Procedure for assessing myeloperoxidase and inflammatory mediator responses in hairless mouse skin.

A preparation procedure for making multiple inflammatory biomarker measurements from the same skin tissue was assessed. The backs of euthymic hairless mice were exposed to sulfur mustard (HD) vapor for 6 min. Animals were euthanized 24 h following exposure, dorsal skin tissue was excised and 12-mm, full-thickness biopsy punches of the exposed skin sites were taken. Specimens were snap-frozen, crushed to a powder using a biopulverizer unit, solubilized in buffer and centrifuged. Supernatant was assayed for pro-inflammatory cytokines and the acute-phase reactive protein, serum amyloid P (SAP). Myeloperoxidase (MPX), which is indicative of neutrophil infiltration into the skin, was associated with the pellet fraction. Results indicate an elevation of interleukin-6, SAP and MPX in mouse skin tissue specimens 24h following HD vapor exposure. The tissue preparation procedure allows the use of a single skin specimen to make multiple inflammatory endpoint measurements requiring different preparation processes, and it will be used in subsequent studies to characterize further the inflammatory nature of HD-exposed skin tissue.

Therapeutic approaches to dermatotoxicity by sulfur mustard. I. Modulaton of sulfur mustard-induced cutaneous injury in the mouse ear vesicant model.

The mouse ear edema model is recognized for its usefulness in studying skin responses and damage following exposure to chemical irritants, and for evaluating pharmacological agents against chemically induced skin injury. We recently modified the mouse ear edema model for use with sulfur mustard (HD) and used this model to study the protective effect of 33 topically applied compounds comprising five pharmaceutical strategies (anti-inflammatories, protease inhibitors, scavengers/chelators, poly(ADP-ribose) polymerase (PARP) inhibitors, calcium modulators/chelators) against HD-induced dermatotoxicity. Pharmacological modulation of HD injury in mouse ears was established by a reduction in edema or histopathology (epidermal necrosis and epidermal-dermal separation) at 24 h following topical liquid HD exposure. Ten of the 33 compounds administered as single topical pretreatments up to 2 h prior to HD challenge produced significant reductions in edema. Five of these ten also produced significant reductions in histological endpoints. Three candidates (olvanil, indomethacin, hydrocortisone) showing protection at 24 h were evaluated further for 'extended protection' at 48 and 72 h after HD challenge and showed significant modulation of edema at 48 h but not at 72 h. Olvanil also showed significant reductions in histology at 48 and 72 h. Olvanil and indomethacin were shown to reduce significantly the edema at 24 h post-exposure when administered topically 10 min after HD challenge, with olvanil additionally protecting against epidermal necrosis. These results demonstrate prophylactic and treatment effects of pharmacological agents against HD-induced skin injury in an in vivo model and support the continued use of the mouse ear vesicant model (MEVM) for evaluating medical countermeasures against HD.

Neuromuscular effects of low level exposures to Sarin, pyridostigmine, DEET, and chlorpyrifos.

A study is being initiated to investigate subtle neurobehavioral effects and neuropathology in rats due to exposure to combinations of low levels of Sarin (GB), N,N-diethyl-m-toluamide (DEET), chlorpyrifos (CPF), and pyridostigmine bromide (PB). A similar study is being initiated in rhesus monkeys to investigate neurophysiologic effects and neuromuscular pathology due to exposure to a combination of GB, DEET, CPF, and PB, along with vaccination with botulinum toxoid. A description of these studies is presented.

Comparison of an in vitro skin model to normal human skin for dermatological research.

