Hydrogen peroxide-mediated, lysyl oxidase-dependent chemotaxis of vascular smooth muscle cells.

Lysyl oxidase (LO), an enzyme secreted by vascular smooth muscle cells (VSMC), initiates the covalent crosslinking of polypeptide chains within collagen and elastin. The present study reveals that purified LO strongly induces directional migration of VSMC in an in vitro assay system. LO-dependent chemotaxis, but not chemokinesis, was abolished by beta-aminopropionitrile, an active site inhibitor of LO, or by catalase, as well as by prior heat denaturation. This indicates that the H(2)O(2) product of amine oxidation by LO is critical to the expression of its chemotactic activity. The results indicate that the chemotactic response requires direct access between LO and a substrate molecule (or molecules) tightly associated with the VSMC. The addition of LO to VSMC elevated the levels of intracellular H(2)O(2), enhanced stress fiber formation, and focal adhesion assembly, is consistent with the induction of the chemotactic response.

Nickel (Ni2+) enhancement of alpha-tubulin acetylation in cultured 3T3 cells.

Stable microtubules (MTs) are well known to contain acetylated alpha-tubulin. Ni2+-induced MT bundling may be accompanied with such tubulin post-translational modification. To explore this possibility as a mechanism of Ni2+-induced cytoskeletal injury, we have examined acetylated alpha-tubulin levels in cultured 3T3 cells by both immunoprecipitation assays and fluorescent staining of MTs using a monoclonal antibody (clone 6-11B-1) specific for acetylated artubulin. Cell extracts prepared from [35S]methionine labeled cultures in the presence or absence of Ni2+ were immunoprecipitated and analyzed by SDS-PAGE followed by autoradiography. A predominant protein band (molecular mass 55 kDa), representing acetylated alpha-tubulin, appeared in Ni2+-treated cells in a dose and time-dependent manner whereas the corresponding labeled protein band was only barely detectable in control cells. Consistent with the immunoblot findings, MTs in control 3T3 cells in the absence of Ni2+ were not labeled by the 6-11B-1 antibody except for some short, discontinuous segments localized in the cell center or the perinuclear MT organizing center area. In contrast, treatment of cells with NiCl2 (2 mM for 20 hr) resulted in, as expected, the formation of MT bundles that were intensely stained by the 6-11B-1 monoclonal antibody specific for acetylated alpha-tubulin. Furthermore, MTs containing acetylated alpha-tubulin in Ni2+-treated cells were resistant to disassembly induced by nocodazole, and at least partially resistant to cold temperature (0 degrees C), which also depolymerizes MTs. Since acetylated alpha-tubulin serves as a marker for the presence of stable MTs, the marked enhancement of alpha-tubulin acetylation in Ni2+-treated cells indicates that stabilization of MTs may be an important mechanism by which Ni2+ induces cell injury since stabilized MTs in turn should favor MT bundling, an unusual form of cytoskeletal perturbation in response to Ni2+ exposure.

Mg2+ antagonism on Ni(2+)-induced changes in microtubule assembly and cellular thiol homeostasis.

As an essential metal for cell metabolism, Mg2+ is known to exert antagonism on Ni2+ genotoxic and other effects. This study examined the influence of Mg2+ on Ni(2+)-induced changes in microtubule (MT) assembly in vitro, cytoplasmic MT organization, cellular glutathione (GSH), and cytoskeletal and cytosolic protein sulfhydryls (PSH). As determined by a turbidity assay at 27 degrees C, Ni2+ enhanced the in vitro MT assembly in a Pipes buffer by shortening the initial lag (nucleation phase) and increasing the rate of polymerization with a higher final plateau. However, presence of 1 mM exogenous MgCl2 abolished the Ni2+ enhancing effect. Exposure of 3T3 cells to 2 mM NiCl2 for 20 hr resulted in perinuclear bundling of MTs and decreases in cytoskeletal and cytosolic PSH and cellular GSH levels. However, coincubation of cells with MgCl2 (1.25-20 mM) added to the culture medium markedly diminished the Ni2+ injury to MT organization. Under these conditions the Ni2+ interference with PSH was blocked such that both the cytoskeletal and cytosolic PSH levels returned to the range of control cells without metal treatment. Treatment of cells with Mg2+ (1.25-5 mM) for 20 hr slightly increased, while with higher Mg2+ doses (> 10 mM) decreased, cellular GSH content. Importantly, in Ni(2+)-treated cultures, addition of Mg2+ (1.25-10 mM) elevated GSH levels to > or = 200% of that in cells treated with Ni2+ alone. Furthermore, these Ni2+ and Mg2+ (1.25-10 mM) treated cells actually maintained GSH levels which were essentially unchanged from the basal level of control cells with no metal treatment. Although Mg2+ replacement of Ni2+ bound to MT proteins could be an important mechanism, cellular GSH may also be a critical factor in Mg2+ antagonism on Ni(2+)-enhanced MT assembly in view of the essential role of tubulin PSH in modulating MT assembly and, in turn, the GSH modulation of PSH.

