Abstract  Some non-protein alpha-amino acids were derivatized with 1-fluoro-2,4-dinitrophenyl-5-L-alaninamide (Marfey's reagent, MR, FDNP-L-Ala-NH(2),) and four of its structural variants FDNP-L-Phe-NH(2), FDNP-L-Val-NH(2), FDNP-L-Leu-NH(2) and FDNP-L-Pro-NH(2). The resultant diastereomers were separated by normal and reversed phase thin layer chromatography (TLC) and reversed phase HPLC. In normal phase TLC, best resolution was obtained with solvent combination of phenol-water (3:1) while in reversed phase TLC mixtures of acetonitrile with triethylammonium phosphate buffer were found successful for resolution of diastereomers. The separation behavior of diastereomers prepared with different reagents was compared. The diastereomers of most of the amino acids prepared with FDNP-L-Leu-NH(2) were best separated while those prepared with FDNP-L-Pro-NH(2) failed to separate in most of the cases. The diastereomers were also separated on a reversed phase C(8) column with gradient elution using mixture of aqueous-trifluoroacetic acid (TFA) and acetonitrile and with detection at 340 nm. The effects of TFA concentration, flow rate and run time on HPLC separation were studied.

Abstract  Serial magnetic resonance imaging (MRI) was used to evaluate the influences of dietary deoxycholic acid (DCA) on the rate of progression of chemically induced hepatocellular neoplasms in rats. Male Fischer-344 rats with established persistent hepatocellular nodules generated by the Solt-Farber protocol were exposed to dietary DCA (0.3%) between 6 and 12 mo of age. Growth of nodules and carcinomas in vivo was measured by morphometric quantification of tumor images obtained every 6 wk. The final stages of neoplastic progression were determined by terminal histopathological examination and by expression and functional evaluation of glutathione S-transferase (GST) isoenzyme phenotypes. Dietary DCA increased the number of hepatocellular neoplasms per rat, accelerated the rate of growth of persistent nodules, and increased the histological progression of liver tumors. Expression of immunoreactive GST subunits Yf, Ya, and Yb1 was induced in early persistent nodules, a pattern that was maintained throughout the study in both basal diet and DCA-fed groups. However, 5% of early nodules and about 75% of advanced neoplasms were partially or completely deficient in GST Yb2 expression in both groups. DCA did not alter the cytosolic activity for the GST substrates 1-chloro-2,4-dinitrobenzene (CDNB) or trans-4-phenyl-3-buten-2-one (tPBO) in tumors or surrounding liver. However, in both groups, CDNB activity was increased in the tumors relative to the surrounding nonneoplastic tissue, whereas activity for tPBO, a substrate more specific for the Yb2 subunit, was reduced in the tumors. All advanced neoplasms were similarly more resistant than surrounding liver to DNA-binding metabolites of aflatoxin B-1 or benzo[a]pyrene. These data demonstrate that DCA can increase the progression of established hepatocellular nodules to larger, more advanced neoplasms but does not preferentially select for a specific GST phenotype. Preferential loss of constitutively expressed GST Yb2 in both basal diet and DCA-fed groups may be an important aspect of progression from resistant nodules to advanced cancers in this model. These studies also demonstrate that serial MRI is a useful tool for measuring the rates of enlargement and patterns of growth in established hepatocellular neoplasms.

Abstract  In order to explore the protective function of human glutathione S-transferase pi (GST-pi) in vitro and in vivo, transfected NIH 3T3 clones were examined in cytotoxicity assays using the carcinogen (+/-)anti-benzo(a)pyrene 7,8-diol-9,10-epoxide (BPDE) or inoculated into nude mice and treated with the carcinogen benzo(a)pyrene (BP) to induce tumor formation. The human GST-pi cDNA under the control of the murine alpha(2)(I)collagen promoter was transfected into NIH 3T3 cells and G418 resistant clones were analyzed by Southern, Northern, Western, and two-dimensional analysis. Clone A2 stably expressed human GST-pi and has 2.5-fold greater activity toward the substrate 1-chloro-2,4-dinitrobenzene and a 1.7-fold increase in LD50 for BPDE in vitro when compared to control-transfected clone G3. This increase in protection, however, did not prevent the formation of BP-induced tumors in vivo.

