Charles F. Zukoski

The Effect of Zinc and Other Metals on the Stability of Lysosomes

Summary The effects of zinc, certain other metals (Cu, Hg, Pb, Cd, Ni and Au), 8-hydroxyquinoline (8-HQ), and various stoichiometric complexes of Zn:8-HQ on the release of β-glucuronidase from lysosomes isolated from rat liver were tested. Zinc and Zn:8-HQ (1:1 and 1:2 complexes) markedly stabilized lysosomes in a concentration-related fashion. The saturated 1:3 complex and 8-HQ alone showed a labilizing effect, while the nonchelating substitute of 8-HQ (8-methoxyquinoline) did not affect lysosomal stability. Cd and Pb also stabilized lysosomes, although they were both less effective than zinc. The distribution of zinc between the membrane and the interior of isolated liver lysosomes is 2:1. After treatment with zinc, the distribution is unchanged but the total content of zinc increased 10-fold. In samples treated with unsaturated complexes of Zn:8-HQ (1:1 or 1:2) the total amount of zinc present is the same as in zinc-treated particles, but the metal is bound exclusively to the membrane fraction. The effect of pH on lysosomal fragility in the presence of zinc was almost opposite to that observed when calcium was tested. The results of this study favor the concept that the stabilizing effect of zinc occurs at the surface of the membrane. The effect does not seem to be related to the function of known phospholipases.

Effect of zinc on lipid peroxidation in liver microsomes and mitochondria.

While studying the role of certain metals and chelating agents in controlling tissue injury, we found that zinc salts stabilize lysosomal membranes in vitro (1, 2): the rupture of these particles with the resulting release of their enzymes was decreased roughly 50% by low (millimolar) concentrations of zinc. The lysis of cells or subcellular structures is frequently attributed to peroxidation of the lipid components of their membranes (3-5). Thus, one possible explanation of this effect of zinc is that it interferes in some way with peroxidation of unsaturated fatty acids in the lysosomal membrane. This hypothesis was evaluated in in vivo and in vitro experiments using carbon tetrachloride to induce lipid peroxidation. The toxic effects of CCl4 in vivo are due to the metabolism of this agent by the liver microsomal drug oxidizing system to the trichloromethyl radical CCl3 (6). This free radical attacks unsaturated lipids in intracellular membranes, oxidizing them and causing membrane distortion. This process terminates in cell necrosis (7). Lipid peroxidation can also be induced in vitro by incubating isolated liver microsomes with CCl4 in the presence of NADPH or a NADPH-generating system (8). We have found that zinc prevents or significantly reduces the CCl4-induced formation of lipid peroxides both in vivo and in vitro. Methods. Male rats (Simonson, 200 ± 15 g) fed standard Purina lab chow diet were injected sc twice weekly with CCl4 at the dose 0.1 ml/100 g. Zinc acetate (5 mg/100 g) was administered daily by intragastric gavage. Control rats were gavaged accordingly with saline. Rats were sacrificed by decapitation after 20 and 34 days. The livers were perfused in situ with 20 ml of ice cold saline and homogenized by hand with all-glass homogenizers in three volumes of Tris-KCl buffer (0.05 M, pH 7.4).

Didemnin B: a new immunosuppressive cyclic peptide with potent activity in vitro and in vivo.

Didemnin B (DB) is a 7-amino-acid, cyclic polypeptide with potent (10−7-10−10M) antiproliferative effects in vivo and in vitro against a variety of viruses and tumor cell lines. Because lymphocyte blastogenesis is essential for many immune responses, DB appeared likely to exert immunosuppressive effects as well. Using primary cultures of murine (Balb/c) splenic mononuclear cells to evaluate this possibility, we found that DB was a potent (IC50=190 ng/ml) inhibitor of lymphocyte protein synthesis, although RNA synthesis and cell viability were unaffected. However, it markedly inhibited blastogenesis stimulated by concanavalin A (IC50=50 pg/ml), lipopolysaccharide (IC50=<100 pg/ml) and alloantigen (IC50=<10 pg/ml) when added to cultures immediately after stimulation. DB added later, at the time of thymidine labeling was much less potent (1/46–1/1430), suggesting that the lymphocyte activation process is particularly sensitive to this agent. Our finding that alloantigen-driven proliferation was exquisitely sensitive to DB (>90% inhibition at 10 pg/ml) led us to test its effects in vivo using the Simonsen parental-to-F1 graft-versus-host reaction (GVHR). Treatment of graft recipients with 0.05, 0.10, and 0.20 mg DB/kg/day for 7 days produced 51%, 40%, and 60% inhibition of splenomegally induced by the GVHR, and treatment with 0.3 mg/ kg/day on days 1, 2, 4, and 6 inhibited 71%. These data show that the in vitro inhibition of alloantigen-driven blastogenesis by DB was reproduced by in vivo treatment as well, even across major histocompatibility differences. This leads us to conclude that DB has potent immunosuppressive activity both in vitro and in vivo.

