Abstract  The many different treatment possibilities for the eradication of warts provide evidence that no single method that is completely effective has been found. Although the various methods described herein are usually successful therapies for warts, they are all associated with treatment failures and side effects. Until the perfect cure for warts is discovered, the physician must evaluate every wart carefully before deciding on a course of action.

Abstract  The acute combined effects of cadmium (Cd) and nickel (Ni) on hepatic monooxygenase activities (ethylmorphine N-demethylase, EMND; aminopyrine N-demethylase, AMND; aniline 4-hydroxylase, AH), cytochrome P-450, cytochrome b5, microsomal heme and reduced glutathione (GSH) levels and glutathione S-transferase (GST) activities toward several substrates (1-chloro-2,4-dinitrobenzene, CDNB; 1,2-dichloro-4-nitrobenzene, DCNB; ethacrynic acid, EAA; 1,2-epoxy-3-(p-nitrophenoxy)-propane, ENPP) were determined and compared with those of Cd or Ni alone in mice. Male adult mice (25-30 g) were administered either a single dose of Cd (3.58 mg CdCl2.H2O/kg, i.p.) 48 hr prior to killing or a single dose of Ni (59.5 mg NiCl2.H2O/kg, s.c.) 16 hr prior to killing. For the combined treatment, the animals received the single dose of Ni 32 hr after the single dose of Cd and were then killed 16 hr later. Cd treatment alone significantly decreased EMND, AMND, and AH activities and cytochrome P-450 and heme levels as compared with controls. Cytochrome b5 level was not altered by Cd treatment. Cd also inhibited GSH level and the GST activities toward CDNB, EAA and ENPP significantly. No significant change was observed in the GST activity for DCNB by Cd. Ni treatment alone, however, decreased the monooxygenase and GST activities studied, and cytochrome P-450, cytochrome b5, heme and GSH levels significantly. Combined treatment significantly depressed the monooxygenase activities and cytochromes and heme levels. GSH level was not significantly altered.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract  Novel approaches to testing of skin sensitizing chemicals have made use of immature dendritic cells (DCs) cultured from different hematopoietic progenitors. These cells resemble Langerhans cells (LCs), which are the most potent antigen presenting cells in the skin. Former research has focused on the phenotypic and functional changes of LCs after application of skin sensitizers. But it has proven difficult to isolate sufficient numbers of LCs from skin. This disadvantage is overcome by cultures of immature DCs providing high numbers of reactive cells. The aim of the present investigation was to test the response of DC cultures established from different blood donors to known sensitizers, an irritant and a vehicle. The sensitizers NiSO(4), dinitrochlorobenzene (DNCB), 2,4,6 trinitrobenzene sulfonic acid (TNBS), alpha-hexylcinnamaldehyde (Cinn) and eugenol (Eu) induced the up-regulation of the co-stimulatory molecule CD86, of intercellular adhesion molecule CD54 and of the HLA-DR antigen. The irritant sodium dodecyl sulfate (SDS) and the vehicle dimethyl sulfoxide (DMSO) had no effect. A high rate of responders within blood donors was found for NiSO(4), TNBS, Cinn and Eu, while DNCB was less effective. The augmentation of surface marker expression in dendritic cells obtained from peripheral human blood seems to be a promising readout in prescreening for strong and moderate sensitizers. This test could thus help to reduce animal numbers for in vivo testing.

