Effect of dosing frequency on ZDV prophylaxis in macaques infected with simian immunodeficiency virus.

The effect of dosing frequency on zidovudine (ZDV) prophylaxis against simian immunodeficiency virus (SIV) infection was examined in long-tailed macaque monkeys (Macaca fascicularis). The results indicate that dosing frequency is extremely important for drug efficacy. The monkeys were divided into three groups based on dosing frequencies of 6-, 8-, or 12-h intervals. All were given a total daily dose of 100 mg/kg of ZDV. The drug was administered subcutaneously starting 24 h before SIV inoculation, and treatment continued for an additional 28 days. With the total daily dose held constant, ZDV was most therapeutic when administered at 12-h intervals, less effective at 8-h intervals, and least effective at 6-h intervals. These results indicate that early ZDV treatment based on infrequent but high dosages may increase the antiretroviral effect of the drug. These findings could serve as a model for ZDV chemoprophylaxis in humans. In cases involving accidental exposure to SIV or human immunodeficiency virus (HIV-1 or HIV-2), immediate, high-dosage therapies may be most therapeutic.

In vitro screening for antiretroviral agents against simian immunodeficiency virus (SIV).

Simian immunodeficiency virus (SIV), which causes an acquired immunodeficiency syndrome in macaques, is a lentivirus that is morphologically, antigenically, genetically, and biologically similar to the human immunodeficiency virus (HIV). Because of these similarities, the SIV model represents a unique opportunity for in vitro and in vivo testing of antiretroviral agents. Since antiretroviral agents may exhibit different properties in different cells in vitro, more than one cell line may be necessary to evaluate the efficacy and modes of action of an antiretroviral agent. Initially we tested ten cell lines for their permissiveness to five SIV isolates. One B-cell line (AA-2) and one T-cell line (HuT 78) were selected to test antiretroviral agents since both were extremely permissive for SIVmac251, an isolate with a high rate of infectivity. Using this optimized in vitro testing protocol, we screened ten antiretroviral agents for their ability to inhibit SIV replication. Six of the compounds completely inhibited SIV viral antigen expression. Based on the selectivity index, 3'-azido-3'-dideoxythymidine, 3'-azido-2',3'-dideoxyuridine, and 3'-fluoro-3'-deoxythymidine appear to be the most efficacious antiretroviral agents against SIVmac251. Several different assays for determining viral antigen inhibition were conducted and the results of these assays were comparable. Our results demonstrate that the SIV in vitro model is a valuable screening tool for determining the efficacy and toxicity of new antiretroviral agents.

Cytoskeletal effects of acrylamide and 2,5-hexanedione: selective aggregation of vimentin filaments.

Several neurotoxic compounds cause aggregation of neurofilaments in peripheral axons. This may represent a primary action of these chemicals or a secondary response to other cellular damage. To distinguish between these possibilities, the effects of acrylamide and 2,5-hexanedione (2,5-HD) on vimentin were examined in PtK2 cultured cells. Vimentin intermediate filaments were chosen because they are closely related, in structure, to neurofilaments. Effects on other components of the cytoskeleton (cytokeratin filaments, microtubules, and microfilaments) were also determined. Both acrylamide and 2,5-HD caused aggregation of vimentin filaments in a concentration-dependent fashion; these effects occurred at a lower concentration than alterations in other cytoskeletal filaments. The effects of both acrylamide and 2,5-HD were reversible, except at high concentrations of 2,5-HD. Crosslinking of cytoskeletal proteins was also examined. High-molecular-weight proteins with vimentin-like immunoreactivity were detected on blots from cells exposed to high concentrations of 2,5-HD. No crosslinked protein was detected after acrylamide treatment. These results suggest that both acrylamide and 2,5-HD cause a primary collapse of vimentin intermediate filaments in cultured cells. The initial redistribution of vimentin filaments occurred without apparent crosslinking of cytoskeletal proteins. The aggregation of vimentin filaments in cultured cells and of neurofilaments in vivo may share a common molecular mechanism.

Selectivity of methyl mercury effects on cytoskeleton and mitotic progression in cultured cells.

Methyl mercury (MeHg) disrupts microtubules, but effects on other cytoskeleton components are not well studied. Dose-effect relationships were used to determine the selectivity of MeHg effects on vimentin intermediate filaments, actin microfilaments, and microtubules. These were examined in PtK2 cells by immunofluorescence. At 0.5 microM MeHg the number of microtubules was reduced. After 1.0 or 2.0 microM MeHg, microtubules in most cells were disassembled except for a few "stable" microtubules. No effects on vimentin or actin filaments were observed except as secondary effects at concentrations of MeHg that caused extensive microtubule disassembly. Antimitotic effects of MeHg are well known. An assay based on fluid pinocytosis was used here to study kinetics of mitotic progression. HeLa cells were arrested at prometaphase with a lengthening of subsequent stages of mitosis. Progression from prophase to prometaphase was not affected. MeHg treatment also increased the number of micronucleated and multinucleated cells. Drugs specific for actin cause similar effects by blocking cytokinesis but the selective action of MeHg on microtubules argues against this mechanism. Data from both interphase and mitotic cultured cells indicate that MeHg acts selectively on microtubules. It further supports the hypothesis that the mechanism of damage of MeHg is related to its antimicrotubule activity.