Downregulation of lysyl oxidase in cadmium-resistant fibroblasts.

Lysyl oxidase, a copper-dependent metalloenzyme, plays a central role in crosslinking of collagen and elastin in the extracellular matrix. Notably, lung lysyl oxidase activity is markedly stimulated in rats exposed to cadmium vapors. To further understand the mechanism of cadmium toxicity, the mRNA expression, synthesis, post-translational processing, and catalytic activity of lysyl oxidase were examined in cadmium-resistant (CdR) cells and the cadmium-sensitive Swiss mouse 3T3 cells from which they were derived. These CdR cells synthesized and accumulated markedly elevated levels of metallothionein, a known marker for cadmium resistance, whereas the expression of lysyl oxidase was reduced considerably. In comparison to the parental, cadmium-sensitive cells, the suppression of enzyme production in the CdR cells was seen at the mRNA level, at the levels of intracellular proprotein production and mature enzyme secreted into the medium, and in terms of total enzyme activity in the culture. The presence of cupric chloride in the culture medium during the incubation of the CdR cells for 16 h significantly enhanced lysyl oxidase activity accumulating in the medium, suggesting that lysyl oxidase deficiency in CdR cells may be related to abnormal copper metabolism.

Alterations in cytoskeletal organization and homeostasis of cellular thiols in cadmium-resistant cells.

To understand further the mechanisms of cadmium toxicity, cytoskeletal organization and homeostasis of cellular thiols were examined in cadmium-resistant cells isolated from Swiss mouse 3T3 cells by incubation in graded concentrations of CdCl2 (Cd2+) in the culture medium. Cd(2+)-resistant cells displayed profound alterations in their cytoskeletal organization characterized by the appearance of many elongated, tadpole-shaped cells with a high density of microtubules (MT) and microfilaments (MF), with the former being mainly distributed along the long axis of the cell. Exposure of Cd(2+)-resistant cells to 50 microM Cd2+ for 16 hr did not cause apparent cytoskeletal perturbations, whereas treatment of parental cells with 5 microM Cd2+ for the same duration produced a severe loss of MT and smeared patches of MF. Thus, the cytoskeleton of Cd(2+)-resistant cells is markedly more preserved and protected against Cd2+ damage than that of their parental counterparts. Cd(2+)-resistant cells contained a higher basal level of protein sulfhydryls (PSH) in both the cytoskeletal and cytosolic fractions than the parental cells. Exposure to 50 microM Cd2+ further increased cellular PSH contents, reaching 192 and 215% of the basal levels for the cytoskeletal and cytosolic fractions, respectively. Although 5 microM Cd2+ exposure also elevated the amounts of PSH in parental cells, the "absolute" values were still below the corresponding basal levels in Cd(2+)-resistant cells. Furthermore, Cd(2+)-resistant cells also exhibited enhanced basal levels of metallothionein and cellular glutathione (GSH), amounting to 19- and 2.1-fold of the parental basal levels, respectively. Thus, the Cd(2+)-resistant cells produced larger quantities of both protein and nonprotein thiol-containing elements than the parental cells. Interestingly, exposure of Cd(2+)-resistant cells to 50 microM Cd2+ also further increased metallothionein and cellular GSH to 178 and 138% of the basal levels, respectively. Based on the affinity of Cd2+ for sulfhydryls as a mechanism of Cd2+ toxicity, we propose that the coordinately increased levels of metallothionein, GSH, and PSH in Cd(2+)-resistant cells would provide a mechanistic basis for the homeostasis of cellular thiols which may collectively contribute to the cytoskeletal preservation by protecting the cytoskeleton from Cd2+ insult.