Abstract  To better guide the development of liquid chromatography/electron capture-atmospheric pressure photoionization-mass spectrometry (LC/EC-APPI-MS) in analysis of low polarity compounds, the ionization mechanism of 19 compounds was studied using dopant assisted negative ion-APPI. Four ionization mechanisms, i.e., EC, dissociative EC, proton transfer, and anion attachment, were identified as being responsible for the ionization of the studied compounds. The mechanisms were found to sometimes compete with each other, resulting in multiple ionization products from the same molecule. However, dissociative EC and proton transfer could also combine to generate the same [M - H](-) ions. Experimental evidence suggests that O(2)(-*), which was directly observed in the APPI source, plays a key role in the formation of [M - H](-) ions by way of proton transfer. Introduction of anions more basic than O(2)(-*), i.e., C(6)H(5)CH(2)(-), into the APPI source, via addition of di-tert-butyl peroxide in the solvent and/or dopant, i.e., toluene, enhanced the deprotonation ability of negative ion-APPI. Although the use of halogenated solvents could hinder efficient EC, dissociative EC, and proton transfer of negative ion-APPI due to their EC ability, the subsequently generated halide anions promoted halide attachment to compounds that otherwise could not be efficiently ionized. With the four available ionization mechanisms, it becomes obvious that negative ion-APPI is capable of ionizing a wider range of compounds than negative ion chemical ionization (NICI), negative ion-atmospheric pressure chemical ionization (negative ion-APCI) or negative ion-electrospray ionization (negative ion-ESI).

Abstract  BACKGROUND: Offspring of asthmatic mothers have increased risk of developing asthma, based on human epidemiologic data and experimental animal models. The objective of this study was to determine whether maternal allergy at non-pulmonary sites can increase asthma risk in offspring.

METHODS: BALB/c female mice received 2 topical applications of vehicle, dinitrochlorobenzene, or toluene diisocyanate before mating with untreated males. Dinitrochlorobenzene is a skin-sensitizer only and known to induce a Th1 response, while toluene diisocyanate is both a skin and respiratory sensitizer that causes a Th2 response. Both cause allergic contact dermatitis. Offspring underwent an intentionally suboptimal protocol of allergen sensitization and aerosol challenge, followed by evaluation of airway hyperresponsiveness, allergic airway inflammation, and cytokine production. Mothers were tested for allergic airway disease, evidence of dermatitis, cellularity of the draining lymph nodes, and systemic cytokine levels. The role of interleukin-4 was also explored using interleukin-4 deficient mice.

RESULTS: Offspring of toluene diisocyanate but not dinitrochlorobenzene-treated mothers developed an asthmatic phenotype following allergen sensitization and challenge, seen as increased Penh values, airway inflammation, bronchoalveolar lavage total cell counts and eosinophilia, and Th2 cytokine imbalance in the lung. Toluene diisocyanate treated interleukin-4 deficient mothers were able to transfer asthma risk to offspring. Mothers in both experimental groups developed allergic contact dermatitis, but not allergic airway disease.

CONCLUSION: Maternal non-respiratory allergy (Th2-skewed dermatitis caused by toluene diisocyanate) can result in the maternal transmission of asthma risk in mice.

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  A new member of the cytochrome P450 superfamily, CYP2S1, has recently been identified in human and mouse. In this paper, we review the data currently available for CYP2S1. The human CYP2S1 gene is located in chromosome 19q13.2 within a cluster including CYP2 family members CYP2A6, CYP2A13, CYP2B6, and CYP2F1. These genes also show the highest homology to the human CYP2S1. The gene has recently been found to harbor genetic polymorphism. CYP2S1 is inducible by dioxin, the induction being mediated by the Aryl Hydrocarbon Receptor (AHR) and Aryl Hydrocarbon Nuclear Translocator (ARNT) in a manner typical for CYP1 family members. In line with this, CYP2S1 has been shown to be inducible by coal tar, an abundant source of PAHs, and it was recently reported to metabolize naphthalene. This points to the involvement of CYP2S1 in the metabolism of toxic and carcinogenic compounds, similar to other dioxin-inducible CYPs. CYP2S1 is expressed in epithelial cells of a wide variety of extrahepatic tissues. The highest expression levels have been observed in the epithelial tissues frequently exposed to xenobiotics, e.g., the respiratory, gastrointestinal, and urinary tracts, and in the skin. The observed ubiquitous tissue distribution, as well as the expression of CYP2S1 throughout embryogenesis suggest that CYP2S1 is likely to metabolize important endogenous substrates; thus far, retinoic acid has been identified. In conclusion, CYP2S1 exhibits many features of interest for human health and thus warrants further investigation.

