Stage-specific effects of defined mixtures of polychlorinated biphenyls on in vitro development of rabbit preimplantation embryos.

PCBs adversely affect various reproductive functions. Little is known about the embryo- toxic effects during the preimplantation period in mammals. In the present study the effects of various mixtures of highly purified PCB-congeners on embryo morphology, blastocyst formation, embryo size and cell proliferation were investigated. For 24 hr, day 3 morulae and day 4 blastocysts were cultured in the presence/absence of coplanar congeners (PCB77, PCB126, and PCB169), non-coplanar congeners (PCB28, PCB52, PCB101, PCB118, PCB138, PCB153, PCB180) or both mixtures in concentrations ranging from 0.3 ng/mL to 60 microg/mL total PCB. The main effects were (1) degeneration of all embryos at 60 microg/mL, (2) reduction of cell proliferation in day 4 embryos only by coplanar PCB; in day 3 embryos, however, by all PCB-mixtures, and (3) reduction of cell proliferation in a non-linear dose response with the strongest impairment caused by the lowest concentration. Cell proliferation was decreased by 0.3 ng/mL coplanar PCB to 50% of the level in control blastocysts. Our results show that purified PCB congeners in the range of 0.3 ng/mL to 30 microg/mL affect the development of preimplantation embryos in a stage-specific and congener-specific manner. This study provides first evidence for an embryotoxic potential of coplanar PCB congeners.

Intranuclear distribution of 2,2',4,4',5,5'-hexachlorobiphenyl and 2,3,7,8,-tetrachlorodibenzo-p-dioxin.

The in vitro intranuclear distribution of the chlorinated aromatic hydrocarbon compounds 2,2',4,4',5,5'-hexachlorobiphenyl and 2,3,7,8-tetrachlorodibenzo-p-dioxin was determined in isolated rat liver cell nuclei. Suspended nuclei were incubated with the 3H-labeled congeners, and the incubation terminated by brief UV irradiation. High-intensity UV irradiation at 254 nm changes the reversible association between macromolecules and ligands into covalent linkages and thus stabilizes the equilibrium distribution. The nuclei were then fractionated with the radioactive congeners covalently linked to the purified fractions. The intranuclear distribution of the model compounds was not uniform. The majority of either chemical was attached to the nuclear envelope and to the chromatin fraction. Much lower amounts were bound to nucleoli. The nuclear matrix was almost devoid of the chemicals. Minute amounts of either compound were detected in association with DNA, none with nuclear RNA. The substantial association of these chlorinated hydrocarbon model compounds with chromatin may bear more general biological relevance and point to detrimental effects on the genetic apparatus. The presented method yields an unequivocal profile of the genuine nuclear distribution of photoactivatable chemicals.

Partial purification and characterization of a rat liver polychlorinated biphenyl (PCB) binding protein.

A protein capable of specifically binding polychlorinated biphenyls (PCB) was partially purified from rat liver cytosol. After labeling with [3H]2,2',4,4',5,5'-hexachlorobiphenyl (6-CB), protein enrichment was guided by monitoring the protein-bound radioactivity through a sequence of purification steps including ion exchange chromatography and preparative gel electrophoresis. In addition, specific binding tests of individual fractions were carried out. An average 100-fold enrichment of the 40 kDa protein was achieved. A variety of ligands were tested in competitive binding studies with 6-CB. Whereas penta- and hexachloro-PCB congeners are high affinity competitors, the 3,3',4,4'-tetrachlorobiphenyl congener does not compete for 6-CB binding. Studies on the species and tissue distribution suggest a prevalence of the binding protein in tissues of the rat. Since the natural physiological ligand of the protein has not yet been identified, the function of the protein can only be speculated on.

Effect of Endosulfan on Azospirillum lipoferum Growth, Morphology, Nitrogenase Activity, and Protein Binding.

The organochlorine Thiodan CE inhibited growth and nitrogenase activity of Azospirillum lipoferum. The active ingredient, Endosulfan, was nonspecifically bound to proteins and mainly adsorbed to the cell envelope with small amounts transported into cytosol. The involvement of the external membrane and cyst formation in protection against hazardous substances is discussed.