Microtubule network and microtubule-associated proteins in leukemic T lymphocytes.

Cytoskeletal changes have been known to occur in cell transformation. Vinca alkaloids which bind to the tubulin dimer and inhibit microtubule (MT) assembly as well as disrupting the MT network and mitotic spindle, have been used as cancer chemotherapeutic agents. It has been proposed that apart from their anti-mitotic activity, these drugs act on the peripheral MT of malignant cells to produce their cytolytic effects. In this paper we demonstrate the presence of an altered cytoplasmic MT network in MOLT-4 and HuT-78 leukemic cells (human T-cell leukemic lines) compared to normal human peripheral blood lymphocytes stimulated with mitogens. In addition, using a selective extraction protocol we have compared microtubule-associated proteins (MAPs) profiles of G1/S synchronized leukemic human T-cells and 20 h mitogen-stimulated human peripheral blood T-cells. We observed a dramatic decrease in the expression of a MAP of apparent molecular weight 52 kDa and pI 5.2 in the leukemic cells synchronized at the G1/S border of the cell cycle. These results suggest that altered MT network morphology and MAP synthesis may be components of the malignant phenotype in the T-lymphocytic leukemias studied here.

Alterations in cytoskeletal protein sulfhydryls and cellular glutathione in cultured cells exposed to cadmium and nickel ions.

To understand the mechanisms of Cd2+ and Ni2+ cytotoxicity, we have studied the effects of these two metal ions on the organization of cytoskeletal elements, microtubules (MT) and microfilaments (MF), cytoskeletal protein sulfhydryls and cellular glutathione (GSH) in cultured 3T3 cells. At a metal ion dose that caused 95% inhibition of DNA synthesis, Cd2+ (10 microM, 16 h exposure) induced MT depolymerization whereas Ni2+ (2 mM, 20 h exposure) elicited MT aggregation and bundling. Under these conditions, Cd2+ and Ni2+ also caused MF aggregation and redistribution. Furthermore, exposure of cells to Cd2+ resulted in a dose-dependent increase in cytoskeletal protein sulfhydryls and cellular GSH levels. In contrast, treatment of cells with Ni2+ resulted in a dose-dependent decrease in cytoskeletal protein sulfhydryls as well as cellular GSH content. Time course studies showed that cells exposed to 10 microM Cd2+ exhibited a biphasic response in regulating their cytoskeletal protein sulfhydryls and cellular GSH, e.g. an initial decrease followed by a steady recovery and overshooting upon prolonged incubation. However, restoration of cytoskeletal protein sulfhydryls occurred approximately 2 h after commencement of cellular GSH recovery in Cd(2+)-treated cells. These results suggest that cellular GSH may play an important role in regulating cytoskeletal protein sulfhydryls. On the other hand, decrease of cellular GSH induced by Ni2+ might facilitate oxidation of cytoskeletal protein sulfhydryls and formation of disulfide bonds between individual MT polymers which would favor MT aggregation in Ni(2+)-exposed cells. In addition, we also demonstrated that elevation of cellular GSH in Cd(2+)-treated cells probably resulted from new GSH synthesis.

Microtubules and microtubule-associated proteins in resting and mitogenically activated normal human peripheral blood T cells.

The binding of an appropriate ligand to its specific receptor on the membrane of T cells triggers a cascade of events involved in T cell activation. An important yet unanswered question is how the mitogenic signals are transmitted through the cytoplasm and into the nucleus. The present study was carried out to determine changes in the microtubule (MT) system, following T cell activation. Fluorescence microscopy was employed to examine the organization of the microtubule network in human peripheral blood T cells in response to four different mitogens (phytohemagglutinin, concanavalin A, anti-CD3, and phorbol 12-myristate 13-acetate). The microtubules increase in length, number, and complexity of distribution 20 h after mitogenic stimulation. Using an in situ direct analysis protocol consisting of selective extraction of cells with detergent and Ca2+, 11 protein species, which fulfill the operational definition of microtubule-associated proteins (MAPs), were identified in resting human T cells. Alterations in the expression of these protein species were studied following mitogenic stimulation. These alterations in MAPs expression were also found in purified blast cell fractions indicating that they were specific changes occurring in activated T cell populations. These observations suggest a role for MT and MAPs in the cascade of human T cell activation.