Abstract  The skin protects our body by producing an efficient barrier membrane, the stratum corneum, from desiccation as well as from various damaging effects of environmental chemicals. Although the skin expresses various cytokines after barrier perturbation, exact cell types producing each cytokine have not been determined. Using a cell culture system, we analyzed the initial responses of various cutaneous cells to treatments simulating epicutaneous stimuli induced by a barrier perturbation of the skin in comparison with those caused by irritant or hapten exposure. We used cultured normal human epidermal keratinocytes (NHEK), human microvascular endothelial cells (HMVEC) and normal human dermal fibroblasts (NHDF). We treated them with the following chemicals and examined their cytokine mRNA levels 6 h later: high osmotic (0.5 molar) NaCl and hydrogen peroxide (H2O2), which simulate desiccation and exposure to high oxygen pressure, respectively, that may take place in vivo after perturbation of the barrier. In addition, we also studied their response to two representive haptens, nickel chloride (NiCl2) and dinitrochlorobenzene (DNCB), and an irritant, sodium dodecyl sulfate (SDS). We found that 0.5 M NaCl treatment increased mRNA levels of proinflammatory cytokines such as IL-1alpha, IL-6 and IL-8 as well as ICAM-1 in NHEK and IL-1alpha, IL-1beta and IL-6 mRNA levels in NHDF. In contrast, H2O2 treatment remarkably increased IL-10, GMCSF and ICAM-1 mRNA levels in NHEK, and IL-6 mRNA levels in HMVEC and NHDF. The exposure to haptens did not induce any remarkable increase in mRNA levels of the proinflammatory cytokines in NHEK. But NiCl2 increased IL-1alpha, IL-6 and IL-8 mRNA levels in HMVEC, while DNCB increased only their IL-6 mRNA levels. By contrast, SDS stimulated all the cell types to increase at least some of these proinflammatory cytokine mRNA levels. Our present data suggest that each skin component cell participates in inflammatory processes of the skin through its distinctive cytokine production profile when the skin barrier is compromized physically or chemically.

Abstract  These studies were conducted to investigate the potential use of a flow cytometric analysis method for the identification and differentiation of chemicals with the capacity to induce irritation, IgE- or T cell-mediated hypersensitivity responses. An initial study investigated the ability of equally sensitizing concentrations (determined by local lymph node assay) of IgE-mediated (Toluene Diisocyanate-TDI) and T cell-mediated (Dinitrofluorobenzene-DNFB) allergens to differentially modulate the IgE+B220+ population in the lymph nodes draining the dermal exposure site. Sodium lauryl sulfate (SLS) was also tested as a nonsensitizing irritant control. Female B6C3F1 mice were dermally exposed once daily for 4 consecutive days, with the optimum time point for analysis determined by examining the IgE+B220+ population 8, 10, and 12 days post-initial chemical exposure. At the peak time point, day 10, the IgE+B220+ population was significantly elevated in TDI (41%), while moderately elevated in DNFB (18%) exposed animals when compared to the vehicle (0.8%), and remained unchanged in SLS (2.2%) exposed animals when compared to the ethanol control (2.5%). Experiments in our laboratory and others have demonstrated that the draining lymph node B220+ population becomes significantly elevated following exposure to allergens (IgE- and T cell-mediated), not irritants, allowing for their differentiation. An existing mouse ear swelling assay was used to identify chemical irritants. Therefore, using the endpoints of percent ear swelling, percent B220+ cells, and percent IgE+B220+ cells, a combined irritancy/phenotypic analysis assay was developed and tested with tetradecane (irritant), toluene diisocyanate, trimellitic anhydride (IgE-mediated allergens), benzalkonium chloride, dinitrofluorobenzene, oxazolone, and dinitrochlorobenzene (T cell-mediated allergens) over a range of concentrations. Based upon the pattern of response observed, a paradigm was developed for continued evaluation: Irritant exposure will result in significant ear swelling without altering the B220+ or IgE+B220+ populations. Exposure to sensitizers (IgE-mediated or T cell-mediated) will increase the B220+ population and the percent ear swelling will remain unchanged or will significantly increase, depending on the irritancy capacity of the chemical. Both the IgE+B220+ and B220+ populations will become elevated at the same test concentration following exposure to IgE-mediated, hypersensitivity inducing allergens. At its peak, the percent of IgE+B220+ cells will be equal to the percent of B220+ cells. The B220+ population will increase at a lower test concentration than the IgE+B220+ population, following exposure to T cell-mediated, hypersensitivity inducing allergens. At its peak, the percent of IgE+B220+ cells will reach less than half that of the percent of B220+ cells. The irritancy/phenotypic analysis method may represent a single murine assay able to identify and differentiate chemicals with the capacity to induce irritation, or IgE-mediated or T cell-mediated responses.

