dinitrobenzenes and Hemolysis

Exposure to 2,4,6-trinitrotoluene (TNT) causes methemoglobin (metHb) formation, hemolysis and negative heme balance in vivo, but the mechanistic details are poorly understood. In the present study, we examined the participation of metabolic activation in TNT-mediated hematotoxicity. Exposure of rats with TNT (300 mg/kg, i.p.) for 4 days resulted in a decrease of hematocrit value coupled to an increase in metHb formation. The red blood cells treated with 4-hydroxylamino-2,6-dinitrotoluene (HADNT), a metabolite of TNT, underwent readily hemolysis in vitro, whereas such a phenomenon was not seen with TNT. Consistent with this, HADNT is active toward metHb formation and the decrease in thiol content of the globin moiety compared with TNT and its metabolites 4-amino-2,6-dinitrotoluene (ADNT) and 4-acetylamino-2,6-dinitrotoluene (AADNT). Furthermore, interaction of purified rat oxyhemoglobin (oxyHb) with HADNT, but not TNT, ADNT, and AADNT, caused a concentration-dependent production of H2O2 and ferrylhemoglobin (ferrylHb) which is a highly oxidizing state formed by reaction of oxyHb with H2O2. Notably, hemin was released during interaction of oxyHb with HADNT. Taken together, these findings suggest that HADNT is an active metabolite that mediates TNT-induced hematotoxicity via formation of prooxidants such as H2O2 and ferrylHb.

Topics: Aniline Compounds; Animals; Cells, Cultured; Dinitrobenzenes; Dose-Response Relationship, Drug; Erythrocytes; Hematocrit; Hemin; Hemoglobins; Hemolysis; Hydrogen Peroxide; Male; Methemoglobin; Nitro Compounds; Oxyhemoglobins; Rats, Inbred F344; Toluene

Antibody contents of IgG1, IgG2a, IgG2b and IgE were measured by enzyme-linked immunosorbent assay (ELISA) in ascites and sera obtained from mice injected with hybridomas producing monoclonal anti-DNP antibodies. In addition, IgG1 and IgE antibodies from sera of immunized mice were also measured by ELISA. Concomitantly, antibody contents were also determined by passive cutaneous anaphylaxis (PCA) in mice for IgG1, IgG2a, IgG2b, by PCA in guinea pigs for IgG2a, by PCA in rats for IgE and by passive hemolysis (PL) for IgG2a and IgG2b. Good correlations were found in the investigated samples between ELISA and the biological determinations.

Topics: Animals; Antibodies; Dinitrobenzenes; Enzyme-Linked Immunosorbent Assay; Hemolysis; Immunoenzyme Techniques; Immunoglobulin E; Immunoglobulin G; Mice; Mice, Inbred BALB C; Nitrobenzenes; Passive Cutaneous Anaphylaxis

For detection of mouse cells producing anti-ARS antibody a modified method using ARS-SRBC is described. For optimal results a suitable source of SRBC must be selected. The number of ARS groups per SRBC required for optimal lysis in PFC assay varies, and ARS-SRBC prepared by the method of Ingraham which are suitable for detection of rabbit-PFC give negative results with the mouse system. ARS-SRBC prepared for the PFC assay are unsuitable for the haemagglutination test and vice versa.

Topics: Animals; Antibody-Producing Cells; Azo Compounds; Binding, Competitive; Cattle; Dinitrobenzenes; Erythrocytes; Female; Guinea Pigs; Hemagglutination Tests; Hemolysis; Hemolytic Plaque Technique; Immunoglobulin Fab Fragments; Mice; Mice, Inbred A; Mice, Inbred C3H; p-Azobenzenearsonate; Rabbits; Serum Albumin; Sheep; Swine

Topics: Animals; Antibody Formation; Dinitrobenzenes; Female; Ficoll; Hemolysis; Immune Tolerance; Immunization; Immunoglobulin E; Immunoglobulin G; Mice; Mice, Inbred A; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred DBA; Nippostrongylus; Nitrobenzenes; Polysaccharides

Topics: Animals; Antibody Specificity; Binding, Competitive; Biopolymers; Complement Inactivator Proteins; Dinitrobenzenes; Hemolysis; Immunoglobulin A; Immunoglobulin G; Mice; Myeloma Proteins; Trinitrobenzenes

The capacity of mouse IgM, IgGl, IgG2 and IgA anti-dinitrophenyl (DNP) antibodies to activate mouse or guinea-pig complement was studied, using a sensitive haemolytic assay and two-dimensional immunoelectrophoresis to detect cleavage of mouse C3. Three monoclonal IgM antibodies, and a heterogeneous IgM fraction, lysed trinitrophenylated erythrocytes in the presence of guinea-pig C, but failed to produce lysis in the presence of mouse C. and only activated mouse C3 very inefficiently. A monoclonal IgGl antibody did not produce haemolysis in the presence of guinea-pig or mouse C, but cleaved mouse C3 via the alternative pathway. Two IgA myeloma proteins (M315 and M460) had similar properties. A heterogeneous IgG2 antibody fraction produced haemolysis in the presence of both mouse and guinea-pig C, and was shown to activate both the classical and alternative pathways of mouse C.

Topics: Animals; Complement C3; Complement Fixation Tests; Complement System Proteins; Dinitrobenzenes; Erythrocytes; Guinea Pigs; Hemocyanins; Hemolysis; Immunoglobulin A; Immunoglobulin G; Immunoglobulin M; Immunoglobulins; Mice; Mice, Inbred BALB C; Rabbits; Sheep; Trinitrobenzenes

An antibody assay is described based on the principle of complement mediated lysis of sheep red blood cells labelled with antigen. The technique provides a sensitive class specific assay enabling antibody in all three major immunoglobulin classes to be quantitated independently. The assay may be performed in tubes allowing precise measurement of antibody concentration, or in microtitre plates which provides a rapid estimation of antibody titre.

Topics: Animals; Antibodies, Anti-Idiotypic; Antibody Specificity; Complement System Proteins; Dinitrobenzenes; Erythrocytes; Hemolysis; Immune Sera; Immunoglobulin M; Ovalbumin; Sheep; Swine

Topics: Animals; Antibody Formation; Coliphages; Dinitrobenzenes; Dose-Response Relationship, Immunologic; Glutamates; Graft Survival; Hemagglutination Tests; Hemolysis; Lysine; Mice; Mice, Inbred BALB C; Mice, Nude; Skin Transplantation; T-Lymphocytes; Transplantation, Isogeneic; Tyrosine