Effects of sodium arsenite on the cytoskeleton and cellular glutathione levels in cultured cells.

The effects of As3+ (NaAsO2) on the microtubule and microfilament organization, cytoskeletal protein synthesis, cytoskeletal and cytosolic (soluble) protein sulfhydryls, and cellular glutathione (GSH) levels were examined in Swiss 3T3 mouse cells. Exposure of cells to 2.5 microM As3+ for 16 hr resulted in apparent cell retraction and loss of thick cables of actin filaments. However, the cells still retained numerous thinner microfilaments distributed in a disorganized manner. Microtubule organization was relatively undisturbed. At higher doses (greater than or equal to 20 microM), As3+ treatment caused a severe loss of microtubules and the remaining dense finer actin filaments formed smearing clusters in perinuclear areas. Treatment of cells with As3+ also induced a dose-dependent inhibition of cytoskeletal protein synthesis. Furthermore, As3+ exposure enhanced cellular GSH synthesis since the elevated cellular GSH content in As(3+)-treated cells could be abolished by treatment with buthionine sulfoximine, an inhibitor of gamma-glutamylcysteine synthetase required for GSH biosynthesis. As determined by the N-[3H]-ethylmaleimide binding assay, As3+ exposure also increased the amount of protein sulfhydryls in both the cytoskeletal and the cytosolic protein fractions. Moreover, a greater increase in protein sulfhydryls occurred in the cytoskeletal fraction than in the soluble fraction. These results indicate that the cytoskeleton could be a cellular target for injury by As3+ exposure. The elevated cellular GSH content induced by As3+ could provide a protective mechanism against further injury from this metal insult.

Growth restraint and differentiation by LPS/TNF-alpha/IFN-gamma reorganization of the microtubule network in human leukemia cell lines.

The microtubule (MT) network of the cytoskeleton has been implicated as a mediator of cellular signal transduction; disorganization of this network may allow for mitogenesis. In previous work, loss of MT network organization in human MOLT4 and HUT78 T-cell leukemias was demonstrated in contrast to an organized "spoke-wheel-like arrangement" in normal human T-lymphocytes. In this study, loss of MT network organization was shown in several representative acute myeloid leukemia (AML) cell lines: KG1 myeloblastic, HL60 promyelocytic, and U937 myelomonocytic cells. Re-organization of the MT network was observed in HL60 and U937 AML cells treated with combined lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). This re-organization paralleled earlier work which showed this combination was effective in inducing monocytic pathway differentiation and growth restraint in HL60 cells, and growth restraint in U937 cells. In contrast, KG1 cells exhibited growth restraint, but did not re-organize with LPS/TNF-alpha/IFN-gamma treatment. These results are consistent with a role for the MT network in mitogenesis. Loss of MT network organization appeared to parallel the neoplastic phenotype in three AML cell lines, whereas MT network re-organization accompanied recovery of growth control in 2 of 3 AML cell lines.

An immunofluorescence study of the effects of ultraviolet radiation on the organization of microfilaments, keratin intermediate filaments, and microtubules in human keratinocytes.

Indirect immunofluorescence microscopy has been used to investigate the ultraviolet (UV) radiation induced disruption of the organization of microfilaments, keratin intermediate filaments, and microtubules in cultured human epidermal keratinocytes. Following irradiation, concurrent changes in the organization of the three major cytoskeletal components were observed in cells incubated under low Ca2+ (0.15 mM) conditions. UV irradiation induced a dose-dependent condensation of keratin filaments into the perinuclear region. This collapse of the keratin network was accompanied by the reorganization of microfilaments into rings and a restricted distribution of microtubules, responses normally elicited by exposure to high Ca2+ (1.05 mM) medium. The UV induced alteration of the keratin network appears to disrupt the interactions between keratin and actin, permitting the reorganization of actin filaments in the absence of Ca2+ stimulation. In addition to the perinuclear condensation of keratin filaments, UV irradiation inhibits the Ca2+ induced formation of keratin alignments at the membrane of apposed cells if UV treatment precedes exposure to high Ca2+ medium. Incubation of keratinocytes in high Ca2+ medium for 24 hours prior to irradiation results in the stabilization of membrane associated keratin alignments and a reduced susceptibility of cytoplasmic keratin filaments to UV induced disruption. Unlike results from investigations with isogenic skin fibroblasts, no UV induced disassembly of microtubules was discernible in irradiated human keratinocytes.

