Developmental model for thalidomide action.
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Animals↗
Biomedical subjects
D Neubert
Publications and source records attributed to D Neubert.
Never-ending tales of the mode of the teratogenic action of thalidomide.
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Abnormalities, Drug-Induced↗
Vulnerability of the endocrine system to xenobiotic influence.
Natural sex hormones are most important factors guaranteeing the homeostasis of male and female sexual functions, including sexual differentiation and reproduction. Main target tissues include bone and skin, cardiovascular system, and possibly central nervous and immune systems. In medicine, synthetic hormonal substances with agonistic and antagonistic properties have been widely used for decades. Therapeutic benefit is the aim, and the many possibilities to interfere with normal or pathological hormonal situations are rather well understood. Synthetic hormonal agonists or (partial) antagonists may exhibit specific affinities to special receptors resulting in a spectrum of organotropies, or they may even induce opposite actions on different targets. Although not a new issue, environmental substances mimicking potentials of sex hormones have recently gained increased attention. There is not need to reinvent the wheel, since most (adverse) effects may be revealed with today's routine procedures used for testing medicinal substances, but some additional testing strategies should be included. Adverse effects of ecohormones may preferentially affect systems other than the human organism (assuming lower exposure and possibly lower susceptibility). Nevertheless, this survey is confined to possible alterations in the mammalian organism, since such effects are best understood from numerous experimental studies and clinical trials.
Animals↗
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCCD) affects keratin 1 and keratin 17 gene expression and differentially induces keratinization in hairless mouse skin.
The environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes chloracne in humans by mechanisms that are as yet poorly understood. Because TCDD is known to affect keratinocyte differentiation in vitro, we have studied TCDD-dependent morphologic changes and the expression of murine keratin 1 (MK1; differentiation associated) and keratin 17 (MK17; presumably hyperproliferation associated) in HRS/J hr/hr hairless mouse skin. TCDD (0.2 microg in acetone) applied topically to the dorsal skin caused epidermal acanthosis and hyperkeratosis of the dermal cysts as well as an involution of the utricles and the sebaceous glands. By means of in situ hybridization with digoxigenin-labeled riboprobes of sections from untreated and vehicle (control)-treated skin, we localized MK1 mRNA to the epidermal spinous cell compartment. MK17 transcripts were detected only in the derivatives of the hair follicle-utricle epithelium and dermal cysts. No spatial overlap was observed between MK1 and MK17 expression. After TCDD application, MK17 was newly expressed in the upper spinous cell layers of the interfollicular epidermis, although it was suppressed in the involuting utricles. In contrast, MK1 expression in the interfollicular epidermis was not affected by TCDD. Furthermore, MK1 expression was induced in the epithelium of the utricle remnants and in some dermal cysts. These data suggest that increased keratinization of the part of the follicular epithelium corresponding to the dermal cyst epithelium of hairless mice most probably explains the pathogenesis of TCDD-induced chloracne. The results demonstrate, furthermore, that TCDD can differentially affect keratinocyte differentiation in vivo as well as in vitro.
Animals↗
Inducibility of cytochromes P-450 by dioxin in liver and extrahepatic tissues of the marmoset monkey (Callithrix jacchus).