Effect of Zinc on the Viability and Phagocytic Capacity of Peritoneal Macrophages

Summary Peritoneal macrophages ioslated from mice treated for four days with low doses of zinc showed higher viability when exposed to the toxic effect of silica particles (1 μ). By pretreatment with high doses of zinc, the cytotoxic effect of silica was increased. The protective effect of low doses of zinc was not found to be related to the interaction of zinc with silica particles and is thought to lie within the cell. Some functions of macrophages, screened by determination of phagocytosis of Staphylococcus albus and rate of phagocytosis, were inhibited in mice treated with both low as well as high doses of zinc. The concentration of zinc in macrophages, and also in platelets and lymphocytes, increased by a factor of 5-10 when the cells were incubated in medium containing zinc. Zinc accumulation in these cells was pH dependent, but was not pH dependent in the mitochondrial-lysosomal fraction.

PATHOPHYSIOLOGY OF ZINC

Publisher Summary This chapter discusses the pathophysiology of zinc. The stability of various biological macromolecules is dependent on or increased by the presence of zinc. Zinc is involved in the polymerization process, and it is the zinc-insulin complex that is stored in granules. Zinc also binds to DNA nucleoside bases and can be used in vitro to reversibly wind and unwind DNA double helices with heating and cooling. Zinc binds quite weakly to complementary bases so that when the DNA is cooled, the double helix can form again in both directions from the crosslink. Zinc ions bind strongly enough to phosphate to stabilize the double helix at lower temperatures. Zinc inhibits electron transport in the mitochondrial respiratory chain at very low concentrations, which suggests a rather effective and specific role of this metal in the control of respiration even in vivo. The membrane of cells and subcellular particles contain lipoprotein glycosaminoglycan macromolecules, which are either integral parts of this structure or are closely associated with the membrane.

Didemnin B - An immunosuppressive cyclic peptide that stimulates murine hemagglutinating antibody responses and induces leukocytosis in vivo

Didemnin B (DB) is a cyclic peptide with potent immunosuppressive activity in vitro and in the murine graft-versus-host-reaction (GVHR), the only measure of in vivo immunity tested in our prior studies. Because continued production of mature leukocytes by bone marrow and an intact antibody response are crucial to defense against infection in immunosuppressed patients, we have evaluated the effects of DB on these processes as well. Anti-sheep red blood cell (SRBC) hemagglutinating antibody (hAb) production was induced by i.p. injection of 5×107 SRBC in CB6F1, mice (5/group) treated with vehicle or DB once/day for six days. Serum was collected on day 7 and hAb titers measured by SRBC agglutination. Control antibody titers were 1/16, while animals receiving DB doses of 0.025, 0.05, 0.10, and 0.20 mg/kg/day yielded titers of 1/37, 1/74, 1/56, and 1/74, respectively. This stimulation of hAb production (4.6 × control) was confirmed by a second experiment. We then studied DB effects (0.1 mg/kg/day x 6 days) on serum hAb titers in separate groups of five mice at 7, 10, 15, and 20 days postimmunization. Control hAb titers were 1/110 on day 7, then dropped to 1/60 on days 10, 15, and 20. DB-treated animals had titers of 1/130 on day 7, and 1/170 on days 10–20. These data show hat DB treatment in vivo causes a persisting increase