Mechanisms of neurotoxicity related to selective disruption of microtubules and intermediate filaments.

The neuronal response to several neurotoxic chemicals includes disruption of the cytoskeleton such as interactions with microtubules and altered distribution of neurofilaments. Methylmercury (microtubule disrupting) and acrylamide and 2,5-hexanedione (neurofilament disrupting) have been used in a cell culture (PtK2) system to distinguish the cytoskeletal targets of these compounds. Methylmercury caused disassembly of microtubules with secondary collapse of vimentin filaments (epithelial cell equivalent of neurofilaments) at higher concentrations; actin filaments were unaltered. This confirms that disruption of actin does not contribute to methylmercury-induced interference with mitosis. In contrast, both acrylamide and 2,5-hexanedione caused a perinuclear redistribution of vimentin filaments with sparing of microtubules. Biochemical studies revealed that 2,5-hexanedione treatment resulted in high molecular weight vimentin-immunoreactive species, presumably by cross-linking of proteins. Selective action of both acrylamide and 2,5-hexanedione on vimentin filaments and the similarity of effects suggest that a common mechanism of damage may occur whereby these compounds act directly on both vimentin and neurofilaments.

Biological monitoring of metals with special references to the early stages of the life cycle.

In summary, the biological monitoring of toxic metals in the early stages of the life cycle has not yet received the attention it deserves. Most metals have simply not been studied at all. Some successful applications with mercury, lead, and cadmium have been reviewed here. But even for these widely studied metals, there is a great need to study physiological models for metal disposition in developing animals.

A novel mitotic spindle pole component that originates from the cytoplasm during prophase.

Several unique aspects of mitotic spindle formation have been revealed by investigation of an autoantibody present in the serum of a patient with the CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, schlerodacytyly, and telangiectasias) syndrome. This antibody was previously shown to label at the spindle poles of metaphase and anaphase cells and to be absent from interphase cells. We show here that the serum stained discrete cytoplasmic foci in early prophase cells and only later localized to the spindle poles. The cytoplasmic distribution of the antigen was also seen in nocodazole-arrested cells and prophase cells in populations treated with taxol. In normal and taxol-treated cells, the microtubules appeared to emanate from the cytoplasmic foci and polar stain, and in cells released from nocodazole block, microtubules regrew from antigen-containing centers. This characteristic distribution suggests that the antigen is part of a microtubule organizing center. Thus, we propose that a prophase originating polar antigen functions in spindle pole organization as a coalescing microtubule organizing center that is present only during mitosis. Characterization of the serum showed reactions with multiple proteins at 115, 110, 50, 36, 30, and 28 kD. However, affinity-eluted antibody from the 115/110-kD bands was shown to specifically label the spindle pole and cytosolic foci in prophase cells.

Glycosaminoglycan synthesis is depressed during mitosis and elevated during early G1.

[35S]Sulfate incorporation was measured in populations of Chinese hamster ovary cells enriched for mitotics, early G1 cells, and interphase monolayers or suspensions. Incorporation was determined by biochemical analysis of extracts and quantitative autoradiography of thick sections. 90% of [35S]sulfate was incorporated into glycosaminoglycan (GAG). Incorporation was depressed fourfold in mitotics and stimulated by from two- to three-fold in early G1 cells relative to mixed interphase cells. GAG synthesis was maintained into late G2. Thus, the rate of GAG biosynthesis was correlated temporally with the detachment and reattachment of cells to substrate. Inhibitors of protein synthesis brought about the rapid arrest of GAG biosynthesis. However, xylosides, which bypass the requirement for core protein, did not bring oligosaccharide sulfation in mitotics to interphase levels. These observations indicate an inhibition of Golgi processing and are consistent with a generalized defect of membrane vesicle-mediated transport during mitosis.

Reproductive and developmental toxicity of metals.

This paper discusses metal exposure in the male, the nonpregnant female, and the maternal-offspring unit. In the first two situations, the primary targets are the gonads. In the mother-offspring unit, consideration must be given to effects on the fertilized ovum, the growth of the embryo, and, finally, to the fetal and perinatal stages. The central nervous system may be especially vulnerable during development. The placenta also undergoes development, and either the placenta or the fetus may be the primary target. In humans, certain metals may cause abortion or other effects on the conceptus. Effects may also be produced by metal exposure both in utero and in the suckling infant. For example, methylmercury gives rise to a range of effects on the central nervous system at doses lower than those producing damage to the mature nervous system. Effects of lead and arsenic are associated mainly with postnatal exposures during infancy and early childhood, but there is reason to believe from animal experiments that some effects may occur from prenatal exposures to certain metal compounds.

Analysis of transferrin recycling in mitotic and interphase HeLa cells by quantitative fluorescence microscopy.