Abstract  Two mutant forms of human glutathione transferase (GST) A1-1 with affinity for metal ions were constructed by introduction of His residues by site-directed mutagenesis. A mutant, 2-His, contained the mutations Lys84Gln, Asp85His and Glu88His, and another, 5-His, contained the mutations Tyr79His, Asn80His, Lys84His, Asp85His and Glu88His. The mutant proteins were obtained in good yields (40-150 mg per 3 l culture) by heterologous expression in Escherichia coli. The mutant enzymes possessed novel binding affinities for Ni(II) and Zn(II) ions, as demonstrated by immobilized metal ion affinity chromatography. The mutant with two novel His residues (2-His mutant) did not bind as tightly to immobilized Ni(II) as did the mutant with five novel His residues (5-His mutant). When tested for affinity to immobilized Zn(II), only the 5-His mutant remained bound to the column. The affinity of the 5-His mutant for Ni(II) ions in solution was determined by binding experiments in an aqueous polymeric two-phase system. Analysis of the binding curve showed two binding sites per enzyme subunit and a dissociation constant of 6.7 +/- 1.6 mu M. The kinetic constants kcat, Km and kcat/Km for the reaction with glutathione and 1-chloro-2,4-dinitrobenzene were determined by steady-state kinetic analysis and the parameter values for the mutant forms were found to be indistinguishable from those obtained for the wild-type GST A1-1. The differences in surface charge in the mutant proteins as compared with the wild-type enzyme did not alter the pH dependence of kcat. The results provide an alternative method for purification of fully active recombinant GST A1-1 by the introduction of novel metal binding sites. The data also showed that two His residues are sufficient for Ni(II) binding.

Abstract  A spectrophotometric method is described for the detection of total glutathione (oxidized and reduced) in plant extracts based on the conjugation of reduced glutathione with 1-chloro-2,4-dinitrobenzene (CDNB) in the presence of dithioerythritol and glutathione S-transferase (EC 2.5.1.18) at pH 6.5. Glutathione S-transferase also supported a reaction with homoglutathione at about 5% of the rate with glutathione but, at equilibrium, the absorbance change for the two substrates was similar. The glutathione content of acidic extracts of wheat (Triticum aestivum var. Condor) roots as determined by the 1-chloro-2,4-dinitro-benzene (CDNB) method was 6.5% less than that obtained by the HPLC method based on the fluorescence of the monobromobimane derivative. The recovery of exogenous glutathione, added to the extracts, exceeded 90%. The method is suited to the specific determination of glutathione in poaecean species which also contain hydroxymethyl-glutathione since glutathione S-transferase reportedly does not support a reaction with this compound.

Abstract  The assessment of the skin sensitising capacity of chemicals is up to now investigated using in vivo animal tests. However there has been an increasing public and governmental concern regarding the use of animals for chemical screening. This has raised the need for the development of validated in vitro alternatives. Langerhans cells are potent antigen-presenting cells that play a crucial role in the development of allergic contact dermatitis. We used CD34(+) progenitor-derived dendritic cells from cord blood as an in vitro alternative for Langerhans cells. The cells were exposed to four contact allergens (nickel sulphate, dinitrochlorobenzene, oxazolone and eugenol) and two irritants (sodium dodecyl sulphate and benzalkonium chloride) for 3, 6, 12 and 24h. Using microarray analyses we revealed a set of 25 genes with an altered gene expression pattern after exposure to allergens and not to irritants. Five out of these 25 genes were selected and their gene expression changes were confirmed with real-time reverse transcriptase polymerase chain reaction. The list of 25 genes represent valuable candidates to be further evaluated for their capacity to predict the sensitizing potential of different classes of chemicals in studies using a more extended set of (non) allergic substances.

Abstract  BIOSIS COPYRIGHT: BIOL ABS. RRM MEETING PAPER MYTILUS-GALLOPROVINCIALIS ORGANOCHLORINES POLYCHLORINATED BIPHENYLS POLLUTANT DETOXIFICATION FAST PROTEIN LIQUID CHROMATOGRAPHY VENICE LAGOON ITALY