EpiDerm, an in vitro human skin equivalent (HSE), was compared to normal human breast skin (NHS) to morphologically and biochemically assess its feasibility for dermatological research. Intralot and interlot variability was studied in day 0, 1, 2, and 3 in vitro cultures and in day 0, 3, 5, and 7 NHS. For NHS, light microscopy (LM) at day 0 showed stratified epidermis which exhibited an increase in vacuoles and dark basal cells as storage increased to 3, 5, and 7 days. Transmission electron microscopy (TEM) revealed typical organelles in the epidermis and a convoluted basement membrane at day 0. With increased storage, vacuoles and paranuclear clefts became numerous, necrosis increased, tonofilaments became less organized, and overall cellular integrity decreased. Biochemical data showed consistent MTT and glucose utilization (GU) through day 5, while lactate production decreased to 75% by day 3. By LM, day 0 HSE consisted of a thick, compact, stratum corneum that sent projections between the stratum granulosum cells. By TEM, the configuration organization, differentiation, distribution, and frequency of the organelles differed slightly from NHS. In addition, the basement membrane of the HSE was not completely differentiated, and the dermis was thin and acellular. Although day 1 and 2 cultures showed little change, day 3 exhibited an overall degeneration. Biochemical analysis showed GU and the lactate production decreased through day 3. In conclusion, the EpiDerm HSE, although exhibiting slight differences, was morphologically and biochemically similar to normal human epidermis and may be a valuable model in assessing the toxicology, metabolism, or pharmacology of nonvesicating compounds.

Cutaneous histopathologic features in weanling pigs after exposure to three different doses of liquid sulfur mustard.

Sulfur mustard (2,2' dichlorodiethyl sulfide, HD) is a chemical warfare agent that is easily produced, and may be used against civilian populations as well as against military troops. However, good therapeutic and prophylactic measures await a better understanding of the pathophysiology of lesions produced by this agent. Because the skin remains is one of the principal routes for HD exposure and damage, the study of HD-induced skin lesions is of major interest. Blister formation is a characteristic of HD-induced skin lesions in humans. Attempts have been made to find an animal model that produces cutaneous microblisters after exposure to the naturally occurring liquid as well as vaporized HD. Weanling pigs were exposed to three different doses of liquid HD. Histopathologic findings showed microblister formation as well as variable apoptosis and/or necrosis of epidermal keratinocytes and vascular endothelium. Pig skin is morphologically similar to human skin. In the pig, the epidermal lipids, the density of hair follicles, the presence of sweat glands, the proliferation kinetics, and the antigenicity are all closer to human skin than are rodent models. All these features may be important in lesions induced by HD, and may mean that the pig is a superior model for studying the pathophysiology of HD-induced cutaneous lesions.

Efficacy of tacrine as a nerve agent pretreatment.

Tacrine (THA) was evaluated in vitro and in vivo as a pretreatment for nerve agent intoxication. In vitro experiments showed that the primary effect of THA was direct inhibition of purified fetal bovine serum acetylcholinesterase (AChE) with a slight effect on slowing the aging rate of nerve agent-inhibited AChE. THA produced significant behavioral effects at doses above 1.7 mg/kg, i.m., in the mouse and 3.4 mg/kg, i.m., in the guinea pig. At the no observable effect level (NOEL) for mice (1.7 mg/kg), THA was effective (P < or = 0.05) in reducing tabun- and soman-, but not sarin-induced lethality in mice. Experiments in the guinea pig showed that at the NOEL (3.4 mg/kg, i.m.) THA was not effective in decreasing lethality due to soman exposure. Since there was significant overlap between pharmacologically effective doses of THA and those which produce behavioral toxicity, THA was not considered a suitable pretreatment for nerve agent intoxication.

Effects of calmodulin antagonists on calcium transport by chorioallantoic membranes of developing chicken embryos.

Sections of chorioallantoic membranes (CAM) from developing chicken embryos were incubated in the presence of calmidazolium (CMZ) or trifluoperazine (TFP). Both the forward (ectodermal to endodermal) and backward flux rates of calcium were higher in the presence of drug. However, the effect was greater in the forward direction and the agents produced a statistically significant increase in ectodermal to endodermal calcium transport. The initial rate of uptake of calcium into CAM sections was increased by TFP, and the efflux of calcium from preloaded sections was increased in the presence of TFP and CMZ.

