Transcriptome-Wide Prediction and Measurement of Combined Effects Induced by Chemical Mixture Exposure in Zebrafish Embryos.

BACKGROUND: Humans and environmental organisms are constantly exposed to complex mixtures of chemicals. Extending our knowledge about the combined effects of chemicals is thus essential for assessing the potential consequences of these exposures. In this context, comprehensive molecular readouts as retrieved by omics techniques are advancing our understanding of the diversity of effects upon chemical exposure. This is especially true for effects induced by chemical concentrations that do not instantaneously lead to mortality, as is commonly the case for environmental exposures. However, omics profiles induced by chemical exposures have rarely been systematically considered in mixture contexts. OBJECTIVES: In this study, we aimed to investigate the predictability of chemical mixture effects on the whole-transcriptome scale. METHODS: We predicted and measured the toxicogenomic effects of a synthetic mixture on zebrafish embryos. The mixture contained the compounds diuron, diclofenac, and naproxen. To predict concentration- and time-resolved whole-transcriptome responses to the mixture exposure, we adopted the mixture concept of concentration addition. Predictions were based on the transcriptome profiles obtained for the individual mixture components in a previous study. Finally, concentration- and time-resolved mixture exposures and subsequent toxicogenomic measurements were performed and the results were compared with the predictions. RESULTS: This comparison of the predictions with the observations showed that the concept of concentration addition provided reasonable estimates for the effects induced by the mixture exposure on the whole transcriptome. Although nonadditive effects were observed only occasionally, combined, that is, multicomponent-driven, effects were found for mixture components with anticipated similar, as well as dissimilar, modes of action. DISCUSSION: Overall, this study demonstrates that using a concentration- and time-resolved approach, the occurrence and size of combined effects of chemicals may be predicted at the whole-transcriptome scale. This allows improving effect assessment of mixture exposures on the molecular scale that might not only be of relevance in terms of risk assessment but also for pharmacological applications. https://doi.org/10.1289/EHP7773.

Formalin-induced changes of NMDA receptor subunit expression in the spinal cord of the rat.

Using RT-PCR, the present study investigated the effects of formalin administration on mRNA expression coding for NMDA receptor (NR) subunits and splice variants in rat lumbar spinal cord. Subsequent to formalin injection (5%; subcutaneously) into the hind paw of Sprague-Dawley rats, the animals exhibited the typical biphasic behavioural pain response. Spinal cord (L3-6) was prepared six hours after formalin injection. In controls, NR1-b predominated over NR1-a, and NR1-2 and NR1-4 exceeded over NR1-1 and NR1-3, respectively. Regarding the NR2 subunit expression in controls, NR2B exhibited the highest expression, followed by decreasing proportions of NR2C, NR2A, and NR2D. Formalin treatment did not affect NR1 splice variant expression but significantly increased and decreased the proportion of NR2A and NR2C, respectively. In summary, the present data demonstrate adaptive changes in the NR subunit expression pattern in rat spinal cord due to formalin injection.

Novel hepatoselective insulin analog: studies with a covalently linked thyroxyl-insulin complex in humans.

OBJECTIVE: To test whether a thyroxyl-insulin analog with restricted access to receptor sites in peripheral tissues displays relative hepatoselectivity in humans. RESEARCH DESIGN AND METHODS: Five normal human subjects received a subcutaneous bolus injection of either N(alphaBl) L-thyroxyl-insulin (Bl-T4-Ins) or NPH insulin in random order. Insulin kinetics, relative effects on hepatic glucose production, and peripheral glucose uptake were studied using euglycemic clamp and stable isotope [D-6,6-(2)H2]glucose) dilution techniques. Blood samples were taken for the determination of total immunoreactive insulin/analog concentrations and for liquid chromatography to assess the protein binding of the analog in the circulation. RESULTS: After subcutaneous administration, Bl-T4-Ins was well tolerated and rapidly absorbed. The analog had a long serum half-life and was highly protein bound (approximately 86%). Its duration of action, as judged by the duration of infusion of exogenous glucose to maintain euglycemia, was similar to that of NPH insulin. The effect of the analogs on hepatic glucose production was similar to that of NPH insulin, indicating equivalent hepatic potency. The analog demonstrated less effect on peripheral glucose uptake than NPH insulin (P = 0.025), had no effect on metabolic clearance rate of glucose, and exhibited a reduced capacity to inhibit lipolysis (P < 0.05). CONCLUSIONS: When injected subcutaneously into normal human subjects, Bl-T4-Ins is well tolerated, quickly absorbed, and highly protein bound, resulting in a long plasma halflife. This analog appears to have a hepatoselective action, and, therefore, has the potential to provide more physiological insulin action than the insulin preparations currently used.