Exposure of HIV-infected cells to phospholipid leads to membrane alterations and selective growth retardation.

The effect of exogenous phosphatidylcholine on structure and function of plasma membranes from HIV-1-producing cells and from their non-infected counterparts was determined. The membrane protein composition was not affected by phospholipid treatment. Membrane fluidity and Ca(2+)-permeability were increased in virus-producing cells and in control cells after lipid treatment. The triacylglycerol content of the plasma membranes was increased in virus-producing cells after lipid treatment, whereas the content of phospholipid and cholesterol was not changed. The increased triacylglycerol content was in accordance with a relatively higher rate of [14C]oleic acid incorporation into triacylglycerols of the virus-producing cells after lipid treatment as shown by metabolic labeling. The results suggest that a latent cytopathic effect of HIV-infection becomes manifest if the cells are exposed to exogenous phospholipid and this may open a way to preferentially eliminate HIV-producing cells.

Specific binding of polychlorinated biphenyls to rat liver cytosol protein.

Specific binding of polychlorinated biphenyls (PCBs) to rat liver cytosol protein has been detected using the 3H-labeled PCB probe 2,2',4,4',5,5'-hexachlorobiphenyl (6-CB). Binding of 6-CB to cytosol protein is displaced by its non-radioactive congener, is of high affinity (Kd approximately 3 nM) and is saturable (maximal binding capacity Bmax approximately 600 pmol/mg protein). 6-CB binding is not found in liver cytosol of animals fed a PCB-supplemented diet (500 ppm PCB for 5 days). Binding is also in vitro inhibited by high concentrations of triglyceride. PCB congeners such as 3,3',4,4',5-pentachlorobiphenyl as well as the thyroid hormones 3,5,3',5'-tetraiodothyronine and 3,5,3'-triiodothyronine (the latter hormone with an order of magnitude lower affinity) compete for the PCB binding site. On the other hand, a number of biochemically important compounds including the PCB core compound biphenyl and the hormone ligands dexamethasone and estradiol, as well as 2,3,7,8-tetrachlorodibenzo-p-dioxin, do not compete for the 6-CB binding site. The data provide the first evidence of specific binding of unmetabolized PCB congeners to distinct binding sites in rat liver cytosol.

Growth inhibition of HIV-1-infected cells and membrane alterations induced by phosphatidylcholine.

Cultured T-lymphoma cell lines H9 and KE37-1 permanently infected with human immunodeficiency virus (HIV-1, strain HTLV-III B) were exposed to phosphatidylcholine (PC) and a PC-containing formulation "Essentiale" (PC-E). PC and PC-E, but not triglyceride, were found to inhibit growth of virus-infected cells. Additionally, the membrane lipid composition of infected and uninfected H9 cells was investigated upon exposure to PC. The HIV-1-infected cells showed a 25% increase in membrane triglyceride content and a 15% increase in membrane phospholipid saturated fatty acids. In the presence of PC, there is a further increase in triglyceride content up to 180% compared with uninfected control cells, suggesting a possible cause for the selective growth inhibition of HIV-1-infected cells by PC. The PC-E dose range effective in vitro for inhibition of HIV-1-infected cell growth falls within the range that can be reached in vivo. Formulations containing PC are well tolerated by humans and might be applicable at an early stage of HIV-1 infection to reduce the number of virus-producing cells.

Photo-induced formation of DNA adducts of 2,2',4,4',5,5'-hexachlorobiphenyl in cultured human cells.