Cytochrome P-450 induction was investigated in the marmoset monkey, a non-human primate, using dioxins as inducing agents. Animals received a single subcutaneous dose of 1.6 nmol tetrachlorodibenzo-p-dioxin or tetrabromodibenzo-p-dioxin/kg body weight. Microsomal fractions were prepared from liver, lung and kidney, and homogenates were prepared from gut and adrenal glands. Anti-peptide antibodies which bind to CYP1A1, CYP1A2, CYP2B6 and CYP3A4 in human were used to identify related forms in the marmoset. The results indicate that CYP1A2 is constitutively expressed in liver, but not in lung, kidney, gut or adrenal gland and that CYP1A1 is not expressed in any of these tissues in untreated animals. Treatment with dioxin induced both CYP1A1 and CYP1A2 in liver, but only CYP1A1 in lung. No induction of CYP1A1 or CYP1A2 was found in kidney, small intestine or adrenal glands. Methoxy-, ethoxy-, pentoxy- and benzoyloxyresorufin O-dealkylases and high affinity phenacetin O-deethylase activities were induced in the liver, whereas ethoxycoumarin O-deethylase and aryl hydrocarbon hydroxylase activities were not affected by dioxin treatment. High-affinity phenacetin O-deethylase and CYP1A2 apoprotein were detected only in liver, consistent with this activity being specifically catalysed by CYP1A2. Furafylline was found to be a competitive inhibitor of methoxyresorufin O-demethylase activity with a Ki of 10 microM. In the lung the induction of CYP1A1 was accompanied by 15- and 23-fold increases in ethoxyresorufin O-deethylase and methoxyresorufin O-demethylase activities, respectively, suggesting that both activities are catalysed by CYP1A1. In contrast, there was no induction of aryl hydrocarbon hydroxylase activity in lung or liver showing that, unlike in many other species, marmoset CYP1A1 does not catalyse this reaction efficiently. The expression, distribution, induction and substrate specificities of marmoset monkey P-450 enzymes differ from the situation found in rodents and other species, demonstrating that caution has to be exercised when making cross-species extrapolations.
Animals↗
Induction of cytochromes P450 by dioxins in liver and lung of marmoset monkeys (Callithrix jacchus).
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Animals↗
Down-regulation of adhesion receptors on cells of primate embryos as a probable mechanism of the teratogenic action of thalidomide.
In spite of ongoing speculation, there has been no evidence that adhesion receptors are expressed on the cells of mammalian embryos. In this report, we provide the first proof that a variety of such receptor (beta 1-, beta 2-, and beta 3-integrins and selectin) are indeed expressed on cells of essentially all primordia of marmoset embryos at early organogenesis (developmental stages 11 to 13, or even earlier). Treatment with low doses (20 or as little as 1 mg/kg body weight) of a highly teratogenic derivative (EM12) of thalidomide, the most notorious human teratogen, triggers a dramatic and statistically highly significant down-regulation of several surface adhesion receptors (e.g. CD11a/CD18, CD49d/CD29, CD61, etc.) on early limb bud cells and on cells of some other primordia during early organogenesis of embryos of a primate (marmoset, Callithrix jacchus). Some of these receptors almost disappear, or they are expressed at a lower epitope density in the exposed embryos. These down-regulations of surface adhesion receptors may be expected to alter cell-cell- and cell-extracellular matrix interactions, and they are suggested to be a long-sought primary mechanism of the teratogenic action of thalidomide-type substances.
Animals↗
Tissue distribution after a single subcutaneous administration of 2,3,7,8-tetrabromodibenzo-p-dioxin in comparison with toxicokinetics of 2,3,7,8-tetrachlorodibenzo-p-dioxin in female Wistar rats.
