Effects of miglyol 812 on rats after 4 weeks of gavage as compared with methylcellulose/tween 80.

Miglyol 812 is a medium-chain triglyceride used in toxicology studies as an excipient to improve test compound solubility/absorption. As part of a larger toxicology study, 15 Wistar Han IGS rats/sex/group were dosed by oral gavage for 4 weeks with 10 mL kg(-1) day(-1) of 100% Miglyol 812 or 0.5% methylcellulose/0.1% Tween 80 in water (MC-T) followed by 4 weeks without treatment to evaluate the potential effects of this excipient in long-term toxicology studies relative to a traditional excipient such as MC-T. Clinical signs evident during the dosing phase included soft and/or mucoid stool in 12/15 males and 11/15 females treated with Miglyol 812 but in no animals treated with MC-T. Animals treated with Miglyol 812 had a 6-7% statistically significant reduction in body weight gain as compared to MC-T-treated animals. Statistically significant changes in clinical chemistry parameters as compared to MC-T included decreased blood urea nitrogen (50% and 29% in males and females, respectively), increased cholesterol (1.6-fold and 1.5-fold in males and females, respectively), decreased total protein (6% and 8% in males and females, respectively), decreased globulins (15% and 11% in males and females, respectively), and increased triglycerides (2.8-fold and 1.7-fold in males and females, respectively). Absolute and relative thymic weights decreased 28% and 24%, respectively, in males, and 18% and 17%, respectively, in females without histological alterations. Histopathology revealed increased alveolar histiocytosis with focal interstitial inflammation in lungs in 5/10 males and 7/10 females treated with Miglyol 812 compared to only 1/10 males and 1/10 females treated with MC-T. All effects were reversible during the recovery period. Results of this study indicate that 100% miglyol 812 produces reversible gastrointestinal effects and decreases in body weight gains along with changes in several serum chemistry parameters. Therefore, it should not be considered innocuous when delivered by oral gavage in long-term rodent toxicology studies.

Dermal carcinogenicity in transgenic mice: effect of vehicle on responsiveness of hemizygous Tg.AC mice to phorbol 12-myristate 13-acetate (TPA).

The Tg.AC mouse is being evaluated for use in short-term carcinogenicity bioassays. Because the dermal test protocol necessitates dissolving test agents we determined the effects of several solvents on responsiveness of hemizygous mice to dermal applications of the classical skin tumor promoter. phorbol 12-myristate 13-acetate (TPA). Mice of both sexes received dermal applications of either acetone (negative control) or TPA in various vehicles [acetone, 100% methanol, 70% and 100% ethanol, DMSO and mixtures of acetone and ethanol (1:1), acetone and DMSO (4:1 and 1: 1). and acetone and olive oil (4:1)]. Negative control animals did not exhibit papillomas. When administered in acetone. ethanolic or methanolic vehicles TPA caused prompt and robust papillomatous responses. TPA was also tumorigenic in all nonalcoholic vehicles, except the acetone-olive oil mixture. Papilloma responses were generally delayed when TPA was applied in the nonalcoholic solvents but the distinction between TPA-dosed and negative control groups was unequivocal. These results show that choice of vehicle may affect the quantitative and qualitative nature of the response of Tg.AC mice to TPA, but 8 of 9 vehicles proved satisfactory for delivery of TPA.

Loss of palindromic symmetry in Tg.AC mice with a nonresponder phenotype.

The Tg.AC transgenic mouse carries the v-Ha-ras oncogene under the control of the zeta-globin promoter and is currently being used in a short-term carcinogenesis assay for safety testing of pharmaceuticals. A subset of hemizygous Tg.AC mice was found to be nonresponsive to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate, which characteristically induces skin papillomas in these mice with repeated dermal applications. We previously showed that responder and nonresponder hemizygous Tg.AC mice carry about 40 copies of transgene but that the nonresponders had lost a 2-kb BamHI fragment containing the zeta-globin promoter sequence. The present restriction enzyme and S1 nuclease digestion experiments strongly suggested that the 2-kb BamHI fragment resulted from the orientation of two transgenes in an inverted repeat formation. Two subsets of nonresponder Tg.AC mice were identified. Restriction enzyme and S1 nuclease digestion experiments suggested that one nonresponder genotype was produced by a large deletion of one or more near complete copies of transgene sequence and the other genotype was produced by a small deletion near the apex of the "head-to-head" juncture of the inverted repeat. Polymerase chain reaction amplification, cloning, and sequencing results confirmed the palindromic orientation of transgene in Tg.AC mice. Our results indicated that, despite the presence of multiple copies of transgene in a direct repeat orientation, loss of symmetry in the palindromic array of transgene sequence results in the loss of the responder phenotype in Tg.AC mice. Mol. Carcinog. 30:99-110, 2001. Published 2001 Wiley-Liss, Inc.