Association of the PCB congener 2,2',4,4',5,5'-hexachlorobiphenyl (6-CB) with cell nuclei has been studied in cultured monolayer human Chang liver cells. Photo-induced formation of covalent bonds determined 6-CB binding to protein of cell nuclei and to DNA. Nuclear binding of 6-CB approached equilibrium after approximately 30 min of incubation. Photo-induced binding in vitro to purified Chang liver cell DNA substantiated direct interaction of the PCB congener with DNA. In monolayer cells, low levels of photo-induced 6-CB DNA adducts could be detected using the very sensitive 32P-postlabeling method. Adduct formation was dependent on 6-CB concentration as well as on incubation time. Highest adduct levels were in the range of 2 X 10(-8). Model reactions in vitro showed photo-induced binding of 6-CB to individual purine deoxyribonucleotide-3'-phosphates. The results demonstrate rapid intracellular movement of the PCB congener into the cell nucleus. The vast majority is associated with nuclear protein, minute amounts of 6-CB are found proximate to the DNA helix as evidenced by photo-induced adducts of purine nucleotides.

Photo-induced binding of 2,2',4,4',5,5'-hexachlorobiphenyl to cultured human cells.

The polychlorinated biphenyl congener 2,2',4,4',5,5'-hexachlorobiphenyl can be photoactivated by brief high-intensity ultraviolet irradiation. Photoactivated intermediates are bound to neighboring biological macromolecules. Properties and stability of hexachlorobiphenyl photobinding were examined with bovine serum albumin, a protein known to strongly bind lipophilic compounds. Photobinding to cultured human Chang liver cells was a function of ligand and cell protein concentration as well as of irradiation time. Binding increased with incubation time, in support of the time course of uptake previously measured in the same system by alternative methods. Separation of cell proteins by gel electrophoresis showed that the distribution pattern of photobinding changed at different rates for different proteins. Photobinding to major cell lipid groups and to individual phospholipids likewise reflected uptake of the compound. Notably, photobinding to phosphatidyl choline was elevated relative to phosphatidyl ethanolamine. Thus, the presented method is suitable to follow up transport and intracellular equilibrium distribution of photoactivatable ligands. As a particular advantage, artefactual redistribution of persistent lipophilic compounds during cell fractionation can be avoided.

Uptake of persistent environmental chemicals by cultured human cells.

Uptake of the persistent environmental chemicals 2,2',4,4',5,5'-hexachlorobiphenyl and 1,1,1-trichloro-2,2-di-(4-chlorophenyl)ethane (the insecticide DDT) by Chang liver cells, an established human cell line, has been investigated. Monolayer cells were incubated with culture medium to which the lipophilic model compounds had been added. The time course of uptake of either compound was biphasic, reaching equilibrium after about 5 hr of incubation. The ratio of DDT:hexachlorobiphenyl uptake was dependent on the presence of serum proteins. Increasing concentrations of serum proteins in the culture medium progressively inhibited uptake. Efflux from the cells was not entirely reversible: 10-20% of the chemicals were not released. Uptake was a linear function of the external concentration of the compounds. Absorptive binding to the outer cell plasma membrane could be determined by removing bound chemicals with fetal calf serum ("back exchange"). With this method, temperature-dependent translocation through the cell plasma membrane could directly be demonstrated. The effect of low temperature as well as the influence of metabolic inhibitors point out the contribution of energy-driven uptake pathways. Demonstration of LDL receptor-like binding protein on Chang liver cells facilitated estimation of the role of receptor-mediated uptake. This route of uptake proved to be of minor importance only, as was transport of the protein-bound chemicals via fluid pinocytosis. The results demonstrate that cellular endocytosis of plasma membrane-bound chemicals constitutes a major uptake pathway for lipophilic chemicals.

The insecticide DDT decreases membrane potential and cell input resistance of cultured human liver cells.

The resting membrane potential, Em, and the cell input resistance, Rinp, of cultured human Chang liver cells were measured using the single electrode 'double-pulse' current clamp technique, following exposure of the cells to the insecticide DDT (20 microM). In control (unexposed) cells, the mean Em was -24 mV, and the mean Rinp was 30 M omega. Neither parameter was significantly impaired after 1 h of cell exposure to DDT. But after 7 and 48 h, the Em was depolarized by 15 and 25 mV, respectively, in parallel with a decrease of the cell input resistance. The strongly time-delayed effect of DDT on Chang liver cell membranes may indicate a mode of interaction different from excitable membranes.