Abstract  A variety of hepatobiliary abnormalities occur in inflammatory bowel diseases (IBDs). The role of tight junction (TJ) in hepatobiliary complications have been well described. The purpose of this study was to investigate the role of inducible nitric oxide (NOS) in alteration of hepatocyte TJ paracellular barrier and in the rapid transcytotic vesicular pathway modification associated with intestinal inflammation. To address this question, we used an experimental model of colitis, induced by dinitrobenzene sulfonic acid (DNBS). When compared to DNBS-treated iNOS wild-type (WT) mice, DNBS-treated iNOS knock out mice (iNOSKO) mice experienced a significant less rate of the extent and severity of the histological signs of colon injury. Colon levels of the pro-inflammatory cytokines tumour necrosis factor, interleukin-1beta and interleukin-6 were also significantly reduced in iNOS-KO mice in comparison to wild-type mice. Liver histology from iNOSKO and wild-type mice iNOSWT did not show any parenchymal and portal tract inflammation at 4 days after DNBS administration. Serum total bilirubin and alanine aminotransferase, were significantly reduced in DNBS-iNOSKO mice vs DNBS-iNOSKO mice. Therefore, we found an increase of tight junctional permeability to lanthanum nitrate (molecular weight, 433) in the livers from DNBS-treated IL-10WT mice, lanthanum accumulated throughout the junctional area up to the most apical region bordering the lumen. Absence of a functional iNOS gene in iNOSKO mice resulted in a significant reduction of apical diffusion of lanthanum after DNBS-induced colitis. Immunofluorescent labeling of frozen liver sections from DNBS-iNOSWT mice showed a significant alteration of the immunolocalization for claudin-1 and zonula occludens (ZO)-1. In contrast, a significant reduced alteration in the localization of the immunosignals for claudin-1 and ZO-1 was observed in the liver from iNOSKO mice after DNBS administration. In conclusion, we suggest that the iNOS may represent an important pathophysiological mechanism of hepatobiliary injuries and cholestasis observed in patients with IBD.

Abstract  BIOSIS COPYRIGHT: BIOL ABS. Murine models for assessment of the contract sensitizing properties of chemicals rely on mouse ear swelling tests (Mest), which are not sensitive enough to detect weak sensitizers. The aim of the present study was to develop in mice and adjuvant-free Mest appropriate for in vivo detection of any type of sensitizer (weak to strong), and useful for in vitro assessment of contact sensitivity (CS). 3 haptens were tested: dinitrochlorobenzene (DCNB), para-phenylenediamine (pPD) and isoeugenol. We compared various protocols for induction of the CS reaction, differing by the site of induction, the number of applications and the concentrations of the 3 haptens. Comparison of the induction site for optimal Cs reaction showed that, in Balb/c mice, the back was a better site of induction than the abdomen. Detection of the sensitizing properties of weak sensitizers (pPD, isoeugenol) was possible using an adjuvant-free protocol, provided that the induction phase comprised hapten app

Abstract  BIOSIS COPYRIGHT: BIOL ABS. Simple and efficient ex vivo/in vitro screening systems for contact allergens are developed for alternative to conventional animal tests. We have previously proposed an ex vivo/in vitro proliferation assay as a first stage screening method with advantages over existing alternatives, using lymph node cells (LNC) from sensitized guinea pigs of the Hartley strain. In this study, we have first confirmed, by histochemical analysis using in vivo bromodeoxyuridine (BrdU) and pyronin staining, that the ex vivo/in vitro LNC proliferation reflects in vivo response of lymph nodes to contact allergens. Furthermore, to improve the LNC assay, we then have investigated several experimental conditions for their influences on the LNC assay, demonstrating that, (1) the subscapular and the cervical LNC responded highly to contact allergens, (2) among three cervical lymph nodes the superficial dorsal cervical lymph nodes were the most reactive, (3) several vehicles alone used for animal se

Abstract  In a previous work, a molecularly imprinted silica (MIS) sorbent was synthesized for the selective extraction of nitroaromatic explosives from real samples. This MIS packed in a cartridge was used for an off-line solid phase extraction procedure mainly based on hydrophobic and π-π interactions. In this work, the MIS was packed in a precolumn to be connected online with a reversed-phase LC system and a diode array detector. For this, the chromatographic conditions were first studied to obtain the separation of 1,3-dinitrobenzene, 1,3,5-trinitrobenzene, 2,4-dinitrotoluene, 2,6-dinitrotoluene, 2,4,6-trinitrotoluene, and tetryl. An optimized procedure dedicated to the selective treatment of aqueous samples was then developed with the MIS for the simultaneous extraction of the nitroaromatic compounds commonly used as explosives. Finally, the four nitrotoluenes were selectively extracted and determined simultaneously with extraction recoveries higher than 90% using the online device composed of the MIS coupled with a diphenyl chromatographic column. The potential of this sorbent was highlighted by its use for the cleanup of simulated post-blast samples.

