Ursula Jacob-Müller

Simple assay of 0.1–1.0 pmol of ATP, ADP, and AMP in single somatic cells using purified luciferin luciferase☆

Abstract The sensitivity of ATP determinations with crude firefly luciferin luciferase is limited by contaminating ATP converting enzymes, which cause a rapid decrease of the ATP level during the assay. Purified luciferase has the advantage of producing an almost constant light intensity proportional to the ATP concentration. Sensitivity and specificity of the ATP assay are, therefore, considerably increased when purified enzyme is used instead of crude extracts of the enzyme. ATP, 0.1–1.0 pmol as well as higher amounts can be determined with commercial preparations of purified and stabilized luciferase. In ADP and AMP measurements with the luciferase assay, problems are arising from the enzymes required for the conversion to ATP, since they are frequently contaminated by low amounts of adenine ribonucleotides. Exclusion of contaminated enzymes and removal of ammonium sulfate from adenylate kinase were the only prerequisites for determinations of 0.1–1.0 pmol of ADP and AMP with purified luciferase. The application of the assay in determinations of ATP, ADP, and AMP in single preimplantation mouse embryos is described.

Polyhalogenated dibenzo-p-dioxins and dibenzofurans and the immune system. 1. Effects on peripheral lymphocyte subpopulations of a non-human primate (Callithrix jacchus) after treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

Monoclonal antibodies were used to analyse the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on peripheral lymphocytes from marmosets (Callithrix jacchus) following injections of single low doses of TCDD. A reduction of the percentage and the total number of lymphocytes among the white blood cells was seen 3 weeks after a single administration of 300 ng TCDD/kg body wt, but not following 167 ng TCDD/kg or less. After treatment of marmosets with the single subcutaneous dose of 10 ng TCDD/kg body wt, we observed an average decrease of about 20% in the percentage of CD4+ cells in the venous blood of treated animals. This change was not seen at the time of maximum absorption (2 weeks after the injection), but was demonstrable after 4 weeks and reached its maximum 15 weeks after the injection. The time course of the changes suggests an indirect effect (possibly via the thymus). Due to the considerable inter- and intra-individual variability in the number of peripheral lymphocytes the effect was less convincing on the basis of the total CD4+ cells per microliter blood. There was a significant concomitant increase in the percentage of cells with the CD8+ marker. A closer analysis of the lymphocyte subpopulation involved revealed a predominant effect on the cells with the CD4CDw29 (“leu 3a+4B4+”) surface marker (“helperinducer cells”). Subsequent to a dose of 10 ng TCDD/kg body wt the percentage of these cells was reduced by 50% or more when compared with the (24) controls. On an absolute basis (number of cells/μl blood) these CD4+CDw29+ cells were clearly reduced in only two out of four marmosets. There was no significant effect on the percentage of the CD4+CD45R+ (“leu3a+2H4+”) subpopulation (“suppressor-inducer cells”). The ratios: CD4+CDw29+/CD4+CD45R+, or CD4+CDw29+/CD8+ appear to be convenient measures for monitoring the effect described. Since the difference between the percentage of CD2+ cells minus the sum of CD4+ plus CD8+ cells was found to be increased following rather high doses (167 ng TCDD/kg body wt or more), this suggests that immature cells (CD2+ CD4-CD8-) are released into the periphery as a result of the exposure to TCDD. Furthermore, a decrease in the percentage of CD20+ (B1+) cells (about 50% when compared with controls) was observed in the treated animals following a single dose of 10 ng TCDD/kg body wt or higher. Subsequent to a dose of 10 ng TCDD/kg body wt the percentage of CD56+ (NKH1+) cells seemed to be slightly increased. The dose-response for the effects observed was rather poor. At present it cannot be decided whether the changes have to be considered as adverse health effects. All of the animals involved were apparently healthy and did not exhibit, e.g. any infections. However, the deviations described certainly represent biologicaleffects induced at very low dose levels. From the data available up till now, a single dose of 10 ng TCDD/kg body wt seems to be the “lowest-observed-effect-level” (LOEL) in the marmoset for all the effects studied. More extensive experiments within the dose range between 1 and 10 ng TCDD/kg body wt are under way in our laboratory. When peripheral lymphocytes of TCDD-treated animals were incubated in vitro with a mitogen (PWM = poke weed mitogen) an exaggeration of thedecrease in the percent of cells with the CD4 surface marker and a concomitant increase in the percentage of CD8+ cells was observed. This seems to be an especially sensitive experimental approach for demonstrating biological effects (but not necessarily adverse health effects) of this class of substances. Such an effect was demonstrated and found to be statistically significant already 2 weeks after a single injection of 10 ng TCDD/kg body wt in comparison to vehicle-treated controls.