2,3,7,8-Tetrachlorodibenzo-p-dioxin stimulation of tyrosine phosphorylation in B lymphocytes: potential role in immunosuppression.

The prototype halogenated aromatic hydrocarbon 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is carcinogenic and toxic in experimental animals. At the cellular level, TCDD toxicity is often expressed as an inhibition or alteration in normal cell maturation. In this respect, we and others have demonstrated that exposure of experimental animals to TCDD causes immunosuppression, including inhibition of B lymphocyte maturation and antibody synthesis. Although the immunological effects of TCDD are well described, little is known about its mechanism of action. In the present studies, it was found that TCDD increases membrane protein phosphorylation, which is, in part, associated with tyrosine-specific phosphorylation in B lymphocytes. This increase in phosphorylation occurred within minutes following TCDD treatment and was not associated with protein kinase C. The increase in tyrosine kinase by TCDD appears to be primarily due to de novo synthesis of new protein, because the protein synthesis inhibitors puromycin and cycloheximide, as well as the transcriptional inhibitor actinomycin D, partially inhibited the effect, although increased activity of preexisting protein cannot be fully dismissed. The dose response for increased phosphorylation by TCDD was identical to that we previously reported for inhibition of antibody synthesis, suggesting that immunosuppression by TCDD may be expressed through alterations in regulatory processes controlled by tyrosine kinases. These studies are discussed in terms of the potential role of TCDD-induced tyrosine phosphorylation in immunosuppression.

Effect of acetazolamide on calcium accumulation by the developing chicken embryo.

The injection of 10 mg of acetazolamide into fertile chicken eggs on the 16th or 17th day of incubation resulted in a decreased accumulation of calcium by the developing embryo. A dose of 1 mg of acetazolamide did not affect embryonic calcium content. Although the eggshell serves as the primary source of calcium for the embryo, acetazolamide did not influence eggshell mobilization as measured by the release of 45Ca from labeled eggshells. Acetazolamide did decrease bone formation resulting in a statistically significant reduction in tibia dry weight and calcium content.

Comparative immunotoxicity of 2,2'-dichlorodiethyl sulfide and cyclophosphamide: evaluation of L1210 tumor cell resistance, cell-mediated immunity, and humoral immunity.

The immunotoxicity of 2,2'-dichlorodiethyl sulfide (sulfur mustard, SM), on humoral and cell-mediated immunity was compared with that of the nitrogen mustard 2-[bis(2-chloroethyl)amino]tetrahydro-2H-1,3,2-oxazophosphorine 2-oxide (cyclophosphamide, CP). SM and CP had similar effects on thymic and splenic weights, spleen cell number, and the formation of antibody producing cells to sheep red blood cells (sRBC) when examined 5 days after exposure, but differed in their effects on body weights. Although there were no differences in the delayed hypersensitivity response to keyhole limpet hemocyanin, CP and SM had different effects in the L1210 tumor cell allograft rejection assay. CP, but not SM, decreased the 28 day survival rate of allogeneic mice exposed to a sublethal L1210 tumor challenge. The differing effects on survival to the L1210 tumor challenge could not be attributed to a direct cytotoxic effect of SM on the L1210 tumor cells as SM did not increase the survival rate or median survival time of syngeneic mice exposed to a lethal L1210 tumor cell challenge. In summary, SM and CP had immunosuppressive effects in the humoral immune assay. Although neither compound suppressed the delayed hypersensitivity response, CP was found to suppress host resistance to L1210 tumor cells.

Pertussis toxin inhibition of anti-immunoglobulin-stimulated proliferation and inositol phosphate formation.