Abstract  The photolysis of the charge-transfer complex of tetranitromethane and 1,2,4,5-tetramethylbenzene in dichloromethane or acetonitrile gives the epimeric 1,3,4,6-tetramethyl-3-nitro-6-trinitromethylcyclohexa-1,4-dienes 8 and 9, in addition to products of nuclear nitration 12 and side-chain modification 10, 11, and 13-18. Similar reactions of 1,2,3,5-tetramethylbenzene gave trans-1,3,5,6-tetramethyl-6-nitro-3-trinitromethylcyclohexa-1,4-diene 30 and two isomeric 'double' adducts 31 and 32, in addition to products of nuclear nitration 27 and side-chain modification 26, 28 and 29. The eliminative rearrangements of adducts 8 and 30 to give re-aromatized products in acetonitrile or [H-2(3)]acetonitrile and in [H-2]chloroform are reported. The photolysis of the charge-transfer complexes of tetranitromethane with either 1,2,4,5-tetramethylbenzene or 1,2,3,5-tetramethylbenzene in 1,1,1,3,3,3-hexafluoropropan-2-ol (HFP) gives a marked increase in the yields of ring-nitration products 12 or 27, respectively, reactions presumed to proceed via a nitrosation-oxidation sequence. Reaction of 1,2,4,5-tetramethylbenzene with excess nitrogen dioxide in HFP also results in extensive ring nitration to give 12 and 2,3,5,6-tetramethyl-1,4-dinitrobenzene (25); the latter compound is seen as arising via the 2,3,5,6-tetramethyl- 1,4-dinitrosobenzene (34). Similar reaction of 1,2,3,5-tetramethylbenzene gives ring-nitration product 27 as the major product. X-Ray crystal structures are reported for 2,4,6-trimethyl-1-(2',2',2'-trinitroethyl)benzene (26) and trans-1,3,5,6-tetramethyl-6-nitro-3-trinitromethyl-cyclohexa-1,4-diene (30). (C) Acta Chemica Scandinavica 1996.

Abstract  The immunosuppressive effect of topical ethacrynic acid (ECA) was tested on both the induction and elicitation phases of contact sensitization in a mouse model. ECA (0.5% in vehicle) reduced the sensitization response by >50% when the sensitizer was either dinitrochlorobenzene (DNCB), oxazalone (OX) or para-phenylenediamine (PPD), and was applied 1 day later to the ECA-pretreated skin site. The immunosuppressive effect of combining ECA with either hydrocortisone or with cis-urocanic acid was also tested. An additive suppressive effect was observed with ECA in both combinations. The effect of ECA (1% in vehicle) on blocking the elicitation phase was also examined in a mouse ear edema assay. ECA was highly effective in preventing the challenge response in mice previously sensitized to either DNCB, OX or PPD. ECA (1% in vehicle) was also tested for its ability to inhibit contact irritation. ECA (1% in vehicle) was highly effective in preventing ear edema due to topically applied skin irritants including arachidonic acid, capsaicin, lactic acid, phorbol myristate acetate, trans-retinoic acid, and sodium lauryl sulfate. ECA may be useful for both prophylaxis and therapeutic treatment of diverse skin conditions including contact dermatitis, eczema, and other related allergic skin disorders.

Abstract  The development of novel in vitro methods to assess risks of allergic sensitization are essential in reducing animal testing whilst maintaining consumer safety. The main research objectives of this study were to identify novel biomarkers to assess the sensitization predictability of chemicals. Phenotypic and cytokine responses of moDCs and MUTZ-3 cells were investigated following application of contact sensitizers; dinitrochlorobenzene (DNCB), cinnamaldehyde (Cin), eugenol (E), isoeugenol (IE), P-phenylenediamine (PPD) and non-sensitizers; salicyclic acid (SA) and sodium lauryl sulphate (SLS). CD86 was up-regulated on MUTZ-3 cells in response to DNCB, Cin and PPD, however, moDCs only modulated CD86 in response to DNCB and E. PDL-1 (Programmed death receptor ligand-1) proved a promising sensitization biomarker in MUTZ-3 cells where up-regulation occurred in response to DNCB, Cin, IE and PPD. Additionally, moDC-expressed PDL-1 was modulated in response to Cin, IE and E thus demonstrating improved sensitizer predictability when compared with CD86. MCP-1 and RANTES were identified as biomarkers of DNCB exposure but MCP-1 did not show any change in expression above controls for the other sensitizers investigated. However, RANTES was increased in MUTZ-3 cells by both DNCB and Cin. Our findings highlight novel biomarkers which, in MUTZ-3 cells, could be taken forward within a multiple biomarker in vitro assay ensuring strong and reliable predictability.