Polyhalogenated dibenzo-p-dioxins and dibenzofurans and the immune system. 2. In vitro effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on lymphocytes of venous blood from man and a non-human primate (Callithrix jacchus).

The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on poke weed mitogen-stimulated proliferation and differentiation of peripheral lymphocytes was studied in vitro with cells from a non-human primate (marmoset monkey,Callithrix jacchus) and from man. Monoclonal antibodies and flow cytometry (FACScan) were used for analysis. The extent of the overall mitogenstimulated proliferation of isolated lymphocytes in vitro from marmoset blood was only slightly reduced in the presence of TCDD compared to the solvent control (0.01% DMSO). However, incubation with TCDD in the culture medium together with the mitogen led to a pronounced decrease in the percentage of the lymphocyte subset with the surface marker CD4, and a concomitant increase in the percentage of CD8+ cells. The lowest concentration found to be effective in vitro was 1 × 10−13 M TCDD (25 fg TCDD/ml). When culturing lymphocytes from human blood of different donors under identical conditions in the presence of TCDD and the mitogen, corresponding effects were observed to those seen with marmoset cells. A closer analysis of the T lymphocyte subsets affected revealed the CD4+ CDw29+ (helper-inducer cells) to be the main target for the action of TCDD. A clear-cut change in the percentage of this subpopulation was induced at concentrations as low as 1 × 10−13 M TCDD. The development of the IL-2-marker in culture was only slightly affected by TCDD, and concentrations of 1 × 10−12 M were required to slightly reduce the number of CD2+CD25+ cells. Special B cells, namely CD20+ (i.e. B1) cells, were found to be especially susceptible to the action of TCDD, and clear-cut effects were seen in several experimental series at 1 × 10−14 M TCDD. The formation of B cells with IgG lambda or kappa chains was also depressed in culture in the presence of TCDD. When the cultures were initiated with whole blood instead of with isolated lymphocyte fractions, the effects described were also observed, but the concentration of TCDD needed seemed to be higher (about 1 × 10−12 M). The mixed lymphocyte culture with human cells provides a simple system for studying the effects of TCDD and similar substances in vitro at low concentrations. For the first time a direct effect of TCDD on peripheral lymphocytes of primates was demonstrated. This in vitro effect on special sub-classes of peripheral lymphocytes from non-human primates represents the biological effect demonstrated with the lowest TCDD concentration ever reported in the literature.

Chlorinated dibenzo-p-dioxins and dibenzofurans and the human immune system. 1. Blood cell receptors in volunteers with moderately increased body burdens