Tissue concentrations of 2,3,7,8-tetrabromodibenzo-p-dioxin (TBDD) and induction of ethoxyresorufin O-deethylase (EROD) were determined in female Wistar rats following a single subcutaneous (s.c.) injection of TBDD. Two sets of experiments were performed in order to study (a) the time course after a single s.c. administration of 600 ng TBDD/kg body wt up to 78 days, and (b) the dose-response seven days after a single s.c. injection of different doses of TBDD (3 to 3,000 ng/kg body wt). The results obtained on toxicokinetics and enzyme induction were compared with those following a single s.c. administration of 300 ng/kg body wt 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Three days after the injection, approximately 93% of TBDD and 90% of TCDD had been absorbed. Fourteen days after s.c. injection less than 1% of the administered dose of both substances remained at the injection site. Three days after a single s.c. injection of 600 ng TBDD/kg body wt and 300 ng TCDD/kg body wt, the maximum tissue concentrations in the liver amounted to (M +/- S.D.) 5.7 +/- 0.8 and 4.7 +/- 0.9 ng/g wet weight, respectively. In adipose tissue, the peak concentration was 3.2 +/- 0.2 ng/g wet weight for TBDD on day 14, and 0.8 +/- 0.1 ng/g for TCDD on day 7. Throughout the study, the concentration ratio in the TCDD-treated group was always at least twice as high as that in the TBDD-treated group. The elimination half-life (t1/2) of TBDD and of TCDD in the liver was 13.3 and 13.6 days, respectively. In the adipose tissue the t1/2 of TCDD was 24.5 days but no reliable t1/2 could be calculated for TBDD (t1/2 = 39.4 days with a 95% confidence interval of 25.9 to 82.4 days). Tissue content of TBDD and TCDD in liver and adipose tissue increased dose-dependently, and the linear regression in a double-logarithmic plot showed a straight line. Time course of the induction of hepatic EROD activity after treatment with 600 ng TBDD/kg body wt was almost identical with that observed following a single dose of 300 ng TCDD/kg body wt. The induction of hepatic EROD activity was linearly correlated in a double-logarithmic plot to the hepatic concentrations of the congeners (both TBDD and TCDD). The slopes of the dose-response curves after administration of TBDD and TCDD were almost parallel for tissue concentrations ranging from 0.1 to 30 ng/g wet weight.
Absorption↗
Thalidomide derivatives and the immune system. 6. Effects of two derivatives with no obvious teratogenic potency on the pattern of integrins and other surface receptors on blood cells of marmosets.
The two thalidomide (Thd) derivatives beta-EM12 and phthalimidophthalimide (Phtpht), which exhibit no obvious teratogenicity, were tested for their ability to induce changes in the pattern of lymphocyte subpopulations, and especially changes in integrin receptors, in marmosets (Callithrix jacchus). Previously, Thd and its highly teratogenic derivative alpha-EM12 had been found to alter the expression of adhesion molecules, such as CD2 (LFA-2) or CD11a/CD18 (LFA-1). None of these typical effects on adhesion receptors were observed following administration of the relatively high daily doses of 50 mg/kg body wt beta-EM12 and Phtpht. Nevertheless, there were some minor effects, such as alterations in the receptor density on peripheral blood mononuclear cells, which were often contrary to the effects induced by Thd. Mainly affected were: CD8 cells, B cells bearing the CD54 receptor and CD4 cells bearing the CD56 (NCAM) surface marker. We observed an increase in the receptor density of CD11c (p150,95) on monocytes with Phtpht but not with beta-EM12. The inability of the two substances with no obvious teratogenic potential to typically modify beta 2-integrin receptors on white blood cells at comparatively high doses is consistent with our hypothesis, that the teratogenicity of Thd may also be linked to alterations in the expression of adhesion molecules.
Animals↗
Studies on the immunoglobulin-E system of the common marmoset in comparison with human data.
In the common marmoset (Callithrix jacchus jacchus) immunoglobulin E (IgE) serum levels and IgE synthesis of peripheral blood mononuclear cells (PBMC) in vitro were investigated in order to look for homologies to the human system. While IgE was not found in marmoset blood plasma with three commercial antihuman IgE-kits with monoclonal antibodies (mAbs), two other kits using polyclonal antibodies against human IgE revealed detectable IgE concentrations of up to 10 kU/liter in plasma samples of 19 out of 21 marmosets. In accord with human data, rhIL-4 showed biological functions under in vitro conditions in PBMC of the New World monkey. Proliferation, measured by 3H-thymidine incorporation, of isolated PBMC of marmosets could be induced by rhIL-4. FACScan analysis showed an enhanced expression of the low affinity IgE receptor CD23 (Fc epsilon RII) on CD20+ B lymphocytes after incubation with rhIL-4. Furthermore, PBMC from marmosets could be stimulated by IL-4 alone or in combination with dexamethasone as well as with lipopolysaccharide (E. coli) to produce IgE in culture. The results indicate that Callithrix jacchus is using an IgE system that is rather similar to that of humans, although not completely identical. Antihuman mAbs and rhIL-4 can be used to investigate IgE regulation in vitro of marmoset PBMC. These data encourage the development of a primate animal model for studying possible modifications of the IgE system under pathological conditions to find new therapeutic strategies in atopic diseases.