Abstract  Recent changes in regulatory requirements and social views on animal testing have accelerated the development of reliable alternative tests for predicting skin sensitizing potential of chemicals. In this study, we aimed to develop a new in vitro skin sensitization assay using reconstructed human epidermis, RhE model, which is expected to have broader applicability domain rather than existing in vitro assays. Microarray analysis revealed that the expression of five genes (ATF3, DNAJB4, GCLM, HSPA6 and HSPH1) related to cellular stress response were significantly up-regulated in RhE model after 6h treatment with representative skin sensitizers, 1-fluoro-2,4-dinitrobenzene and oxazolone, but not a non-sensitizer, benzalkonium chloride. The predictive performance of five genes was examined with eight skin sensitizers (e.g., cinnamic aldehyde), four non-sensitizers (e.g., sodium lauryl sulfate) and four pre-/pro-haptens (e.g., p-phenylenediamine, isoeugenol). When the positive criteria were set to obtain the highest accuracy with the animal testing (LLNA), ATF3, DNAJB4 and GCLM exhibited a high predictive accuracy (100%, 93.8% and 87.5%, respectively). All tested pre-/pro-haptens were correctly predicted by both ATF3 and DNAJB4. These results suggested that the RhE-based assay, termed epidermal sensitization assay (EpiSensA), could be an useful skin sensitization assay with a broad applicability domain including pre-/pro-haptens.

Abstract  The combined effects of micellar and chemical catalysis were studied with a view to improving the features of catalytic kinetic determinations. For this purpose we chose the reaction between N,N-dimethyl-p-phenylenediamine (DPD) and N,N-dimethylaniline (DA) to form Bindschedler's Green leuco base, which is oxidized by hydrogen peroxide in a reaction catalysed by Cu(II). This reaction was found to be accelerated by anionic sodium dodecylsulphate (SDS) and cationic dodecyltrimethylammonium bromide (DTAB) micelles. Several advantages were gained in relation to the analytical features of the kinetic photometric determination of Cu(II) when the reaction was developed in the presence of micelles compared to that occurring in an aqueous medium. Such advantages include a lower detection limit, higher sensitivity, precision and solubility of DPD and DA, and substantially increased selectivity in some cases. A detailed study of the parameters which influence both reactions is reported. Some observations on the effect of SDS and DTAB on the reaction are also commented on.

Abstract  In quantitative NMR (qNMR) selection of an appropriate internal standard proves to be crucial. In this study, 25 candidate compounds considered to be potent internal standards were investigated with respect to the ability of providing unique signal chemical shifts, purity, solubility, and ease of use. The (1)H chemical shift (delta) values, assignments, multiplicities and number of protons (for each signal), appropriateness (as to be used as internal standards) in four different deuterated solvents (D(2)O, DMSO-d(6), CD(3)OD, CDCl(3)) were studied. Taking into account the properties of these 25 internal standards, the most versatile eight compounds (2,4,6-triiodophenol, 1,3,5-trichloro-2-nitrobenzene, 3,4,5-trichloropyridine, dimethyl terephthalate, 1,4-dinitrobenzene, 2,3,5-triiodobenzoic acid, maleic acid and fumaric acid) were qualified using both differential scanning calorimetry (DSC) and NMR spectroscopy employing highly pure acetanilide as the reference standard. The data from these two methods were compared as well as utilized in the quality assessment of the compounds as internal standards. Finally, the selected internal standards were tested and evaluated in a real case of quantitative NMR analysis of a paracetamol pharmaceutical product.