Cytoskeletal injury induced by hexavalent chromate.

To understand the mechanisms of toxicity of hexavalent chromate (K(2)CrO(4), Cr(6+)), the effects of this metal ion on the organization of microtubules (MTs) and microfilaments (MFs), DNA synthesis, cytoskeletal protein synthesis, cytoskeletal protein sulphhydryls (-SH groups), and cellular glutathione (GSH) levels in 3T3 cells were examined. Exposure of cells to Cr(6+) for 16 hr resulted in a dose-dependent inhibition of DNA synthesis with 50% inhibition occurring at 16.6 mum. Treatment of cells with 3.13 mum-Cr(6+) for 16 hr resulted in a slight cell retraction and, in some cells, MT bundling, without much effect on the morphology of dense MFs. At 25 mum, Cr(6+) caused disruption of the cell sheet, depolymerization of MTs, particularly those in the peripheral areas, and redistribution of MFs, which assumed a smearing morphology. Exposure to 100 mum-Cr(6+) induced severe thinning of MTs and loss of MFs. Although doses of 3.13 mum-Cr(6+) or less slightly enhanced the level of cytoskeletal protein synthesis (e.g. 38% increase at 3.13 mum-Cr(6+)), Cr(6+) at 6.25 mum or more produced a dose-dependent inhibition of cytoskeletal protein synthesis (50% inhibition at 11.25 mum). Exposure of cells to Cr(6+) for 16 hr resulted in a dose- and time-dependent increase of cellular GSH level. Furthermore, the elevated cellular GSH induced by Cr(6+) was diminished by treatment with buthionine sulphoximine (BSO), a potent inhibitor of GSH biosynthesis. In addition, depletion of GSH by BSO increased cell sensitivity to Cr(6+) insult and aggravated Cr(6+)-induced cytoskeletal perturbation. However, Cr(6+) treatment of cells did not significantly affect the amount of cytoskeletal protein sulphhydryls. These results suggest that cytoskeletal injury may be an important part of the mechanism for Cr(6+) toxicity. The cytoskeletal damage may result directly from the inhibition of cytoskeletal protein synthesis rather than from the interaction between Cr(6+) and cytoskeletal protein sulphhydryls.

An immunofluorescence study of the calcium-induced coordinated reorganization of microfilaments, keratin intermediate filaments, and microtubules in cultured human epidermal keratinocytes.

Indirect immunofluorescence microscopy has been used to investigate the coordinated reorganization of microtubules, microfilaments, and keratin intermediate filaments in cultured human epidermal keratinocytes following a switch from low-Ca++ (0.15 mM) medium to high-Ca++ (1.05 mM) medium. A dramatic reorganization occurs concurrently in the three major cytoskeletal components shortly after the calcium switch. The most prominent features are the alignment of keratin filaments at the plasma membranes of apposed cells, the induction of microfilament rings, the restriction of microtubules to the area within the boundaries of the microfilament rings, and the alignment of actin bundles at cell borders. Additional changes are observed in terminally differentiated cells. This is the first report that describes simultaneous changes in the organization of the three major cytoskeletal components of epidermal keratinocytes. Cytochalasin D and demecolcine (colcemid) studies were performed to determine whether the organization of microtubules, microfilaments, and keratin filaments, as well as the calcium-induced reorganization of these cytoskeletal elements, may be dependent on the existence of structural relationships between them. These studies demonstrate that the disruption of microfilaments results in the formation of a latticelike keratin network, with a close association of actin and keratin being maintained. The formation of keratin filament alignments occurs even in the absence of intact microfilaments. In addition, it was found that the Ca(++)-induced reorganization of microfilaments and keratin filaments is not dependent on an intact microtubule network. Furthermore, the reorganization of actin into concentric rings can be dissociated from changes in the organization of keratin filaments.

Disruption of cytoplasmic microtubules by ultraviolet radiation.