In- Vitro Hepatotoxicity of Three Dichlorobenzene Isomers in Human Liver Slices

1 The cytotoxicity of dichlorobenzenes in cultured rat liver slices has previously been shown to be strain specific and biotransformation related. 2 In order to extrapolate animal models to humans, the dichlorobenzenes were incubated with human liver slices to try to clarify their hepatotoxic potential in man. 3 The degree of hepatotoxicity observed with the dichlorobenzenes depended on whether Waymouths or Krebs-Henseleit was used as the incubation medium. 4 All three dichlorobenzenes (1 mM) produced no significant differences from control when incubated in Waymouths medium. However, in the Krebs-Henseleit buffer there was a substantial increase in cytotoxicity. 5 In both incubation mediums the dichlorobenzene isomers exhibited the following rank order 1,3-DCB > 1,2-DCB > 1,4-DCB. 6 1,2-dichlorobenzene hepatotoxicity was blocked by metyrapone, 1,3-dichlorobenzene toxicity was blocked by SKF 525-A and neither one of these inhibitors could block the 1,4-dichlorobenzene cytotoxicity. 7 The use of human liver tissues to evaluate potential toxicants merits consideration since the hepatotoxicity of xenobiotics and drugs in man is the ultimate question.

Zinc in Erythrocyte Ghosts

Summary Proteins and phospholipids but not zinc were released into the medium containing hemoglobin-free ghosts from dog erythrocytes incubated in peroxidation-inducing agents (ascorbic acid, Fe2+, O2). The content of zinc in the pure ghost depends on the purification procedure; using phosphate buffer it amounted to 62 ± 22 μg/g protein, and using barbital buffer-extracted ghost it was three times higher. Although most of the zinc was linked to the lipid phase of the membrane, there is a certain protein moiety of membrane proteins binding more zinc than the other protein fractions. Among various fractions of lipids, zinc is linked mainly to phospholipids and this linkage is not broken by phosphate buffer. It is concluded that zinc is part of the erythrocyte ghost, linked to protein as well as lipid phase. This work is supported in part by NIH Research Grants AM 16489, ES 00790, and ES 01570. The professional assistance of Mrs. Waltraud W. Nichols is highly appreciated.

Identification of prolactin receptors in hepatic nuclei

Prolactin is a trophic hormone which may act directly at the hepatocyte nucleus. In this study, specific prolactin binding sites were sought in purified rat liver nuclei. Saturable and specific, high affinity 125I-prolactin binding sites were demonstrated to be on or within the nucleus. Prolactin binding was competitively inhibited by rat and ovine prolactins but not by rat growth hormone. Using immunogold electron microscopy, we detected prolactin receptors throughout the nucleus, in association with heterochromatin. Furthermore, endogenous immunoreactive prolactin was demonstrated to be within hepatic nuclei. We conclude that rat liver nuclei possess prolactin binding sites which likely participate in hormone-directed growth processes.

A specific binding site in Nb2 node lymphoma cells mediates the effects of didemnin B, an immunosuppressive cyclic peptide

Didemnin B (DB) is a cyclic depsipeptide with a variety of biologic effects, including potent antiviral, antitumor, and immunosuppressive activities. Although its mechanism of action has been attributed to inhibition of DNA and protein synthesis, the exact cellular site of interaction has not been previously defined. Since DB is strongly antiproliferative in Nb2 node lymphoma cells, we investigated potential DB binding sites in these cells, using [3H]-DB (2.7 mCi/mg) as the radiolabeled ligand. Time course studies with Nb2 cells showed that steady state [3H]-DB binding was attained after 4 h. Scatchard analysis with resting cells yielded a Kd of 180 nM (200 ng/ml), and 7 x 10(6) binding sites/cell. The IC50 of DB inhibition of ongoing protein and DNA synthesis in Nb2 cells, measured 24 h after prolactin (PRL) stimulation, was also in the range of 100 ng/ml. Didemnin analogs, with alterations at critical amino acid residues, inhibited the synthesis of DNA and protein and competed with [3H]-DB binding with the same rank order of potency. This implies that this binding site may mediate the inhibition of macromolecule synthesis. Subcellular fractionation of [3H]-DB labeled Nb2 cells revealed that specific binding occurred predominantly in the 100,000 g cytosolic fraction. Comparison with cyclophilin and the FK506 binding protein, both cytosolic receptors, suggests that the DB binding site may also belong to the family of immunophilins.