Animals↗
Enoxacin is an inducer of CYP1A2 in rat liver.
The induction of cytochrome P450 by enoxacin, ciprofloxacin, and ofloxacin was investigated in female Wistar rats. Animals were treated orally with daily doses ranging from 10 to 400 mg enoxacin per kg body wt, 400 mg ciprofloxacin, or 400 mg ofloxacin per kg body wt for up to 7 days. Activities of methoxyresorufin O-demethylase (MROD) and ethoxyresorufin O-deethylase (EROD) were determined fluorimetrically in hepatic microsomes. MROD activity was increased 2.6-fold after treatment with 100 mg enoxacin per kg body wt for 7 days. Lower doses of enoxacin did not induce MROD activity significantly. Antipeptide antibodies directed specifically against different rat cytochrome P450 enzymes demonstrated that CYP1A2, but not CYP1A1, was induced in rats treated with enoxacin. After ciprofloxacin or ofloxacin treatment, no induction of MROD or EROD activity was observed. Neither ciprofloxacin nor ofloxacin caused any change in CYP1A1 or CYP1A2 apoprotein levels. Further investigations with antipeptide antibodies showed that there was no induction of CYP2B1, CYP2B2, CYP2E1, CYP3A1, CYP3A2, CYP4A1, or CYP4A2 following treatment with enoxacin, ciprofloxacin, or ofloxacin. It is concluded that enoxacin, but not ciprofloxacin or ofloxacin, is an inducer of CYP1A2 in rat liver.
Animals↗
Four generation reproductive toxicity study with 2,3,7,8-tetrachlorodibenzo-P-dioxin (TCDD) in rats. I. Toxicokinetic variations in dams and offspring.
A multigeneration study on the reproductive toxicity of TCDD in rats was conducted. In this paper, the results of extensive pharmacokinetic evaluations are presented. The time course of tissue concentrations within the framework of a multigeneration study was investigated, using radioactive labeled 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a substance with a long elimination half-life. So far, long term exposure to TCDD has generally been conducted by administering the same daily doses via the feed. Since the half-life of TCDD in rats is several weeks, the concentration of the test substance can be predicted to change continuously during such a study. Therefore we intended to expose the animals to a constant tissue concentration by using a loading dose/maintenance dose approach. To achieve this, the animals were treated with initial loading doses of 50, 120 or 250 ng TCDD/kg body wt. Based on the elimination half-life of 3 weeks and a planned dosing interval of 7 days, the weekly maintenance doses were calculated to be 20% of the loading dose. During the postnatal phase of rapid growth, this dosing schedule was insufficient to keep the tissue concentration of TCDD constant. It was necessary to administer a second loading dose and to increase the weekly maintenance dose to 40% of the loading dose. While it was possible to control the tissue concentrations in the F0 generation, a considerably larger variation was observed during the different developmental stages of the F1 generation. The fluctuations could be reduced by using a complex dosing schedule, but even with that it was impossible to achieve completely steady levels in liver and adipose tissue.(ABSTRACT TRUNCATED AT 250 WORDS)
Adipose Tissue↗
Thalidomide and the immune system. 4. Down-regulation of the CD26 receptor, probably involved in the binding of HIV components to T cells in primates.
Thalidomide (Thd) is capable of down-regulating the CD26 receptor on CD4+ lymphocytes after treatment of healthy volunteers. Similar effects are observed when marmosets (Callithrix jacchus) are treated with Thd. The Ta1 epitope of the CD26 receptor has recently been shown to bind the HIV-1 Tat trans-activating protein, and CD26 has also been suggested to be a coreceptor for the binding of the V3 loop of the gp120 HIV envelope protein. This might provide a hint for possible therapeutic interventions.