Abstract  This paper presents precise sensitization test data of 15 chemicals with a wide spectrum of sensitization potencies, and proposes a new protocol and criteria for quantitative evaluation of sensitization potencies of chemicals. The tests were performed according to the design of Magnus-son and Kligman, changing the application concentrations for induction as well as for challenge phases. 3-dimensional relationships between mean response (or sensitization rate), induction and challenge concentrations were found in all chemicals tested. The following 2 values are proposed as a quantitative measure of sensitization potency: (a) the minimum induction concentration that induces a positive response; (b) the challenge concentration that induces a mean response approximately equal to 1.0 among the animals applied with the highest concentration for induction. Both values coincided with each other within the range of 1 order of magnitude in every compound except 2. The values varied by 5 orders or more of magnitude among the compounds, showing a wide variation of sensitization potencies among chemicals. A good correlation was found for every chemical between the value of sensitization potency thus obtained and the residual levels in causative products in human cases of allergic contact dermatitis. A new experimental protocol for obtaining values (a) and (b) is proposed.

Abstract  Keratinocytes mount immune responses through the secretion of a variety of inflammatory cytokines, soluble proteins and reactive oxygen species (ROS). However, the role of ROS in keratinocytes in response to allergens and irritants has not yet been elucidated. In this study, we investigated the (i) ROS production; (ii) potential sites of ROS production; (iii) expression of cell surface molecules; (iv) secretion of cytokines; and (v) ROS-dependent protein carbonylation in chemical-treated human keratinocyte cell line (HaCaT) cells. Treatment of HaCaT cells with 2,4-dinitrochlorobenzene (DNCB) and benzalkonium chloride (BKC) increased ROS levels in a time- and dose-dependent manner, as determined with dichlorodihydrofluorescein diacetate (CM-H(2) DCFDA), without reducing cell viability. Potential sources of ROS production were evaluated with pretreatment of diphenylene iodonium (DPI), an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase; rotenone, an inhibitor of the mitochondrial electron transport chain complex or allopurinol, a xanthine oxidase inhibitor. The DNCB-induced ROS was related to both NADPH oxidase and mitochondrial electron transport chain complex. Conversely, BKC-induced ROS was related to NADPH oxidase only. Western blotting using an anti-DNP antibody revealed ROS-dependent protein carbonylation in response to DNCB but not BKC. Both DNCB and BKC increased the secretion of IL-1α from HaCaT cells; however, ROS production as well as other changes, except DNCB-induced secretion of IL-1α, was not inhibited by antioxidants. Although the role of ROS in keratinocytes in response to chemicals was inconclusive, our results suggest that the characteristics of ROS produced by keratinocytes in response to chemicals might differ.

Abstract  Existing data indicate that the increase of il-1beta gene expression can be a promising marker of Langerhans cells activation after exposure to contact sensitizers. In this study, we were interested in development of an alternative in vitro screening test detecting such sensitizers. Two IL-1beta reporter constructs containing the enhanced green fluorescent protein (GFP) gene and mouse IL-1beta promoter fragments of varying lengths (-500 bp and -4093 bp) were used for transient transfections of J771A.1 murine monocyte-macrophage cells. As a result of the transfections performed using Lipofectamine reagent we did not observe any GFP fluorescence after stimulation of the cells with LPS as well as known sensitizers (potassium tetrachloroplatinate, dinitrochlorobenzene and nickel sulfate). Low transfection efficiency of J774A.1 cells (less than 0.1%) was confirmed using control plasmid containing GFP gene under the control of cytomegalovirus promoter. The fact that, using the same conditions, we were able to transfect murine fibroblasts 3T3-L1 with the control plasmid very efficiently, may support the theory of high metabolic activity of macrophages being responsible for the extremely low transfection efficiency. These data suggest limited suitability of J774A.1 cell line for transient transfections using cationic liposomes.