Novel hepatoselective insulin analogues: studies with covalently linked thyroxyl-insulin complexes.

To explore the possibility that insulin analogues designed to have restricted access to peripheral tissues may display relative hepatoselectivity in vivo, Nalphabeta1-thyroxyl-insulin (B1-T4-Ins) and Nalphabeta1-thyroxyl-aminohexanoyl insulin (B1-T4-AHA-Ins) were synthesized. These insulin analogues bind thyroid hormone binding proteins to form high molecular weight complexes. Effects of intravenous infusions of B1-T4-Ins; B1-T4-AHA-Ins; combined thyroxine binding globulin (TBG) and B1-T4-Ins and combined TBG and B1-T4-AHA-Ins were compared with those of insulin infusion in hyperinsulinaemic euglycaemic clamp protocols in anaesthetized beagles (n=4 and n=3 for combined TBG infusions). Glucose turnover rates were measured using D-[3-3H]glucose infusion. With all 5 protocols the rate of glucose disappearance (Rd) was increased and the rate of endogenous glucose production (Ra) decreased from basal level 13.53+/-0.60 micromol kg(-1) min(-1)(p<0.05). Insulin-like activity for Ra and Rd was calculated as the area between the basal values of each variable and the subsequent values plotted graphically against time (AUC). For insulin, B1-T4-Ins, B1-T4-AHA-Ins, combined infusions of TBG+B1-T4-Ins, and TBG+B1-T4-AHA-Ins, respectively, AUC for Rd values were 6.30+/-0.69, 3.35+/-0.53, 4.40+/-0.64, 2.82+/-0.40 and 3.46+/-0.95 (mmol kg(-1)), all analogue infusions being different from insulin (p<0.05). AUC for Rd was further reduced by addition of TBG to B1-T4-AHA-Ins (p<0.05). In contrast the effect of all analogues on AUC for Ra was similar to that of insulin. These observations are compatible with the suggestion that insulin analogues which bind to thyroid hormone binding proteins retain access to hepatic insulin receptors which primarily control Ra. The reduced peripheral insulin-like effect (Rd) could be due to reduced transcapillary access to peripheral insulin receptor sites.

Value of superoxide dismutase for prevention of multiple organ failure after multiple trauma.

In a prospective, randomized trial, recombinant human superoxide dismutase (rhSOD, 3000 mg/day, Grünenthal, Aachen, Germany) or placebo was given intravenously during 5 days after multiple injuries (Injury Severity Score [ISS] > or = 27; 24 patients). Manifestation of multiple organ failure (MOF) and posttraumatic inflammatory response were evaluated over 14 days. No side effects were noted by continuous infusion of rhSOD, which allowed high SOD plasma levels (24.77 +/- 9.43 mg/L) compared with controls (0.03 +/- 0.02 mg/L). Multiple organ failure was attenuated by rhSOD treatment in respect to cardiovascular and pulmonary functions. Additionally, intensive care therapy was shortened from 30 days (Q25: 15; Q75: 37) to 21 days (Q25: 12; Q75: 41). A secondary increase of inflammatory mediators (e.g., C-reactive protein, polymorphonuclear [PMN]-elastase, phospholipase A2), as observed at the end of the first week in the placebo group, was reduced by rhSOD therapy. The results reveal an attenuation of organ failure after trauma, most likely by decreasing the release of inflammatory mediators and reduction of leukocyte-mediated organ injury. These preliminary results, while promising, need to be confirmed in a larger number of patients.

[Glucose homeostasis in mucoviscidosis]. / Untersuchung zur Glukosehomöostase bei Mukoviszidose.