A variety of receptor agonists activate cells by stimulating polyphosphoinositide hydrolysis. Increasing evidence supports the concept that receptor-stimulated phosphoinositide hydrolysis is mediated by a guanosine triphosphate binding protein, which in some cell systems is inhibited by pertussis toxin through ADP-ribosylation. The cross-linking of membrane immunoglobulin by antigen or anti-Ig stimulates phosphoinositide hydrolysis resulting in the formation of inositol phosphate and diacylglycerol which act as second messengers in initiating B lymphocyte activation. In this report, we demonstrate that anti-Ig-stimulated inositol phosphate formation is enhanced by the nonhydrolyzable guanosine triphosphate analogue, GppNHp, in permeabilized B lymphocytes and also inhibited by pretreatment of intact cells with pertussis toxin. This latter effect is associated with the pertussis toxin-catalyzed ADP-ribosylation of a 41-kDa membrane protein which is of the same molecular weight as the guanosine triphosphate binding protein reported to mediate receptor-stimulated phosphoinositide hydrolysis in other cellular receptor systems. B lymphocyte proliferation induced by agents such as lipopolysaccharide and PMA plus calcium ionophore, which activate cellular proliferation without stimulating phosphoinositide breakdown, is not inhibited by pertussis toxin. We conclude that anti-Ig activation of B lymphocytes contains pertussis toxin- and guanosine triphosphate-sensitive components which are involved in regulating phosphoinositide breakdown and initiating cellular activation.

Suppression of B cell function by methotrexate and trimetrexate. Evidence for inhibition of purine biosynthesis as a major mechanism of action.

Methotrexate (MTX) is a widely used drug in the treatment of a variety of human neoplasms. Trimetrexate (TMQ) is a lipid-soluble quinazoline derivate of MTX that, unlike MTX, is not dependent upon membrane folate transport for cellular entry. A number of studies have demonstrated that MTX and, more recently, TMQ possess potent immunosuppressive properties. To examine the cellular events associated with the immunomodulatory effects of anti-folates on humoral immunity, a murine B cell maturation model was used. In vitro, MTX and TMQ reduced the number of antibody-forming cells to SRBC, as well as IgM production. B cells stimulated with anti-Ig demonstrated a dose-related suppression in [3H]UdR incorporation after addition of either drug, suggestive of a decrease in de novo DNA synthesis. B cell activation events preceding S phase were also suppressed by both anti-folates, as evidenced by inhibition of RNA synthesis. However, neither drug affected surface expression of Ia Ag nor inositol phosphate accumulation. Addition of TdR caused a slight non-significant increase in the antibody-forming cell response in the presence of 10(-7) M MTX. However, addition of hypoxanthine or adenine, but not guanine, resulted in complete restoration. Timed addition revealed that the ability of MTX to suppress antibody responses was diminished if added after 48 h of culture, similar to the reversal of this suppression mediated by hypoxanthine. Cell cycle analysis of LPS-stimulated B lymphocytes demonstrated that both drugs modulated events preceding, as well as during, the S phase. The present studies suggest that although drug-induced impairments in dTMP biosynthesis may be responsible for deficient lymphoid proliferation, anti-folate-induced impairment in purine biosynthesis is a major mechanism in anti-folate-induced suppression of humoral immunity.

Biochemical events associated with inhibition of B-cell proliferation by phorbol diesters.

Phorbol myristate acelate (PMA), a potent tumor promoter, has a variety of effects on cells of the immune system resulting in altered patterns of cell proliferation and differentiation. Although PMA is mitogenic or co-mitogenic for human lymphocytes and murine T-cells, it inhibits proliferation of murine B-cells stimulated by LPS or anti-Ig. PMA, however, does not inhibit the ability of LPS or anti-Ig to activate B-cells, as evidenced by increased Ia antigen expression and RNA synthesis. In the present studies it was shown that inhibition of DNA synthesis by PMA coincided with qualitative and quantitative changes in phosphorylated proteins. In particular, PMA treatment resulted in a unique profile of phosphoproteins independent of LPS or anti-Ig treatment. Inhibition of DNA synthesis occurred over a wide range of PMA concentrations. At concentrations up to 10(-9) M, inhibition of proliferation correlated with decreased phosphatidylinositol turnover and decreased intracellular Ca2+ levels, suggesting that PMA affects the phosphoinositide signal transduction pathway. However, at PMA concentrations less than 10(-10) M, inhibition of anti-Ig- and LPS-mediated proliferation occurred without inhibition of the phosphoinositide transduction signal. At these concentrations, PMA-induced inhibition of DNA synthesis was highly sensitive to recombinant IL-2. These data suggest that the antiproliferative effects of PMA on B-cells stimulated by LPS or anti-Ig may be mediated by two mechanisms. At high concentrations, PMA causes a feedback regulation of the phosphoinositide-dependent messenger system, while at lower concentrations, PMA alters the response to specific growth factors. Since PMA induces unique phosphoproteins and both of these events can be regulated by protein phosphorylation, it is possible that these unique phosphoproteins are responsible for the antiproliferative effects of PMA.