Abstract  Glutathione S-transferases (GSTs) are a family of multifunctional proteins playing important roles in detoxification of harmful physiological and xenobiotic compounds in organisms. In our study, a gene encoding a GST from Proteus mirabilis strain V7, gst(Pm)-4, was cloned and conditionally expressed in Escherichia coli strain BL21(DE3). The purified Gst(Pm)-4 protein, with an estimated molecular mass of approximately 23 kDa, was able to conjugate 1-chloro-2,4-dinitrobenzene and bind to the GSH-affinity matrix. Real-time reverse transcriptase PCR suggested that mRNA level of gst(Pm)-4 was increased in the presence of CdCl2, CuCl2, HgCl2 and PbCl2, respectively. Correspondingly, overexpression of gst(Pm)-4 in the genetically engineered bacterium Top10/pLacpGst exhibited higher heavy metal resistance compared to the control Top10/pLacP3. Another genetically engineered bacterium Top10/pBATGst, in which the DNA encoding Gst(Pm)-4 protein was fused with the DNA encoding Pfa1-based auto surface display system, was built. Top10/pBATGst could constitutively express the chimeric Gst(Pm)-4 and anchor it onto the cell surface subsequently. Almost 100% of the Hg2+ within the range of 0.1-100 nM was adsorbed by Top10/pBATGst, and 80% of the bounded Hg2+ could be desorbed from bacterial cells when pH was adjusted to 6.0. Thus, Top10/pBATGst can be potentially used for efficient treatment of Hg2+-contaminated aquatic environment. (C) 2013 Elsevier B.V. All rights reserved.

Abstract  Glutathione-transferase (GST) systems in human nasal mucosa were purified and characterized. Nasal membranes were removed from five males and five females during therapeutic surgery. Samples were minced, homogenized, and centrifuged to obtain supernatant fractions for GST assay and subsequent enzyme purification by affinity chromatography and isoelectric focusing. Characterization was performed by SDS/polyacrylamide gel electrophoresis (PAGE), Western blot analysis, and GST assay. GST activity for the ten samples in a 1-chloro-2,4-dinitrobenzene substrate ranged from 47 to 113 nanomoles per minute per milligram (nmol/min/mg) protein (mean, 76.8nmol/min/mg). Mean GST activity in Sprague-Dawley-rat nasal mucosa was 18.0nmol/min/mg protein. Pooled fractions passed through an affinity column increased in specific activity to 17 micromoles/min/mg protein. Isoelectric focusing indicated that 85 percent of GST activity was explained by an anionic peak at pH of 4.5, while remaining peaks were near neutral or basic; the acidic peak was identified as class pi GST. The affinity bound fraction contained a GST subunit which migrated at 28,000Mr, and could not be identified as either a pi, mu, or alpha isoenzyme. The authors suggest that this isoenzyme may be specific to nasal mucosal tissue.

Abstract  2,6‐Dinitro‐1‐iodobenzene and 2,4‐dinitro‐1‐iodobenzene were deiodinated with tributyltin hydride at different temperatures using various addition modes. The product ratios of 1,3‐dinitrobenzene and the corresponding tributylstannyldinitrobenzene compounds were determined by NMR in order to evaluate the optimum conditions for impending tritiation experiments. Copyright © 1995 John Wiley & Sons, Ltd.