In patients with cystic fibrosis (CF) of the pancreas an endocrine imbalance especially of insulin secretion due to progressive structural abnormalities of the pancreas must be expected. 30-75 percent of CF-patient exhibit impaired oral glucose tolerance tests (oGTT). Deterioration of the glucose homeostasis leads to a secondary diabetes mellitus that mimics a type II diabetes in the early stage, in the later course of disease it resembles a type I diabetes with absolute insulinopenia. In this study glucose homeostasis was investigated after an oral glucose load with 1.75 g glucose/kg bodyweight. Glucose, C-peptide and insulin were measured during 180 minutes. 32 nondiabetic CF-patients were studied. 16 patients revealed an impaired oral glucose tolerance according to the criteria of the National Diabetes Data Group. 6 patients showed a normal glucose tolerance and 10 patients with normal fasting and 120 minute glucose concentrations were hyperglycemic at midtest determinations. Impaired oGTTs were observed in malnourished CF-patients in a higher rate than in normal weight patients. A delayed and exceeded C-peptide and insulin response to the oral glucose load was determined with deteriorating glucose tolerance. Glucose values did not drop to fasting values at the 180 minute determination in cases of impaired oral glucose tolerance.

Time-dependence of biological activity induced by covalent insulin-receptor complexes in rat adipocytes.

Lipogenesis in isolated adipocyte preparations is stimulated when photosensitive insulin derivatives are attached covalently to specific receptors. This response was compared quantitatively with that to reversibly associated insulin, and it was shown that both covalent and reversible insulin-receptor complexes behave very similarly. The extent of stimulation of lipogenesis was studied as a function of time. Cells were incubated in buffer for various times before addition to vials containing 0 (basal) or 10 ng of monocomponent insulin/ml (maximal) and [U-3H]glucose. After 60 min, the toluene-soluble [3H]lipids were measured. The maximal stimulation induced by reversibly bound insulin was virtually constant over a period of 4 h. In contrast, adipocytes to which N alpha B2-(2-nitro-4-azidophenylacetyl)-des-PheB1-insulin had been covalently attached at the start of the experiment showed a loss of stimulation with time when incubated at 37 degrees C. This loss was decreased in the presence of lysosomotropic agents such as chloroquine at concentrations (approx. 200 microM) that had very little or no effect on the basal and maximal lipogenesis rates. A simple method was used to transform the measured rate of loss of stimulation into a rate of loss of effective units. A half-time of 80 min was calculated for the effective covalent insulin-receptor units in adipocytes at 37 degrees C at pH 7.4. This is very close to values reported by others for the internalization of covalent complexes in these cells, suggesting that this may be the causative event for the deactivation of the insulin-receptor unit. The inhibitory effect of chloroquine on the deactivation may indicate that the insulin-receptor complex can function even after internalization.

Biological action and fate of photoaffinity-labelled insulin-receptor complexes.

Covalent linking of two photoactivatable insulin derivatives, B2-(2-nitro,4-azidophenylacetyl)-des-PheB1-insulin and B29-(2-nitro,4-azidophenylacetyl)-insulin to viable rat adipocytes gives a system, which contains a fixed stoichiometry between hormone and receptor. The biological signal of prolonged lipogenesis has been used to study several aspects of insulin binding and action: the role of the site of the crosslink between insulin and receptor, recognition of bound photoinsulin by anti-insulin antibodies, the half-life of the biologically active complex, the pH-dependence of the biological signal, and the possible role of internalization. Furthermore, the effect of trypsin on the insulin receptor, as well as the insulin-receptor complex, has been investigated and a refined model of the receptor is presented.

Effects of covalently linked insulin dimers on receptor kinase activity and receptor down regulation.

Certain covalently linked insulin dimers have previously been found to have a greater ability to bind to the insulin receptor than to stimulate lipogenesis in adipocytes. The present report presents data indicating that the same insulin dimers also have a greater ability to bind to the receptor than to stimulate the kinase activity of the insulin receptor. In particular, one such covalently linked insulin dimer had less than 1% the potency of native insulin in stimulating the receptor kinase although it could bind to the solubilized receptor with 30% the potency of native insulin. In contrast, this dimer could down regulate the insulin receptor with approximately 30% the potency of native insulin. These results suggest that stimulation of the receptor kinase may require more than simple occupancy of the receptor binding site whereas down regulation of the receptor may require only the binding of ligand to the receptor.