Abstract  The tumor promoting activity of 2,4-dinitrofluorobenzene (70348) (DNFB) was studied in mice. DNFB at concentrations of 0, 0.05, 0.1, or 0.2% was applied to the shaved backs of female SENCAR-mice once or twice, the second dose given 3 days after the first. Other mice were similarly treated with 2 micrograms (microg) 12-O-tetradecanoylphorbol-13-acetate (TPA). The mice were killed 24 or 48 hours after the last dose and skinned. The treated skin areas were examined for histological changes and analyzed for ornithine-decarboxylase (ODC) activity. The RNA was extracted and analyzed for expression of mRNA for ODC, c-fos, and c-jun. Mice were topically administered 0 or 1microg TPA and administered 0 or 0.2% DNFB 3 days later. They were killed 15 to 48 hours later and the treated skin areas were removed and assayed for protein-kinase-C (PKC) and ODC activity. Partially purified PKC was incubated with 0 or 1microg per milliliters DNFB or TPA for up to 48 hr. The effects on PKC activity were determined. SENCAR-mice were initiated topically with 0 or 10 nanomoles 7,12-dimethylbenz(a)anthracene (DMBA) and then treated with 0, 0.1, or 0.2% DNFB or 0 or 1microg TPA twice weekly for 27 weeks. They were monitored for skin tumor development. DNFB given once at 0.1 or 0.2% induced mild epidermal hyperplasia. Two doses induced pronounced hyperplasia. DNFB caused a small increase in ODC activity relative to that of TPA. The second dose caused a large increase in ODC activity. DNFB and TPA significantly increased mRNA coding for ODC, c-fos, and c-jun. DNFB did not activate ODC in-vitro. In-vivo, DNFB alone significantly decreased PKC activity after 15 hours. By 48 hours, PKC activity had returned to the control value. In combination with TPA, ODC activity was increased by DNFB to a greater extent than by DNFB treatment alone. In DMBA initiated mice, 0.1 and 0.2% DNFB induced tumors in 65 and 85% of the mice, respectively. The tumor multiplicities were 2.0 and 3.2 tumors per mouse, respectively. TPA produced a 100% tumor incidence. The authors conclude that DNFB induces many of the same responses as TPA including cellular inflammation and proliferation and skin tumor promotion. The effects are independent of the PKC pathway.

Abstract  1. The acute combined effects of cadmium (Cd) and nickel (Ni) on rat hepatic glutathione S-transferase (GST) activities toward the substrates 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB) and ethacrynic acid (EAA) were determined and compared to those of Cd or Ni alone. 2. Male adult rats (225-275 g) were administered either a single dose of Cd (3.58 mg CdCl2.H2O/kg, i.p.) 72 hr prior to sacrifice or a single dose of Ni (59.5 mg NiCl2.6H2O/kg, s.c.) 16 hr prior to sacrifice. For the combined treatment, animals received the single dose of Ni 56 hr after the single dose of Cd and they were killed 16 hr later. 3. Cd treatment alone did not produce any changes in the hepatic GST activities toward the substrates studied. 4. Ni treatment alone, however, significantly increased hepatic GST activity toward EAA whereas it was ineffective on GST activities for CDNB and DCNB. 5. Combined treatment of metals did not alter hepatic GST activities toward the substrates CDNB and DCNB. Hepatic GST activity for EAA, however, was significantly increased by the combined treatment. Nevertheless, the combined treatment did not augment the increase in GST activity for EAA noted by Ni treatment alone.

Abstract  Background: A variety of hepatobiliary abnormalities have been described in patients with chronic inflammatory bowel diseases (IBDs). The purpose of this study was to investigate the role of endogenous IL- 10 in alteration of hepatocyte TJ paracellular barrier and in the rapid transcytotic vesicular pathway modification associated with intestinal inflammation.

Materials and methods: To address this question, we used an experimental model of colitis, induced by dinitrobenzene sulfonic acid (DNBS). When compared to DNBS-treated IL-10 wild-type (IL-10WT) mice, DNBS-treated IL- 10 knock-out mice (IL- 10KO) mice experienced a higher rate of the extent and severity of the histological signs of colon injury.

Results: Colon and liver levels of the pro-inflammatory cytokines tumour necrosis factor, interleukin-1beta,6 and interleukin-6 were also greatly enhanced in IL-10KO mice in comparison to wild-type mice. Liver histology from IL-10KO and IL-10WT did not show any parenchymal and portal tract inflammation at 4 days after DNBS administration. Serum total bilirubin and Alanine aminotransferase, were significantly increased in DNBS-IL-10KO mice vs. DNBS-IL-10KO mice.