alpha-Naphthoflavone antagonism of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced murine lymphocyte ethoxyresorufin-O-deethylase activity and immunosuppression.

Suppression of murine humoral immunity by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown to occur in vivo and in vitro. Studies have indicated that suppression of humoral immunity is mediated by the Ah receptor. Data presented in this paper demonstrate that alpha-naphthoflavone (ANF) and beta-naphthoflavone (BNF), like TCDD, bind to rat and murine hepatic and murine splenocyte cytosolic Ah receptor. Furthermore, BNF induces cytochrome P1-450 monooxygenase activity as measured by ethoxyresorufin-O-deethylase (EROD) in murine spleen cells to the same extent as TCDD. In contrast, ANF predominantly acts to antagonize TCDD induction of splenocyte EROD activity. Examination of humoral immunity in vitro demonstrated that BNF, like TCDD, is suppressive. Whereas ANF is suppressive at cytotoxic concentrations, lower concentrations of ANF antagonize the suppressive effect of TCDD. Antagonism by ANF of TCDD-induced EROD activity and suppression of humoral immunity occur at similar concentrations. These data suggest that ANF blocks TCDD suppression of B lymphocyte differentiation by competing with TCDD for binding to the Ah receptor. Since the mechanism of TCDD toxicity is not fully understood, probes such as ANF may be of great use in examining the role of the Ah receptor in mediating toxicity.

1-amino-3,7,8-trichlorodibenzo-p-dioxin: a specific antagonist for TCDD-induced myelotoxicity.

It was recently reported that suppression of murine bone marrow hematopoiesis is a very sensitive indicator for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity (1). We report here that a structural analog of TCDD, 1-NH2-3,7,8-trichlorodibenzo-p-dioxin (NH2-TriCDD), is a specific and effective antagonist for TCDD-induced myelotoxicity and enzyme induction. When administered to mice or added directly into culture at a 100-fold excess, relative to TCDD, NH2-TriCDD completely abrogated the ability of TCDD to inhibit granulocyte-macrophage progenitor cells (CFU-C) formation, an indicator of hematopoiesis. Further, NH2-TriCDD inhibited TCDD-induced activation of cytochrome P1-450 monooxygenase activity. Studies designed to measure specific binding of TCDD to the cytosolic Ah receptor indicated that NH2-TriCDD effectively inhibited binding of TCDD to the receptor by acting as a competitive antagonist (Ki = 0.72 nM).

Effects of heterocyclic sulfonamides on bone metabolism.

Sulfonamide inhibitors of carbonic anhydrase attenuate parathyroid hormone and vitamin D3 induced bone resorption. Parathyroid hormone stimulates bone cell accumulation of cAMP in the presence of concentrations (10(-7) - 10(-3) M) of acetazolamide (an inhibitor of carbonic anhydrase) that are antagonistic to bone resorption. Bone cell acid phosphatase activity and lactic acid production are stimulated by parathyroid hormone or vitamin D3. These cellular responses are not influenced by the presence of acetazolamide (10(-4) M). Bone cells incubated in the presence of parathyroid hormone reveal an increase in carbonic anhydrase like activity. This activity is lost upon heating a supernatant of the bone cell population or upon treatment of the supernatant with appropriate concentrations of acetazolamide.