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Evaluation of the contact allergic potential of 2,4-dinitrothiocyanobenze
Poster Session 2L. lmmunotoxicity matic Mutation and Recombination Test (SMART) of the fruitfly Drosophila, PAs show strong genotoxic effects which correlate in potency with hepatotoxicity in mammals. PAs occur in nature as free base alkaloids and as N-oxides. In the SMART approach, the N-oxides of indicinc and retrorsine are less genotoxic than the parent alkaloids. We can increase the cytochrome P450 (CYP) levels in the SMART assay by the introduction of a mutant CYP-synthesis control gene. This decreases to some extent the genotoxic effects of the PAs monocrotaline and retrorsine, but increases those of retrorsine-Noxide. The results suggest that in biotransformation of PAs in vivo a CYP-dependent detoxifying excretion pathway counterbalances the bioactivation pathway.
Keywords: in vivo genotoxicity; drosophila; pyrrolizidine alkaloids; pharmacokinetics; high-level cytochrome P450
P2L. Immunotoxicity I
OF MURINE NICKEL-SPECIFIC T CELL IP2L-271 I ISOLATION HYBRIDOMAS I
Christian V. Vult~e *, Peter Griem, Ernst Gleichmann. Division of
Immunology, Medical Institute of Environmental Hygiene, Diisseldorf, Germany The molecular mechanism of allergies to heavy metals, such as nickel, is still elusive. This is the first report on the isolation of murine nickel-specific T cell hybridomas. Female C57BL/6 mice were immunized with nickel compounds plus mouse serum albumin (MSA) which is known to be a nickel-binding protein. MSA was preincubated with either Ni(II)CI2 or Ni(IV)(OH)20 which exerts strong oxidizing effects on organic compounds. Neither Ni(II)C12 nor Ni(IV)(OH)20 led to the isolation of T cell hybridoma clones specific for nickel-altered MSA, i.e. the metal-protein complex used for priming. Following immunization with Ni(II)CI2 plus MSA, one clone was found that recognized splenic antigen-presenting cells (APC) without MSA in the presence of Ni(II), i.e. unknown nickel-altered self-protein. After immunization with Ni(IV)(OH)20pretreated MSA, however, 61 clones recognizing APC plus Ni(II) were obtained, suggesting that a higher oxidation state of Ni is involved in T cell priming. For challenging of established clones though Ni(II) proved sufficient. Since the clones obtained did react to Ni(II) plus unknown self-proteins, but failed to react to the original nickel-MSA complexes, the role of MSA in T cell priming to nickel is obscure. Almost all of the clones failed to react to Ni(lI) when B-cell hybridoma line LB27.4, instead of spleen cells, were used as APC. This indicates that LB27.4 cells do not spontaneously present those self-peptides in the context of which Ni(II) is recognized by the T cells. Hence, LB27.4 might be a tool to identify the self-proteins altered by nickel.
Keywords: nickel allergy; murine T cell hybridomas I
ANTIOXlDANT STATUS OF RAT LUNG AND IP2L-2721 THE SOME BAL.PARAMETERS 24 HOURS AND 3 I
MONTHS AFTER EXPOSURE TO ASBESTOS-AMOSITE
Marta Hurb,4nkov~i, Zuzana Kov~i~ikovd, Altbeta Kaiglov~, Emil Ginter. Institute of Preventive and Clinical Medicine,
Department of Respiratory Toxicology, Bratislava, Slovak Republic This study was designed to evaluate some lung parameters and lung antioxidant status 24 hours and three months after intratracheal instillation of amosite to rats. The mechanisms of asbestos related diseases have not been clearly defined yet. It is believed that in these patho-
75
logical processes are involved - in addition to the others - reactive oxygen intermediates as well. The bronchoalveolar lavage (BAL) was performed and some parameters in BAL or lung tissue [the number of leukocytes (Le)/ml, the number of alveolar macrophages (AM)/ml, viability and phagocytic activity of AM, AM/granulocytes (GR) ratios in lavage fluid, levels of alkaline phosphatase (AP), lactate dehydrogenase (LDH) and total amount of protein] were investigated. Additionally, the lung antioxidant status was examined by markers such as ascorbic acid (AA), retinol, t~-tocoferol, glutathione peroxidase (GSH-Px) and lipid peroxides (LPO) in our study. The results of exposed groups were compared to the results of control animals. 24 hours after exposure 11 of 15 parameters (increase of Le, GR, LPO, AP, LDH, proteins in BAL and decrease of number and viability of AM in BAL, AA in BAL and tissue as well as GSH-Px in tissue) were significantly changed. 3 months after exposure only 6 parameters were statistically significantly changed, namely: increased phagocitic activity, AP, and decreased antioxidant parameters such as AA in BAL, retinol in tissue, t~-tocopherol and GSH-Px in lung tissue. Decreased parameters of antioxidant status - also 3 months after exposure - may signalize presumable pathological changes in lung tissue. The results of our study indicate that these antioxidant parameters can play an important role in lung tissue disease development after exposure to asbestos.