Animals↗
Chlorinated dibenzo-p-dioxins and dibenzofurans and the human immune system. 2. In vitro proliferation of lymphocytes from workers with quantified moderately-increased body burdens.
Lymphocyte proliferation responses were studied in workers with moderately increased body burdens of 2,3,7,8-tetrachlorodibenzo-p-dioxin and other polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDDs/PCDFs, calculated as International Toxicity Equivalencies [I-TE]). Mitogens (pokeweed mitogen [PWM], phytohemagglutinine [PHA], concanavalin A [Con A]), as well as an anti-human monoclonal antibody against CD3 were used as proliferation stimulators in vitro. Additionally, the feasibility of using the lymphocyte response to tetanus toxoid was assessed, and the response to this recall-antigen was included in this trial. No decrease in the capacity of 3H-thymidine incorporation was observed with any of the proliferation stimulators in the group of volunteers with the increased TCDD-body burden when compared with volunteers exhibiting TCDD-concentrations in blood fat within the reference range. Regression analysis revealed a slight trend towards an increase for 3H-thymidine incorporation during the stimulation with PHA only. It can be concluded from our data that moderate increases in the TCDD- or I-TE-body burdens do not induce any medically significant changes in the capacity for proliferation of lymphocytes, measured as 3H-thymidine incorporation.
Adult↗
Proliferative capacity of marmoset lymphocytes after tetanus vaccination and lack of 2,3,7,8-tetrachlorodibenzo-p-dioxin to reduce a booster effect.
Marmosets (Callithrix jacchus) were vaccinated with tetanus toxoid and boostered 3 months and 1 year following the initial immunization. During this period, the proliferative response of lymphocytes (3H-thymidine incorporation) to the recall antigen was measured in vitro in blood samples 7 times. The experimental procedure proved to be suitable to monitor a defined but complex function of the immune system, and to assess possible substance-induced alterations with minimal stress or discomfort for the non-human primates. As a first example, a possible interference by a single very small dose (100 ng/kg body weight) of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) given at the time of the second booster was evaluated. No reduction in the in vitro response of the lymphocytes to recall antigen was observed under the experimental conditions used, and the extent of the 3H-thymidine incorporation was not significantly different in the groups. When the ratio of the responses between the first and the second booster was taken as a measure, there was a slight but statistically significant increase in this ratio for the lymphocytes of the TCDD-treated marmosets over that of reference animals. The limitations of these attempts to develop a test system and evaluate a substance-induced effect, and possible improvements of the test, e.g. with multivaccination, are discussed. It is suggested to use this approach also after routine multivaccination in children to assess possible substance-induced effects on immunological variables. This would allow an excellent comparison of experimental and clinical data obtained in primates with an identical technology.
Animals↗
Characterization of cytochromes P-450 purified from untreated and 14C-2,3,7,8-tetrachlorodibenzo-p-dioxin--treated marmoset monkeys: identification of the major form as a possible orthologue of P-450 1A2.
Three purified cytochrome P-450 (P-450) forms obtained from liver microsomes of marmoset monkeys induced with 14C-2,3,7,8-tetrachlorodibenzo-p-dioxin (14C-TCDD) were characterized. Comparison of electrophoretic and spectral properties and reconstituted methoxy-and ethoxyresorufin O-dealkylase (MROD and EROD) activities with those of forms isolated from untreated marmosets indicated that one of these (form F) is likely constitutive. Another form (D) had MROD and EROD activities which were 100 and 15 times those observed for form F. A form having biophysical properties similar to those of form D was also found in untreated animals. A third form (C) had an appreciable capacity to bind its inducing agent and showed a TCDD-to-P-450 molar ratio for detergent-free solutions of 0.66 +/- 0.13 to 1. In immunoblot analyses of these forms with antibodies raised against specific peptide sequences derived from rat P-450 1A1 and 1A2, the only positive reactions seen were those for untreated and inducible forms D with anti-rat 1A2. This provides evidence that the main or sole P-450 1A form in marmoset liver microsomes is 1A2, as in humans, and that this is inducible by TCDD.