Therefore, we found an increase of tight junctional permeability to lanthanum nitrate (molecular weight, 433) in the livers from DNBS-treated IL-10WT mice; lanthanum accumulated throughout the junctional area up to the most apical region bordering the lumen. Absence of a functional IL-10 gene in IL-10KO mice resulted in a significant augmentation of apical diffusion of lanthanum after DNBS-induced colitis. Immunofluorescent labelling of frozen liver sections from DNBS-IL-10KO mice, immunolocalization for and claudin-1 and ZO-1 resulted in a significant alteration in the localization of the immunosignals for claudin- I and ZO- I after DNBS administration in comparison with DNBS-IL-10WT.

Conclusion: In conclusion, we suggest that the absence of IL-10 may represent an important pathophysiological mechanism of hepatobiliary injuries and cholestasis observed in patients with IBD.

Abstract  The murine local lymph node assay (LLNA) has been developed as an alternative to guinea pig models for the assessment of the contact sensitization potential. However, there is a need to develop a non-radioisotopic endpoint for the LLNA because of the radioisotopic method's requiring the use of special facilities. In this study, we investigated to evaluate the populations of intracellular cytokine producing cells and to analyze the expression of mRNA levels in the lymph node (LN) cells following allergen and irritant. Female Balb/c mice were treated by the topical application on the dorsum of both ears with strong sensitizers, 2,4-dinitrochlorobenzene (DNCB) and toluene diisocyanate (TDI) and a strong irritant, sodium lauryl sulfate (SLS), once daily for 3 consecutive days. The lymph node cells were harvested 72h after the final treatment. The analysis of intracellular cytokine cell in LN cells was performed with a flow cytometry. Mice were treated with DNCB and TDI showed a preferential increase in the percentage of CD4+IL-2+ cells compared with vehicle and irritant-treated mice. There was an increase in CD4+IFN-g+ cells of mice treated with DNCB and TDI, but no significant increases were observed in mice treated with SLS. Mice were treated with DNCB and TDI showed an increase in the percentage of CD4+IL-4+ cells compared with vehicle and irritant-treated mice. There was an increase in the mRNA level for interleukin 4 (IL-4) in mice treated with DNCB and TDI, but no significant increases were observed in mice treated with SLS. These results suggest that the population of interferon-gamma (IFN-g+) and IL-4+ cells on CD4+ cells and the mRNA expression for IL-4 in lymphocytes could be selectively modulated in allergen-treated mice.

Abstract  Although p38 mitogen-activated protein kinases (MAPK) play a crucial role in the activation of monocyte-derived dendritic cells (MoDC) by contact sensitizers, the upstream signals of p38 MAPK remain undetermined. To examine whether sensitizers induce redox or oxidative stress in dendritic cells (DC), which subsequently stimulate p38 MAPK, we measured the ratio of the oxidized (GSSG) versus reduced (GSH) form of cellular glutathione in MoDC stimulated with five sensitizers including NiCl2 and 2,4-dinitrochlorobenzene (DNCB) and three non-sensitizers including sodium dodecyl sulfate using colorimetric assays. All the sensitizers, but none of the non-sensitizers at sublethal concentration, reduced the GSH/GSSG ratio, which was accompanied by phosphorylation of p38 MAPK. Treatment with the antioxidant, N-acetyl-L-cysteine, which suppressed the reduction of the GSH/GSSG ratio, abrogated both the phosphorylation of p38 MAPK and the augmentation of CD86 expression. A similar response pattern was observed in THP-1 macrophage-monocyte cells. Unexpectedly, however, formaldehyde (HCHO) reduced the GSH/GSSG ratio in MoDC, but not in THP-1. This finding, in conjunction with the observation that DNCB and NiCl2 reduced the GSH/GSSG ratio at different kinetics, indicated that the sensitizers reduced the GSH/GSSG ratio by a different mechanism. These data suggest that the GSH/GSSG imbalance plays a crucial role in triggering DC maturation by sensitizers.