Keywords: amosite; bronchoalveolar lavage parameters; antioxidant status
P2L-2731
EVALUATION OF THE CONTACT ALLERGIC POTENTIAL OF 2,4-DINITROTHIOCYANOBENZENE
Rebecca J. Dearman *, Jennifer Hilton, David A. Basketter 1, Ian Kimber. Zeneca Central Toxicology Laboratory, Alderley Park,
Macclesfield, Cheshire, SKIO 4TJ, UK; I Unilever Environmental Safety Laboratory, Sharnbrook, Bedfordshire, MK44 1LQ, UK It has been reported by some investigators that 2,4-dinitrothiocyanobenzene (DNTB) causes contact sensitization [1] while others have found it to be a non-sensitizing tolerogen [2]. The sensitizing properties of DNTB have been re-evaluated in comparison with 2,4-dinitrochlorobenzene (DNCB), a potent contact allergen. A local lymph node assay was performed according to the standard protocol [3] using BALB/c mice. Exposure to 1% DNTB or 1% DNCB resulted in positive responses of comparable magnitude. Additional groups of mice were exposed topically to chemical over a 2 week period. Draining lymph nodes were excised, a single cell suspension prepared and the production of the cytokines interleukins 4 and 10 (IL-4 and IL-10) and interferon y (IFN-y) were measured by enzyme-linked immunosorbent assays. Both chemicals induced the production by draining LNC of high concentrations of IFN-y, but only low levels of IL-4 and IL-10, consistent with the selective activation of T helper cell type 1 (Thl) responses and skin sensitizing activity. The data demonstrate that, in common with DNCB, the type of immune response associated with skin sensitizing chemicals is induced by exposure to DNTB. It is not appropriate therefore to regard DNTB as a negative control in assessments of contact allergenicity. [1] Kimber, Iet al, Int. Arch. Allergy Appl. Immunol, 81,258-264, 1986 [2] Iijima, I and Katz, SI, Invest. Dermatol., 81,325-330, 1983 [3] Kimber, Iet al, Toxicology,93, 13-31, 1994.
Keywords: contact allergy; LLNA; DNTB; cytokines
Keywords: in vivo genotoxicity; drosophila; pyrrolizidine alkaloids; pharmacokinetics; high-level cytochrome P450
P2L. Immunotoxicity I
OF MURINE NICKEL-SPECIFIC T CELL IP2L-271 I ISOLATION HYBRIDOMAS I
Christian V. Vult~e *, Peter Griem, Ernst Gleichmann. Division of
Immunology, Medical Institute of Environmental Hygiene, Diisseldorf, Germany The molecular mechanism of allergies to heavy metals, such as nickel, is still elusive. This is the first report on the isolation of murine nickel-specific T cell hybridomas. Female C57BL/6 mice were immunized with nickel compounds plus mouse serum albumin (MSA) which is known to be a nickel-binding protein. MSA was preincubated with either Ni(II)CI2 or Ni(IV)(OH)20 which exerts strong oxidizing effects on organic compounds. Neither Ni(II)C12 nor Ni(IV)(OH)20 led to the isolation of T cell hybridoma clones specific for nickel-altered MSA, i.e. the metal-protein complex used for priming. Following immunization with Ni(II)CI2 plus MSA, one clone was found that recognized splenic antigen-presenting cells (APC) without MSA in the presence of Ni(II), i.e. unknown nickel-altered self-protein. After immunization with Ni(IV)(OH)20pretreated MSA, however, 61 clones recognizing APC plus Ni(II) were obtained, suggesting that a higher oxidation state of Ni is involved in T cell priming. For challenging of established clones though Ni(II) proved sufficient. Since the clones obtained did react to Ni(II) plus unknown self-proteins, but failed to react to the original nickel-MSA complexes, the role of MSA in T cell priming to nickel is obscure. Almost all of the clones failed to react to Ni(lI) when B-cell hybridoma line LB27.4, instead of spleen cells, were used as APC. This indicates that LB27.4 cells do not spontaneously present those self-peptides in the context of which Ni(II) is recognized by the T cells. Hence, LB27.4 might be a tool to identify the self-proteins altered by nickel.