Abstract  DA-9601 (Stillen™) is a novel anti-peptic formulation prepared from the ethanol extracts of Artemisia asiatica possessing anti-oxidative, anti-allergic and anti-inflammatory activities. However, their effect on atopic dermatitis (AD) has not been studied yet. In this study, we report that topical application of DA-9601 suppressed house dust mite extract (Dermatophagoides farinae extract, DFE) and 2, 4-dinitrochlorobenzene (DNCB)-induced AD-like skin lesions in BALB/c mice model. We established atopic dermatitis model in BALB/c mice by repeated local exposure of DFE/DNCB to the ears. Repeated alternative treatment of DFE/DNCB caused AD-like lesions. DA-9601 reduced AD-like skin lesions based on ear thickness and histopathological analysis, and serum IgE levels. DA-9601 inhibited mast cell infiltration into the ear and elevation of serum histamine in AD model. In addition, DA-9601 suppressed DFE/DNCB-induced expression of IL-4, IL-13, IL-31, and TNF-α in the ears. Taken together, our results showed that topical application of DA-9601 exerts beneficial effects in animal model of AD, suggesting that DA-9601 might be a candidate for the treatment of AD.

Abstract  The seed coat extract of Tamarindus indica, a polyphenolic flavonoid, has been shown to have antioxidant properties. The present studies investigated the inhibitory effect of the seed coat extract of T. indica on nitric oxide production in vitro using a murine macrophage-like cell line, RAW 264.7, and in vitro and in vivo using freshly isolated B6C3F1 mouse peritoneal macrophages. In vitro exposure of RAW 264.7 cells or peritoneal macrophages to 0.2-200 mug/mL of T. indica extract significantly attenuated (as much as 68%) nitric oxide production induced by lipopolysaccharide (LPS) and interferon gamma (IFN-gamma) in a concentration-dependent manner. In vivo administration of T. indica extract (100-500 mg/kg) to B6C3F1 mice dose-dependently suppressed TPA, LPS and/or IFN-gamma induced production of nitric oxide in isolated mouse peritoneal macrophages in the absence of any effect on body weight. Exposure to T. indica extract had no effect on cell viability as assessed by the MTT assay. In B6C3F1 mice, preliminary safety studies demonstrated a decrease in body weight at only the highest dose tested (1000 mg/kg) without alterations in hematology, serum chemistry or selected organ weights or effects on NK cell activity. A significant decrease in body weight was observed in BALB/c mice exposed to concentrations of extract of 250 mg/kg or higher. Oral exposure of BALB/c mice to T. indica extract did not modulate the development of T cell-mediated sensitization to DNFB or HCA as measured by the local lymph node assay, or dermal irritation to nonanoic acid or DNFB. These studies suggest that in mice, T. indica extract at concentrations up to 500 mg/kg may modulate nitric oxide production in the absence of overt acute toxicity. (C) 2003 Elsevier Ltd. All rights reserved.

Abstract  A wide range of industrial chemicals can induce respiratory allergic reactions. Hence, there is an urgent need for methods identifying and characterizing the biological action of chemicals in the lung. Here, we present an easy, reliable alternative method to measure lung function changes ex vivo after exposure to chemical allergens and compare this to invasive in vivo measurements after sensitization with the industrial chemicals trimellitic anhydride (TMA) and 2,4-dinitrochlorobenzene (DNCB). Female BALB/c mice were sensitized epicutaneously with the respiratory allergen TMA and the contact sensitizer DNCB. The early allergic response to TMA and DNCB was registered in vivo and ex vivo on day 21 after inhalational challenge with dry standardized aerosols or after exposure of precision-cut lung slices (PCLS) to dissolved allergen. Airway hyperresponsiveness (AHR) to increasing doses of methacholine (MCh) was measured on the next day in vivo and ex vivo. Bronchoalveolar lavage (BAL) was performed for immunological characterization of local inflammation. TMA-sensitized mice showed AHR to MCh in vivo (ED(50): 0.06 microg MCh vs. 0.21 microg MCh in controls) and in PCLS (EC(50): 0.24 microM MCh vs. 0.4 microM MCh). TMA-treated animals showed increased numbers of eosinophils (12.8 x 10(4) vs. 0.7 x 10(4)) and elevated eotaxin-2 concentrations (994 pg/ml vs. 167 pg/ml) in BAL fluid 24 h after allergen challenge. In contrast, none of these parameters differed after sensitization with DNCB. The present study suggests that the effects of low molecular weight allergens, like TMA and DNCB, on ex vivo lung functions tested in PCLS reflect the in vivo situation.