Interaction of the insecticide 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane with the beta-receptor adenylate cyclase system in Chang liver cell membranes.

Interaction of the insecticide 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)-ethane (DDT) with beta-receptor binding and adenylate cyclase activity of biological membranes has been studied. Following exposure of cultured Chang liver cells to DDT, maximal binding of the catecholamine antagonist [125I]-iodohydroxybenzylpindolol (HYP) to isolated cell membranes was decreased by 30% whereas the dissociation constant remained unchanged. Both basal activity and maximal isoproterenol-stimulated activity of adenylate cyclase were not altered. The isoproterenol concentration required for half-maximal stimulation of the enzyme was increased about 2-fold as was the agonist concentration required for half-maximal displacement of the antagonist HYP at the beta-receptor binding site. Thus, coupling efficiency of hormone-stimulated adenylate cyclase activity was not influenced by the presence of DDT in these membranes. The data show that interaction of DDT with the beta-receptor adenylate cyclase complex is restricted to the receptor component. Enzyme activity is directly linked to changes of agonist binding at the beta-receptor. Interference of DDT with signal transduction via 'fluidization' of membrane lipids has not been detected.

Differential effects of environmental chemicals on liposomal bilayers. Fluorescence polarization and pesticide-lipid association studies.

The thermal dependence of the fluorescence polarization of 1,6-diphenylhexatriene was recorded upon interaction of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) and some other pesticides with dipalmitoylphosphatidylcholine liposomes. Differential effects on the gel-crystalline phase were observed. Most substances decreased probe polarization; pentachlorophenol caused an increase of this parameter. The DDT-induced change of polarization was also dependent on the vesicle concentration thus indicating the influence of light scattering. The amount of DDT and pentachlorophenol residing in the lipid bilayer was determined to confirm the localization in the membrane. Correlation with the effects on probe polarization was obtained. The difference in response of the fluorescent probe to the presence of foreign molecules in the lipid bilayer may reflect different modes of interaction.

Interaction of DDT (1,1,1-trichloro-2,2-bis(p-chlorophenyl)-ethane with liposomal phospholipids.

The influence of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) and several other pesticides on the physical state of membrane phospholipids was investigated using model lipids. The thermal dependence of fluorescence intensity of the probe parinaric acid in dipalmitoylphosphatidylcholine liposomes and lipid vesicles of mixed composition were recorded. DDT was incorporated into the liposomal bilayer. The insecticide lowered the phase transition temperature and broadened the temperature range of the transition. The effects were concentration-dependent. The results may be interpreted as a sort of blurred and facilitated phase transition of bilayer lipids caused by intercalation of DDT between fatty acyl chains of membrane phospholipids.

Specificity and binding capacity of human blood serum for tetrahydropterins.

Binding behaviour of labelled biopterin, 6,7-dimethylpterin, 2,4-diamino-6,7-dimethylpteridin, and of their 5,6,7,8-tetrahydro derivatives was studied with human serum proteins. Binding capacity of serum proteins is in the range of 5 x 10-10--10-3 M without saturation for all these pteridines. Preincubation or competition with tetrahydrobiopterin does not influence binding of tetrahydro-6,7-dimethylpterin in these concentrations. The bulk of pteridines is bound through unspecific adsorption. Only at concentrations of less than 0.8 x 10-8 M does high affinity binding seem to be possible, corresponding to about 300 pmol/g protein, whereas physiological biopterin concentration is near 2 x 10-8 M. The binding proteins are very sensitive to ageing and lose their capacity during purification, whereas unspecific binding to serum proteins is only weakly influenced by alteration of salt concentration, pH, or temperature. Attempts to partially purify the binding proteins by ion exchange, dextran gel (Sephadex G-200), or affinity chromatography, demonstrate a specificity of tetrahydropterins for the alpha2-macroglobulin fraction. Due to the high lability of this protein fraction and of pteridine binding, purification of a protein which specifically binds tetrahydrobiopterin was not achieved.