Keywords: nickel allergy; murine T cell hybridomas I
ANTIOXlDANT STATUS OF RAT LUNG AND IP2L-2721 THE SOME BAL.PARAMETERS 24 HOURS AND 3 I
MONTHS AFTER EXPOSURE TO ASBESTOS-AMOSITE
Marta Hurb,4nkov~i, Zuzana Kov~i~ikovd, Altbeta Kaiglov~, Emil Ginter. Institute of Preventive and Clinical Medicine,
Department of Respiratory Toxicology, Bratislava, Slovak Republic This study was designed to evaluate some lung parameters and lung antioxidant status 24 hours and three months after intratracheal instillation of amosite to rats. The mechanisms of asbestos related diseases have not been clearly defined yet. It is believed that in these patho-
75
logical processes are involved - in addition to the others - reactive oxygen intermediates as well. The bronchoalveolar lavage (BAL) was performed and some parameters in BAL or lung tissue [the number of leukocytes (Le)/ml, the number of alveolar macrophages (AM)/ml, viability and phagocytic activity of AM, AM/granulocytes (GR) ratios in lavage fluid, levels of alkaline phosphatase (AP), lactate dehydrogenase (LDH) and total amount of protein] were investigated. Additionally, the lung antioxidant status was examined by markers such as ascorbic acid (AA), retinol, t~-tocoferol, glutathione peroxidase (GSH-Px) and lipid peroxides (LPO) in our study. The results of exposed groups were compared to the results of control animals. 24 hours after exposure 11 of 15 parameters (increase of Le, GR, LPO, AP, LDH, proteins in BAL and decrease of number and viability of AM in BAL, AA in BAL and tissue as well as GSH-Px in tissue) were significantly changed. 3 months after exposure only 6 parameters were statistically significantly changed, namely: increased phagocitic activity, AP, and decreased antioxidant parameters such as AA in BAL, retinol in tissue, t~-tocopherol and GSH-Px in lung tissue. Decreased parameters of antioxidant status - also 3 months after exposure - may signalize presumable pathological changes in lung tissue. The results of our study indicate that these antioxidant parameters can play an important role in lung tissue disease development after exposure to asbestos.
Keywords: amosite; bronchoalveolar lavage parameters; antioxidant status
P2L-2731
EVALUATION OF THE CONTACT ALLERGIC POTENTIAL OF 2,4-DINITROTHIOCYANOBENZENE
Rebecca J. Dearman *, Jennifer Hilton, David A. Basketter 1, Ian Kimber. Zeneca Central Toxicology Laboratory, Alderley Park,
Macclesfield, Cheshire, SKIO 4TJ, UK; I Unilever Environmental Safety Laboratory, Sharnbrook, Bedfordshire, MK44 1LQ, UK It has been reported by some investigators that 2,4-dinitrothiocyanobenzene (DNTB) causes contact sensitization [1] while others have found it to be a non-sensitizing tolerogen [2]. The sensitizing properties of DNTB have been re-evaluated in comparison with 2,4-dinitrochlorobenzene (DNCB), a potent contact allergen. A local lymph node assay was performed according to the standard protocol [3] using BALB/c mice. Exposure to 1% DNTB or 1% DNCB resulted in positive responses of comparable magnitude. Additional groups of mice were exposed topically to chemical over a 2 week period. Draining lymph nodes were excised, a single cell suspension prepared and the production of the cytokines interleukins 4 and 10 (IL-4 and IL-10) and interferon y (IFN-y) were measured by enzyme-linked immunosorbent assays. Both chemicals induced the production by draining LNC of high concentrations of IFN-y, but only low levels of IL-4 and IL-10, consistent with the selective activation of T helper cell type 1 (Thl) responses and skin sensitizing activity. The data demonstrate that, in common with DNCB, the type of immune response associated with skin sensitizing chemicals is induced by exposure to DNTB. It is not appropriate therefore to regard DNTB as a negative control in assessments of contact allergenicity. [1] Kimber, Iet al, Int. Arch. Allergy Appl. Immunol, 81,258-264, 1986 [2] Iijima, I and Katz, SI, Invest. Dermatol., 81,325-330, 1983 [3] Kimber, Iet al, Toxicology,93, 13-31, 1994.
Keywords: contact allergy; LLNA; DNTB; cytokines