Ultraviolet (UV) irradiation of cultured human skin fibroblasts causes the disassembly of their microtubules. Using indirect immunofluorescence microscopy, we have now investigated whether damage to the microtubule precursor pool may contribute to the disruption of microtubules. Exposure to polychromatic UV radiation inhibits the reassembly of microtubules during cellular recovery from cold treatment. In addition, the ability of taxol to promote microtubule polymerization and bundling is inhibited in UV-irradiated cells. However, UV irradiation of taxol-pretreated cells or in situ detergent-extracted microtubules fails to disrupt the microtubule network. These data suggest that damage to dimeric tubulin, or another soluble factor(s) required for polymerization, contributes to the disassembly of microtubules in UV-irradiated human skin fibroblasts.

Studies on the mechanisms of Ni2(+)-induced cell injury: I. Effects of Ni2+ on microtubules.

Cytoskeletal perturbations have been associated with exposures to a variety of toxic agents as well as a number of human pathological conditions. We have observed dramatic alterations in the organization of microtubules (MT), a major component of the cytoskeleton, in 3T3 cells exposed to Ni2+. Severe perinuclear bundling and aggregation of MT occurred in both a time- and dose-dependent fashion, and this MT damage was reversible upon removal of Ni2+ from the culture media. To understand the mechanism of the Ni2(+)-induced MT change, we investigated the effect of Ni2+ (0.01 to 3.0 mM) on in vitro tubulin polymerization. Ni2+ at lower concentrations (0.01 to 1.0 mM) had little or no significant effect on the kinetics of MT polymerization. In contrast, in the presence of 1.5 to 2.0 mM Ni2+, a significant promoting effect on both the rate and the final extent of polymerization was observed. However, at Ni2+ concentrations higher than 2.0 mM, such stimulatory effect on the rate and the final extent of tubulin polymerization declined. Furthermore, the promoting effects of Ni2+ on MT polymerization were accompanied by a significant decrease in the lag period. Electron microscopic examination of samples of the polymerization product showed that MT, polymerized in the presence of 2.0 mM Ni2+, appeared more numerous and shorter (1.10 +/- 1.02 microns) than those of control (3.81 +/- 2.29 microns; p less than 0.005). This was probably a direct result of an increase in the number of initiation centers in the presence of Ni2+ as a consequence of the decreased critical concentration (7%, p less than 0.05) necessary for polymerization to occur. Our results suggest that Ni2+ may exert its toxic effect on MT in cultured cells by altering the normal kinetics of MT polymerization.

Disruption of keratin intermediate filaments by ultraviolet radiation in cultured human keratinocytes.

Fluorescence microscopy has been utilized to investigate the effects of UV irradiation on the organization of keratin intermediate filaments in normal human epidermal keratinocytes. Sun lamp irradiation induced the condensation of keratin intermediate filaments into the perinuclear region and inhibited the reorganization of keratin filaments normally induced by Ca2+. Exposure to UVC appeared to disrupt keratin filaments similarly, whereas UVA had no discernible effect.

Alterations of cellular characteristics of a human ovarian teratocarcinoma cell line after in vitro treatment with retinoids.

Differentiation of the human teratocarcinoma derived cell line. PA-1, with retinoids was examined at concentrations (10(-6)-10(-8) M) that did not exhibit an antiproliferative effect during log-phase growth. Treatment with naturally occurring retinoic acid or certain synthetic retinoids (13-cis retinoic acid, Ro10-9359, and Ro13-7410), while not significantly altering the log-phase growth rate, decreased the saturation cell density and mitotic indices after confluence. Retinoid treatment also induced changes in cell morphology, which appear to be related to reorganization of microtubules and microfilaments. Following retinoid treatment, the expression of cell glycoproteins (of 162 kDa, 152 kDa, 143 kDa. and 51 kDa) was altered. Treated cells also exhibited decreased expression of alkaline phosphatase, as well as an increased capacity for intercellular communication as evidenced by gap-junctional transfer of the phosphorylated toxic intermediate of 6-thioguanine to HPRT- cells. Treatment with retinoic acid dramatically reduced the quantity of shed plasma membrane material and altered its composition.

Microtubule disassembly induced by sensitizing halogenated nitrobenzene derivatives.