Abstract  The hormone-dependence of malignant melanoma is brought to mind by some epidemiological facts (rarely found in children, more frequently in females, higher frequency in case of prolonged use of oral contraceptives from an early age). On the other hand, estrogen receptors can be found in about 30 p. 100 of malignant melanomas. Starting from this hypothesis of a sub-population of hormone-dependent malignant melanomas, it seemed of interest to us to study the efficiency of an anti-estrogen, namely Tamoxifen (Nolvadex) in the management of metastatic malignant melanoma. It was used at a 40 mg daily dose-regimen on four patients (three post-menopausal females, one male) with multiple estrogen positive cutaneous metastases. In post-menopausal women, two cases of total regression were observed, associated with a distinct increase in progesterone receptors while under treatment and with a return to the initial stage when the treatment was stopped. This phenomenon can be explained by a double self-contradictory effect of the drug, already well known in other hormone-dependent cancers such as those of the breast or the endometrium, namely an anti-estrogen result on the tumoral growth and an estrogen result as attested by the synthesis of progesterone receptors. Certain malignant melanomas therefore behave as endocrine-dependent tumours and may so answer an anti-hormone treatment through a competitive fixation on estrogen-receptors. Our clinical results are in agreement with such a theory since Tamoxifen is at best effective on cutaneous metastases of post-menopausal women. The overall efficiency on all metastases is of the order of 10.6 p 100 (complete or partial regression).(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract  After administration of beta-naphthoflavone and Clophen A50 to juvenile rainbow trout, activities of hepatic cytochrome P-450-dependent deethylation of 7-ethoxyresorufin was increased 172- and 49-fold, respectively. Glutathione transferase activity towards 1-chloro 2,4 dinitrobenzene and UDP glucuronosyltransferase activities towards p-nitrophenol, 1-naphthol and testosterone were increased 1.4 to 3.0-fold by beta-naphthoflavone or Clophen A50. However, significant increases of the rate of glucuronidation of 1-naphthol by Clophen A50 and of testosterone by both Clophen A50 and beta-naphthoflavone were only determined when the activities were measured in digitonin activated microsomes. Epoxide hydrolase activity was not affected by beta-naphthoflavone or Clophen A50. The time course of induction of the various xenobiotic metabolizing enzymes exhibited different patterns. 7-Ethoxyresorufin-O-deethylase activity reached peak values 3 and 7 days after the administration of beta-naphthoflavone and Clophen A50, respectively. The rate of induction of glutathione transferase activity and UDP glucuronosyltransferase activities towards p-nitrophenol and 1-naphthol were relatively slow and did not reach distinct peak levels. These activities were still on maximum levels 4-6 weeks after the treatment. Glucuronidation of testosterone reached peak values 1 week after treatment with both beta-naphthoflavone and Clophen A50. The dissimilar patterns of induction of the cytochrome P-450-dependent activities and the various conjugation activities may indicate that these xenobiotic metabolizing enzymes are differently regulated in the rainbow trout liver.

Abstract  The removal of nitroaromatics from polluted water is difficult due to their high stability to conventional treatment methods. This paper presents a method for the destruction of 1,3-dinitrobenzene and 2,4-dinitrotoluene in aqueous solutions. The compounds are shown to be stable to reaction with ozone, even under ultrasonic activation. The use of ultrasound enhances the rate of electrochemical reduction but the overall rate of reaction is still slow. However, the simultaneous application of ultrasound and ozonation to the electrochemical reaction allows virtually complete destruction of the compounds in short times. The effect is attributed to the ultrasonic enhancement of the electrochemical process giving intermediates that are susceptible to ozone oxidation. While further analytical work is needed to deduce the exact contributions of the various possible degradation mechanisms, the work demonstrates the synergies that can be gained by using combined techniques for the destruction of these difficult compounds.