Using monoclonal antibodies (mAbs) and flow cytometry, we studied a variety of surface receptors on lymphocyte subpopulations of workers with moderately increased body burdens of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and of other polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/PCDF), expressed here as International-Toxicity Equivalencies (I-TE). The hypothesis to be tested was whether or not humans exhibit a similar susceptibility to PCDDs/PCDFs with respect to the surface receptors found previously to respond to small doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in Callithrix jacchus. These are: helper-inducer (memory) T cells (CD4+CD45R0+CD45RA-CD29highCD11a+), CD20+ B cells, and cytotoxic T cells (CD8+CD56+/CD57+). Furthermore, 68 triple-labellings with mAbs were performed on the cells of each volunteer to possibly generate further hypotheses. It was evaluated whether any of the variables might be used as a biomarker of effects for this class of compounds. There were two main goals: (1) to evaluate whether workers with a moderately increased PCDD/PCDF-body burden [25-140 ppt TCDD or 104-522 ppt I-TE in blood fat] exhibit changes in the surface receptors of white blood cells, as observed in previous studies in non-human primates, and (2) to clarify whether persons at the upper range [10-23 ppt TCDD or 30-90 ppt I-TE in blood fat] of the body burden reference values of a not particularly exposed population show detectable deviations in these immunological variables, when compared with persons at the lower and medium range [1-3 ppt TCDD or 9-29 ppt I-TE] of these body burden reference values. Regression analysis of our data revealed slight trends for some of the biomarkers (e.g. CD45R0+). With one exception, these were all increases. None of the alterations observed are of medical relevance. The slight increase in the percentage of CD4+CD45R0+ cells remained significant even after covariant analysis taking age-related changes into account. Altogether, the data do not provide any evidence to support an assumption that moderately increased body burdens of PCDDs/PCDFs in adults induce decreases in the cellular components of the human immune system. Adult humans certainly are less susceptible to this action of PCDDs/PCDFs than adolescent Callithrix jacchus.

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.

Chlorinated dibenzo-p-dioxins and dibenzofurans and the human immune system: 3. Plasma immunoglobulins and cytokines of workers with quantified moderately-increased body burdens

The concentrations of immunoglobulins (IgA, IgD, IgG, IgM) and of several cytokines were measured in the plasma of volunteers with clearly, but moderately, increased body burdens of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/PCDF), using monoclonal antibodies and an enzyme-linked immuno-sorbant assay. Two groups of workers with different body burdens of PCDD/PCDF were studied: (trial I) persons with mainly 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and (trial II) persons with mainly penta- and hexachlorinated dibenzofurans (P5CDF/H6CDF) in their blood fat. Including the reference group, 158 volunteers were investigated. A slight but statistically significant decrease was observed in the plasma concentration of IgG1 in persons exposed to TCDD, but not in persons exposed to P5CDF/H6CDF. When the data of both groups were pooled and a multi-regression analysis against international TCDD toxicity equivalencies (I-TEq, NATO/CCMS) was performed, taking several confounding factors into account, no influence of the dioxin exposure could be revealed. There were no changes in the plasma concentrations of the other immunoglobulins studied. In the same volunteers, no deviation from the reference range was found for the concentrations of the cytokines: IL-1alpha, IL-1beta, IL-6 and TNFalpha in blood plasma.

Expression of lactate dehydrogenase isozyme 5 (LDH-5) in cultured mouse blastocysts in the absence of implantation and outgrowth

Extensive extraction studies with Triton X-100 revealed only LDH-1 (B4) but no trace of LDH-5 (A4) in one-cell and two-cell mouse and rat embryos. The LDH isozyme pattern of preimplantation mouse embryos changes from the maternally inherited B subunit isozyme (LDH-1) to a pattern dominated by LDH-5 when mouse blastocysts are cultured under conditions that prevent hatching but allow trophoblast giant cell transformation. During differentiation of mouse blastocysts in vitro, implantation is therefore not essential for the appearance of the A subunit form of LDH (LDH-5) coded for by the embryonic genome. Mechanisms controlling the expression of LDH-5 in mouse blastocysts during in vivo development are discussed.

Some Data on the Pattern of Lymphocyte Subsets in Blood During the Perinatal Period

As a prerequisite for studies of possible effects of prenatal exposure on lymphocytes in newborn, the knowledge of peculiarities of lymphocyte patterns in the blood of normal, unexposed newborn is essential. In our special case, information on both the human blood and the experimental model to be used is necessary.