Animals↗
Long-term interleukin-6 administration stimulates sustained thrombopoiesis and acute-phase protein synthesis in a small primate--the marmoset.
Interleukin-6 (IL-6) has been ascribed significant roles in both hematopoiesis and the immune response, although its contribution to host defence as a whole is poorly understood. Because short-term IL-6 treatment was previously shown to stimulate megakaryocytopoiesis, we investigated the effect of long-term administration of IL-6 on megakaryocytopoiesis and other systemic parameters in nonhuman primates. We chose a small primate, the marmoset (Callithrix jacchus), which enabled long-term administration at high doses. Recombinant human IL-6 (rhIL-6) administered at doses of up to 1,000 micrograms/kg/d over 4 and 9 weeks caused a sustained twofold to threefold increase of thrombocyte counts, peaking at 4 weeks. Thrombocyte counts declined thereafter, despite continuing IL-6 administration. The number of bone marrow megakaryocytes at 4 and 9 weeks was not increased compared with controls, but the ploidy grade was augmented, suggesting that IL-6 effects are restricted to mature megakaryocytes in vivo. An acute-phase protein response was observed within 24 hours after the first IL-6 administration and reached a maximum after 1 week of IL-6 administration at 25 micrograms/kg. Serum C-reactive protein, haptoglobin, and ceruloplasmin were increased, whereas albumin and transferrin levels declined. The acute-phase protein response was not associated with any morphologic evidence of hepatocellular damage. The increased levels of Ig and soluble IL-2 receptor in the serum levels reflected systemic immunostimulation. There was no evidence of renal mesangioproliferative pathology. Antibodies against rhIL-6 developed within 2 weeks, continuously increasing during the course of the study. High titers of neutralizing antibodies appeared concomitantly with the decrease in platelet counts and decline in acute-phase proteins. Therefore, despite the pleiotropic effects of IL-6 observed in vitro, long-term administration of IL-6 caused a selective and sustained stimulation of thrombopoiesis in marmosets that was only ablated by the appearance of neutralizing antibodies, and high doses were well tolerated in marmosets. A long-term targeting of IL-6 to cells of the megakaryocytic lineage, without evoking general toxicity, confirms the potential therapeutic usefulness of rhIL-6 for the chronic treatment of thrombocytopenic patients.
Acute-Phase Proteins↗
Embryotoxic effects of thalidomide derivatives in the non-human primate callithrix jacchus. IV. Teratogenicity of micrograms/kg doses of the EM12 enantiomers.
The dose-response of the teratogenic potency of the thalidomide (Thd) derivative EM12 was evaluated in the common marmoset (Callithrix jacchus). The smallest daily dose found to be effective was 30 micrograms EM12/kg body wt. This is the lowest dose of a Thd derivative ever reported to induce severe skeletal abnormalities. Ten micrograms EM12/kg body wt may be considered the no-observed-adverse-effect-level (NOAEL) under the experimental conditions chosen. The teratogenic potencies of the two EM12 enantiomers were tested at 100 micrograms/kg body wt, the dose which just induces an almost 100% effect in the case of the racemate. The S(-)-EM12 was found to induce typical severe limb abnormalities such as amelia, phocomelia, and radius aplasia, and none of the exposed fetuses were devoid of skeletal defects. In contrast, only few and minor skeletal defects were observed after application of the R(+) enantiomer. Although a pronounced teratogenic potency of the R(+)-EM12 can now largely be excluded, these low-dose studies are not sufficient to completely rule out any teratogenic potential of this enantiomer, since racemisation to small amounts of the S(-) form may occur in vivo. Further studies with Thd derivatives which are unable to racemise are necessary to prove the assumed complete ineffectiveness of the R(+) enantiomers.
Abnormalities, Drug-Induced↗