The potency of certain halogenated nitrobenzene derivatives to induce allergic contact dermatitis has been demonstrated in guinea-pigs and in man. As the first step towards understanding the mechanisms of cellular injury induced by these sensitizers, we have studied by fluorescence microscopy the effects of 13 halogenated (nitro)benzene derivatives on microtubule organization in mouse 3T3 fibroblasts and human AG1522 skin fibroblasts. Untreated cells have numerous microtubules distributed in a network fashion throughout the cytoplasm and extending to the cell periphery. Exposing cells for 3 hr to micromolar doses of halogenated nitrobenzene derivatives, which induce allergic contact dermatitis in guinea-pigs and man, resulted in a dose-dependent disassembly of microtubules. In contrast, incubation with 10-70 times higher doses of seven halogenated (nitro)benzene derivatives that do not sensitize guinea-pigs or man, had no discernible effect on microtubule organization of both cell types under identical assay conditions. Thus, for the 13 halogenated (nitro)benzene derivatives tested in this study, a 100% positive correlation exists between the sensitizing capacity as determined by in vivo tests on guinea-pigs and man (Landsteiner and Jacobs, 1936) and the ability to cause microtubule disassembly in cultured cells. These results may provide the basis for developing an in vitro screening assay for identifying other potential halogenated nitrobenzene sensitizers. In addition, these studies represent a new approach to investigating the mechanisms of contact sensitivity induced by simple chemicals.

Distinct cytoskeletal injuries induced by As, Cd, Co, Cr, and Ni compounds.

The risks of metal compounds to human health are highlighted by the ubiquity of exposure and their persistence in the environment. Although compounds of As, Cd, Co, Cr, and Ni are known or "reasonably anticipated" to be carcinogenic to humans and/or experimental animals, the cellular targets of these health hazards and the underlying mechanisms of their carcinogenicity are still unclear. We show in this report that dramatic, time- and dose-dependent cytoskeletal perturbations, especially in the distribution and organization patterns of microtubules and microfilaments, two of the principal components of the cytoskeleton, occurred in 3T3 cells upon exposure to these metal salts. Each metal salt appeared to induce a different, typical pattern of cytoskeletal injury, probably reflecting the specificity of action of each metal ion. These results suggest that the cytoskeleton can indeed act as a target for injury by epigenetic carcinogenic metal compounds in the environment. These findings should help our efforts to understand the mechanisms of action of metal compounds at the subcellular and molecular levels.

Relationship between 1-chloro-2,4-dinitrobenzene-induced cytoskeletal perturbations and cellular glutathione.

Exposure of 3T3 cells to micromolar doses of 1-chloro-2,4-dinitrobenzene, a substrate for glutathione-S-transferase, resulted in a rapid depletion of total cellular glutathione accompanied by disassembly of microtubules as visualized by fluorescence microscopy. However, prolonged incubation resulted in cellular recovery from 1-chloro-2,4-dinitrobenzene insult as evidenced by a steady rise in total cellular glutathione accompanied by microtubule reassembly to their normal organization 5 hours after treatment. To evaluate the role of total cellular glutathione in modulating the 1-chloro-2,4-dinitrobenzene-induced cytoskeletal perturbation, we used 1-chloro-2,4-dinitrobenzene and/or buthionine sulfoximine, an effective irreversible inhibitor of glutathione synthesis, to manipulate cellular glutathione levels. Incubation of 3T3 cells with 2.5 microM 1-chloro-2,4-dinitrobenzene and 250 microM buthionine sulfoximine for 5 hours resulted in a complete depletion of total cellular glutathione accompanied by essentially complete loss of microtubules and marked alterations in the density and distribution pattern of microfilaments. Buthionine sulfoximine enhanced markedly the extent and duration of cellular glutathione depletion and the severity of microtubule disruption of 3T3 cells over the level achieved by 1-chloro-2,4-dinitrobenzene treatment alone. Furthermore, buthionine sulfoximine also prevented the restoration of cellular glutathione content and microtubule reassembly that normally were evident 5 hours after 1-chloro-2,4-dinitrobenzene treatment. Exposure of 3T3 cells to 50 microM 2-cyclohexene-1-one, which depletes free glutathione by conjugation, resulted in a complete depletion of total cellular glutathione content without altering the microtubule organization. These results suggest that the total glutathione content may be important for cellular recovery from 1-chloro-2,4-dinitrobenzene-mediated cytoskeletal injuries, and that microtubule disassembly observed in 1-chloro-2,4-dinitrobenzene-treated cells probably results from depletion of cellular glutathione coupled with binding to tubulin and/or other microtubule components.