Evidence concerning the mechanism of insulin-receptor interaction and the structure of the insulin receptor from biological properties of covalently linked insulin dimers.

Covalently linked insulin dimers have been prepared by cross-linking two insulin monomers with a flexible suberoyl chain at either the B1 phenylalanine or the B29 lysine residue. Binding potencies of dimers determined by inhibition of binding of 125I-insulin to isolated rat liver plasma membranes or adipocytes were 2.5-7-fold greater than their abilities to stimulate lipogenesis in adipocytes. Rates of liver plasma-membrane-associated degradation of labelled insulin and dimers, measured by gel filtration, were similar at 37 degrees C. Binding and lipogenesis potencies of dimers prepared by substitution of each monomeric half of an asymmetrical dimer with desoctapeptide insulin, an almost inactive derivative, implicated the B1-cross-linked monomeric half as predominantly interacting with the insulin receptor. These results suggest that (1) dimers bind univalently to a bivalent insulin-receptor complex, in which the two individual binding subunits are arranged with anti-parallel symmetry and (2) the mechanism by which insulin binds and initiates its biological responses requires a conformational change within the insulin-receptor complex and/or in the insulin molecule for full biological expression.

Preparation and properties of covalently linked insulin dimers.

The synthesis of six isomeric insulin dimers, linked through selected amino groups of the monomers by a dicarboxylic acid, is described. Symmetrical dimers were obtained by direct crosslinking of N,N-bis(methylsulfonyl-ethoxycarbonyl)insulins with the bis(p-nitrophenyl) esters of dicarboxylic acids. The synthesis of asymmetrical dimers was achieved by use of Msc-protected insulin active ester intermediates. N epsilon-B29,N epsilon B29'-Insulin dimers containing oxalyl, suberoyl and dodecanedioyl crosslinks were produced. N alpha B1,N epsilon B29'-Insulin dimers were suberoyl and dodecanedioyl crosslinks were synthesized; all other dimers were synthesized with suberoyl crosslinks. The positions of crosslinks were determined by sulfitolysis, tryptic digestion, electrophoresis and quantitative end-group determination. The dimers showed potencies between 1-60% that of insulin on a weight basis in stimulating lipogenesis in isolated fat cells. The potencies are considerably lower than the relative binding affinities determined with isolated fat cells.

[Preparation of N,N-bis(methylsulphonylethoxycarbonyl)insulins (author's transl)]. / Darstellung von N,N-Bis(methylsulfonylethoxycarbonyl)insulinen.

The preparation of N,N-bis(methylsulfonylethoxycarbonyl)insulins is described. In an aequeous buffer at pH 5.8 selectivity of the reaction of insulin with 20 equivalents of N-(methysulfonylethoxycarbonyloxy)succinimide (Msc-ONSu) leads very specifically to N alpha A 1,-N alpha B 1-(Msc)2 - insulin. The product can be isolated in a yield of 60%. Using N alpha A 1-citraconylinsulin the N alpha B 1, NEB29-(Msc)2 -insulin can be prepared in a yield of 40% based on insulin.

Crosslinked insulins: preparation, properties, and application.

Crosslinked insulins have proved to be valuable for structure-function studies and as proinsulin models. In the first part of the paper, a short review of the literature on analytical investigations, the preparation of A1-B1- and A1-B29-crosslinked derivatives, their biological activities in vivo and in vitro, and CD-spectral properties is given. The results of reduction/reoxidation studies with insulin derivatives containing irreversible and cleavable crosslinks are summarized. In the second part, new A1-B29-crosslinked monomers and 3 symmetrical dimers, linked between A1-A'1, B1-B'1 and B29-B'29, are described, as well as some results of tritium-labelling and of enzymatic degradation experiments with A1-B29-linked insulins.

[Aminolysis and racemization of activated esters with the use of high temperatures (author's transl)]. / Aminolyse und Racemisierung aktivierter Ester bei Anwendung höherer Temperaturen

The reactivities in aminolysis of the benzyl, phenyl, p-nitrophenyl and pentachlorophenyl esters of benzyloxycarbonylphenylalanine were ascertained at different temperatures up to 90 degrees C and the occurrence of racemization investigated. It turned out that the protective function of the benzyloxycarbonyl group in relation to the racemization was conserved, even at the high temperatures used in the peptide synthesis.