Skin sensitization in silico protocol.

The assessment of skin sensitization has evolved over the past few years to include in vitro assessments of key events along the adverse outcome pathway and opportunistically capitalize on the strengths of in silico methods to support a weight of evidence assessment without conducting a test in animals. While in silico methods vary greatly in their purpose and format; there is a need to standardize the underlying principles on which such models are developed and to make transparent the implications for the uncertainty in the overall assessment. In this contribution, the relationship between skin sensitization relevant effects, mechanisms, and endpoints are built into a hazard assessment framework. Based on the relevance of the mechanisms and effects as well as the strengths and limitations of the experimental systems used to identify them, rules and principles are defined for deriving skin sensitization in silico assessments. Further, the assignments of reliability and confidence scores that reflect the overall strength of the assessment are discussed. This skin sensitization protocol supports the implementation and acceptance of in silico approaches for the prediction of skin sensitization.

Oral Reference Dose for ethylene glycol based on oxalate crystal-induced renal tubule degeneration as the critical effect.

Several risk assessments have been conducted for ethylene glycol (EG). These assessments identified the kidney as the primary target organ for chronic effects. None of these assessments have incorporated the robust database of species-specific toxicokinetic and toxicodynamic studies with EG and its metabolites in defining uncertainty factors used in reference value derivation. Pertinent in vitro and in vivo studies related to one of these metabolites, calcium oxalate, and its role in crystal-induced nephropathy are summarized, and the weight of evidence to establish the mode of action for renal toxicity is reviewed. Previous risk assessments were based on chronic rat studies using a strain of rat that was later determined to be less sensitive to the toxic effects of EG. A recently published 12-month rat study using the more sensitive strain (Wistar) was selected to determine the point of departure for a new risk assessment. This approach incorporated toxicokinetic and toxicodynamic data and used Benchmark Dose methods to calculate a Human Equivalent Dose. Uncertainty factors were chosen, depending on the quality of the studies available, the extent of the database, and scientific judgment. The Reference Dose for long-term repeat oral exposure to EG was determined to be 15 mg/kg bw/d.

Maternal autoantibodies from preeclamptic patients activate angiotensin receptors on human mesangial cells and induce interleukin-6 and plasminogen activator inhibitor-1 secretion.

BACKGROUND: Preeclampsia affects 3-5% of all pregnancies. It is a major cause of maternal and fetal morbidity and mortality. Recent studies demonstrate that autoantibodies against the angiotensin II type 1 (AT(1)) receptor are present in the serum of preeclamptic patients. In this study, we investigated the role of AT(1) receptor-agonistic autoantibody (AT1-AA) regarding interleukin-6 (IL-6) and plasminogen activator inhibitor-1 (Pai-1) secretion in human mesangial cells. METHODS: The study included ten patients: five severely preeclamptic and five normotensive pregnant women. Immunoglobulin-G (IgG) was purified from each individual. The presence of AT1-AA was determined based on its ability to stimulate an increase in the contraction rate of rat neonatal cardiomyocytes. Primary human mesangial cells were chosen to study IgG-induced secretion of IL-6 and Pai-1. Losartan and epitope peptides were used to determine whether AT1-AA interaction with AT(1) receptor was associated with stimulation of IL-6 and Pai-1 secretion and was mediated through AT(1) receptor activation. RESULTS: The IgG from preeclamptic patients stimulated an increased contraction rate in rat neonatal cardiomyocytes. The IgG from preeclamptic patients induced the AT(1) receptor-specific secretion of IL-6 and Pai-1 from human mesangial cells at a significantly higher level than that achieved with IgG from normotensive patients. Competition with an epitope peptide suggested that the AT(1) receptor was stimulated by AT1-AA. CONCLUSIONS: Our findings suggest that a maternal autoantibody with the ability to activate AT(1) receptors may account for the development of renal damage seen in preeclamptic patients.

Maternal autoantibodies from preeclamptic patients activate angiotensin receptors on human trophoblast cells.

OBJECTIVES: Recent evidence indicates that preeclampsia is associated with the presence of autoantibodies capable of activating the angiotensin II receptor, AT1. We sought to evaluate the role of AT1 agonistic autoantibodies (AT1-AA) in two major features of preeclampsia-increased plasminogen activator inhibitor-1 (PAI-1) production and shallow trophoblast invasion. METHODS: This study included 38 pregnant patients, 20 of whom had severe preeclampsia and 18 normotensive individuals. Immunoglobulin (Ig)G was purified from these individuals, and the presence of AT1-AA was determined based on its ability to stimulate an increase in the contraction rate of cultured rat neonatal cardiac myocytes. Immortalized human trophoblasts were chosen to study PAI-1 production and secretion after treatment with IgG from normotensive and preeclamptic women. An in vitro Matrigel invasion assay was used to test the effect of AT1-AA on the invasive properties of human trophoblasts. Losartan and cyclosporin A were used to determine whether the AT1-AA-induced stimulation of PAI-1 secretion is through the AT1 receptor and the calcineurin-nuclear factor of activated t-cells (NFAT)-dependent pathway. RESULTS: The results show that IgG from 18 of 20 severely preeclamptic women stimulated increased cardiomyocyte contraction rates of 20-40 beats per minute. A significant stimulation of PAI-1 secretion from human trophoblasts was observed with IgG from the same 18 of 20 patients with severe preeclampsia. Of IgG obtained from 18 normotensive pregnant patients, only two showed a relatively low level of biologic activity in the cardiomyocyte contraction and PAI-1 secretion assays. Activation of AT1 receptors by AT1-AA was blocked by losartan (an AT1 receptor antagonist) and by a seven amino acid peptide corresponding to a sequence present on the second extracellular loop of the AT1 receptor. Activation of AT1 receptors by AT1-AA resulted in decreased trophoblast invasiveness as determined by the in vitro Matrigel invasion assay. Additional data indicate that AT1 receptor activation by AT1-AA is followed by the downstream activation of the calcium-dependent calcineurin-NFAT signaling pathway leading to increased PAI-1 gene expression. CONCLUSION: Our findings suggest that maternal autoantibody with the ability to activate AT1 receptors may account for two features of preeclampsia, increased PAI-1 production and shallow trophoblast invasion.