Abstract  Drug-induced allergic reactions (DIARs), including allergic hepatitis, cutaneous reactions, and blood dyscrasias, are unpredictable and can be life threatening. Although current studies suggest that DIARs are caused by immunogenic drug-protein adducts, it remains unclear what factors determine the susceptibility to DIARs. We hypothesized that most individuals may be resistant to DIARs in part because they become immunologically tolerant to drug-protein adducts in the liver, an organ with tolerogenic properties. Because animal models of DIARs are elusive, we tested this hypothesis using a murine model of 2,4-dinitrochlorobenzene (DNCB)-induced delayed type hypersensitivity reaction that is mediated by immunogenic 2,4-dinitrophenylated (DNP)-protein adducts. Intravenous pretreatment of mice with DNP-BSA led to its accumulation in hepatic Kupffer cells (KC) and induced immunological tolerance to subsequent DNCB sensitization. Tolerance could be abrogated by prior depletion of KC or induced in naive mice by transferring a T cell-depleted, KC-enriched fraction of liver nonparenchymal cells from mice tolerized 1 month earlier by DNP-BSA pretreatment. These findings implicate KC as a primary and sustained inducer of tolerance against DNP-protein adducts and suggest a similar role in modulating allergic reactions against drug-protein adducts. Perhaps genetic and/or environmental factors affecting the activities of these cells may play a role in determining individual susceptibility to DIARs.

Abstract  The present review paper summarizes the data available in the literature concerning dinitrophenolic compounds and evaluates male reproductive toxicity in experimental animals. Gavage and feeding doses of 2-sec-butyl-4,6-dinitrophenol (dinoseb; CAS No. 88-85-7) manifested testicular toxicity, and 4,6-dinitro-o-cresol (DNOC; CAS No. 534-52-1) showed similar but weaker testicular toxicity in laboratory animals. Consecutive doses of dinoseb and DNOC by gavage seemed to induce spermatotoxicity by disturbing spermiogenesis or the maturation process of sperm in the epididymis, and the most probable target cells of spermatotoxicity were thought to be testicular spermatids in rats. Prolonged exposure to dinoseb and DNOC in the diet also induced testicular toxicity in rats. However, the feeding dose of dinoseb irreversibly affected the early stage of spermatogenesis and produced infertility in rats. On the other hand, 2,4-dinitrophenol (DNP; CAS No. 51-28-5) did not show testicular toxicity in laboratory animals according to available literature. Further studies in laboratory animals with nitrophenolic compounds are required for clarification of their testicular toxicity and for risk assessment in humans.

Abstract  The formation of the glutathione S-conjugate of monochlorobimane (GSH- bimane) in human colon adenocarcinoma cells was identified by HPLC- fluorimetry and its transport from the cells was found to be temperature-sensitive, saturable and ATP-dependent. The apparent K(m) and Vmax values were 2.4 +/- 0.5 nmol GSH-bimane/10(6) cells and 0.5 +/- 0.1 nmol GSH-bimane/min per 10(6) cells, respectively. This active transport of GSH-bimane was inhibited by low micromolar concentrations of classical uncouplers of oxidative phosphorylation, namely carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), carbonylcyanide m-chlorophenylhydrazone (CCCP) and 2,4-dinitrophenol (DNP). The efflux of GSH-bimane was competitively inhibited by chlorambucil (CMB) and 1-chloro-2,4-dinitrobenzene (CDNB), two other substrates of GST. This study demonstrates the presence and kinetic measurements of the glutathione S-conjugate export (GS-X) pump in human colon cancer cells, an export pump whose function has been implicated in the phenomenon of multidrug resistance.

Abstract  To assess possible maternal hepatic and reproductive effects of this uncharged, low molecular weight, lipophilic chlorinated benzene 0, 100, 300 and 1000 mg/kg/day of 1,2,3,4-tetrachlorobenzene (TCB) was orally administered to pregnant rats on days 9-13 of gestation and the animals were killed on day 14 of pregnancy. Phenobarbital and beta-naphthoflavone were administered to other pregnant rats as positive hepatic controls. Maternal mortality (7/19 rats) was increased and body weight gain was greatly decreased in the 1000 mg/kg/day TCB group. Liver to body weight ratio and hepatic microsomal protein content were unaffected by any TCB treatment. On day 14 maternal NADPH-cytochrome c reductase activity was increased at 1000 mg/kg/day, while the maternal hepatic microsomal cytochrome P-450 content was significantly induced by both 300 and 1000 mg/kg/day of TCB. Microsomal N-demethylation of aminopyrine was increased from 2.6 to 4.0 and 4.5 nmol/mg protein/min at doses of 300 and 1000 mg/kg TCB, respectively. However, maternal hepatic microsomal ethoxyresorufin O-deethylase activity was not consistently increased by TCB. Hepatic glutathione S-transferase activity towards 1,2-dichloro-4-nitrobenzene was increased only by the 1000 mg/kg/day TCB treatment. The rate of microsomal p-nitrophenol and phenolphthalein glucuronidation was increased by TCB administration. Embryonic growth was adversely affected by TCB treatment. Yolk sac diameter, embryonic crown-rump length, and head length were all decreased by treatment with 300 mg/kg/day TCB. This TCB treatment did not significantly elevate the number of dead or abnormal embryos.