Recent findings suggest that membrane vesicle transport during mitosis may be generally inhibited. To test this, we examined the kinetics of uptake and exocytosis of RITC-transferrin in mitotic and interphase HeLa cells. We used quantitative image-intensification fluorescence microscopy to analyze the content of ligands in single cells. This technique was validated by comparison of 3H or RITC-transferrin release from interphase cells determined by microscopy or radiometry. Both methods gave a t1/2 of release of 5-6 min. The uptake of RITC-transferrin was depressed in mitotics. More importantly, we monitored the exocytosis of label during mitosis. Labeled mitotics were obtained by the progression of interphase cells into mitosis during a 50 min incubation with RITC-transferrin. After 30 min chase with unlabeled transferrin, the intensities of interphase cells approached background, whereas those of mitotic cells remained nearly constant. Thus both exocytosis and endocytosis of transferrin were exocytosis and endocytosis of transferrin were blocked during mitosis.

Mitotic arrest in the developing CNS after prenatal exposure to methylmercury.

Methylmercury is toxic to both the mature and the developing nervous system. One mechanism of its effects on the developing neonatal cerebellum is its interference with cell production by mitotic arrest. To investigate whether this mechanism is active in the prenatal CNS, fetuses exposed to methylmercury were compared to control fetuses 24 hours or 48 hours after an 8 mg/kg dose to their dams. By the first sacrifice time, levels of Hg203 in fetuses approached the level in the dam, and by the second sacrifice time methylmercury-exposed fetuses weighed significantly less than controls. Four regions of the developing brain were studied to evaluate methylmercury effects on mitotic activity. General measures such as mitotic index, number of proliferative cells, and thickness of the proliferative zone were not reduced by treatment in any region at either sacrifice time. In contrast, each region showed evidence of methylmercury effects on the pattern of mitosis. Exposed fetuses had increased numbers of early mitotic figures, decreased numbers of late mitotic figures, or a decrease in the proportion of cells reaching late mitosis. Thus, neurons produced during gestation, like those produced postnatally, appear to be sensitive to methylmercury's antimitotic action. Whether the arrest of these cells leads to a permanent reduction in neuron number, as it does in neonates, remains to be investigated.

Differential responses to methylmercury exposure and recovery in neuroblastoma and glioma cells and fibroblasts.

The effects of methylmercury (MeHg) on cytoplasmic microtubules in cultured neuroblastoma cells, glioma cells, and fibroblasts were compared. Neuroblastoma cells appeared to be more sensitive to disruption of microtubules by MeHg than the glioma cells or fibroblasts; cellular concentrations of mercury after MeHg were also higher in neuroblastoma cells. Recovery of microtubule structure was monitored in cells after removal of MeHg; addition of the chelating dimercaptosuccinic acid (DMSA) increased reassembly of microtubules. During MeHg treatment and early recovery, microtubule integrity was dependent on cellular mercury concentrations. However, after prolonged DMSA exposure, mercury appeared to reenter the cell, without causing dissociation of microtubules.

Persistent, differential alterations in developing cerebellar cortex of male and female mice after methylmercury exposure.

Developing animals have long been believed to be more sensitive to methylmercury toxicity than adults, but the reasons for differential effects are not well understood. In the present study, 2-day-old mice received a single per os dose of 4 mg Hg/kg methylmercury and were sacrificed 24 h or 19 days later. This resulted in a mean brain concentration of 1.8 micrograms Hg/g tissue on day 3 and less than 0.1 micrograms Hg/g on day 21. Compared to littermate vehicle controls, the methylmercury-treated mice exhibited a significant reduction in cell numbers in 1 of 4 regions of the developing cerebellar external granular layer 24 h after treatment. Although the mitotic index over the same 4 regions was not significantly altered by methylmercury treatment, the total number of mitotic figures per section of cerebellum was significantly reduced in the treated group. The ratio of late mitotic figures to total mitotic figures was significantly reduced, indicating mitotic arrest. Both of these antimitotic effects were greater in males than females. Cerebellar structure was also examined 19 days after methylmercury treatment. The number of cells in the molecular layer and thickness of the molecular layer and internal granular layer were significantly reduced in males; the number of Purkinje cells in both sexes and all measures in females remained unaltered. This suggests that early cell loss results in persistent reductions in cell number. Although the basis for the differential effect in males and females is not known, the antimitotic effect of methylmercury is most likely the mechanism underlying the reduced cellularity in treated animals.

Interaction of methylmercury with microtubules in cultured cells and in vitro.

The effects of methylmercury (MeHg) on cytoplasmic microtubules in cultured fibroblasts and on the in vitro polymerization of microtubules were examined. MeHg caused disruption of cellular microtubules in a concentration- and time-dependent manner. Addition of the metal-chelating agent, dimercaptosuccinic acid (DMSA), both prevented and reversed the effect of MeHg. Comparisons of the cellular levels of mercury and microtubule integrity indicated that microtubules dissociated at levels higher than 0.6 microgram Hg/mg protein. In vitro polymerization was also directly inhibited by MeHg; this effect was prevented by the addition of DMSA.