of T-cell development in AKR mice. These data support the hypothesis that the lymphocytes in the immature DP stage of T-cell development are susceptible to the induction of AKR lymphomagenesis. The presence of V beta 6+ lymphoma cells indicates that the lymphomagenesis is accompanied by a defective clonal deletion of cells expressing a possible autoreactive TCR.