Abstract  The morphology of glassy carbon surfaces was investigated by the atomic force microscope (AFM) method. Multi-wall carbon nanotubes (MWCNTs) were purified and investigated with a scanning electron microscope (SEM) and a transmission electron microscope (TEM). An electrochemical technique based on the glassy carbon electrode (GCE) or MWCNT-modified GCEs was used for the detection of nitroaromatic compounds (NACs), namely 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitrobenzene (TNB), 2,4-dinitrotoluene (2,4-DNT), and 1,3-dinitrobenzene (1,3-DNB). MWCNT-modified GCEs were more sensitive than GCEs to TNB, 2,4-DNT, and 1,3-DNB, with the detection limit down to b level, whereas the modified GCEs showed lower sensitivity to TNT. In varying degrees, the accumulation of nitro compounds can be promoted by MWCNT-modified GCEs in the detection process, a property which can be attributed to the large surface area and graphene-sheet structure of MWCNTs.

Abstract  The effect of aging on hepatic drug conjugation in 5- to 6-, 12- to 13- and 22- to 23-month-old female Fischer 344 rats was examined. The overall disposition of acetaminophen including the formation and elimination kinetics of its sulfate and glucuronide metabolites were used as in vivo probes. The effects of aging on selected in vitro drug metabolizing enzyme activities and on the pattern of phenol and bile salt sulfotransferase isoenzymes were also determined. Aging decreased the total clearance of acetaminophen and the partial clearance of acetaminophen to acetaminophen sulfate by 36 and 47%, respectively. Increasing age also resulted in a reduced partial clearance of acetaminophen to the glucuronide- (24%) and to the glutathione-derived conjugates (29%). UDP glucuronosyltransferase activity toward 1-naphthol, morphine and testosterone was unaffected by advanced age, whereas there was a significant correlation between increased age and increased UDP glucuronosyltransferase activity toward estrone. Cytochrome P-450 concentration and glutathione-S-transferase activity toward 1-chloro-2,4-dinitrobenzene were unchanged by aging. Oxidative demethylase activity toward p-nitroanisole was decreased 18% and sulfotransferase activities toward p-nitrophenol, acetaminophen and glycolithocholate were decreased 27, 12 and 12%, respectively, in the 22- to 23-month-old rats, compared to the 5- to 6-month-old animals. In contrast to the age-related feminization in the pattern of sulfotransferase isoenzyme activities that occurs in male rats, there was no effect of aging on the pattern of phenol and bile salt sulfotransferase isoenzyme activities in female rats.

Abstract  The mutagenicities of 26 nitro-aromatic compounds and their 10 related compounds for Salmonella typhimurium TA 100 and TA 98 were tested both with and without (mammalian) S-9 mix. The mutagenicities of the products formed by exposing pyrene to NO2 and and SO2 gases were tested for TA 98 without S-9 mix. Nine of 10 polynucleararomatic compounds without the nitro groups were nonmutagenic for both TA 100 and TA 98 with or without S-9 mix. Of 26 nitro-aromatic compounds, 24 were mutagenic for TA 100 and/or TA 98 with or without S-9 mix. All of 6 carcinogenic nitro compounds tested were mutagenic in this test. The specific mutagenic activities of nitroaromatic compounds became higher as the numbers of fused rings and nitro groups increased. The specific mutagenic activities of nitro-aromatic compounds with 3 or 4 fused rings were higher without S-9 mix than with S-9 mix for both TA 100 and TA 98. The reaction of pyrene with NO2 gas (nitration) was enhanced by SO2 gas.