Dermal carcinogenicity in transgenic mice: relative responsiveness of male and female hemizygous and homozygous Tg.AC mice to 12-O-tetradecanoylphorbol 13-acetate (TPA) and benzene.

Assessment of the carcinogenic potential of chemical agents continues to rely primarily upon the chronic rodent bioassay, a resource-intensive exercise. Recent advances in transgenic technology offer a potential resource conserving approach to carcinogen detection. Incorporation of oncogenes with known roles in the development of neoplasms into the genomes of laboratory rodents may provide new models with the potential of quickly and accurately separating carcinogenic from noncarcinogenic chemicals. The insertion of the v-Ha-ras oncogene into the genome of FVB/N mice imparts the qualities of genetically initiated skin in the transgenic mouse line designated as Tg.AC. The skin of either hemizygous (animals carrying the transgene on 1 allele) or homozygous (transgene copies on both alleles) Tg.AC mice promptly responds to the application of nongenotoxic carcinogens, such as the classical tumor promoting phorbol esters, with the development of squamous papillomas. Tumor production generally begins after 8-10 applications of 2.5 micrograms/mouse (3 times/wk) of 12-O-tetradecanoylphorbol 13-acetate (TPA). Maximal tumor response is usually in evidence within 20 wk. If this transgenic mouse line is to be useful in the identification of carcinogenic chemicals, experimental protocols must be systematically optimized. Experiments were conducted to compare the relative responsiveness of male and female hemizygous and homozygous Tg.AC mice to the dermal application of TPA and the known human leukemogen, benzene. Results revealed shipment-related variabilities in the relative responsiveness of hemizygous male and female mice to the application of the proliferative agent. Homozygous mice of both sexes were more reliable and uniform in responsiveness to both TPA and benzene. Therefore, our standard protocol for the conduct of bioassays with the Tg.AC mouse line specifies the use of homozygous males and/or females.

Interaction of lymphocytes with Kupffer cells from halothane-exposed guinea pigs.

BACKGROUND: The purpose of this study was to investigate lymphocyte adhesion to Kupffer cells as a component of an immune-mediated mechanism for halothane hepatitis. METHODS: Kupffer cells were isolated from guinea pigs exposed to 1.0% halothane/40% oxygen and cultured with various synthetic antigens (trifluoroacetyl-protein adducts or hepatocyte homogenate from halothane-exposed animals). Latex beads were also added to Kupffer cell cultures to determine if activation of these macrophages would result in an increased cellular adhesion. Lymphocytes which had been surfaced-labeled with biotin were added to treated Kupffer cells, and lymphocyte adhesion was determined using a streptavidin-peroxidase reagent for colorimetric detection. RESULTS: Trifluoroacetyl-lysine, trifluoroacetyl-rabbit albumin or guinea pig albumin did not induce lymphocyte adhesion. Latex beads also had no effect on cellular adhesion. A noticeable increase in lymphocyte adhesion to Kupffer cells previously treated with either trifluoroacetyl-guinea pig albumin or hepatocyte homogenate was observed. Stimulation of lymphocytes with phorbol myristate acetate did not have an effect on adhesion. Addition ofantimajor histocompatibility complex II antibody had a significant inhibitory effect on lymphocyte adhesion to Kupffer cells treated with trifluoroacetyl-guinea pig albumin or homogenate. CONCLUSION: These results demonstrate that the halothane trifluoroacetyl-guinea pig albumin antigen and hepatocyte homogenate enhances the adhesion of lymphocytes to cultured Kupffer cells and that this interaction involves major histocompatibility complex II expression on stimulated Kupffer cells. The interaction between Kupffer cells which present specific trifluoroacetyl-antigens and lymphocytes from halothane-exposed animals may play an important role in the pathogenesis of halothane hepatitis.

Kupffer cells from halothane-exposed guinea pigs carry trifluoroacetylated protein adducts.

The anesthetic, halothane, is bioactivated by the liver cytochrome P450 system to trifluoroacetyl-chloride, which can readily acylate liver protein. Covalent binding of the trifluoroacetyl moiety may result in hapten formation leading to the induction of an immune response and ultimately halothane hepatitis. In this study the presence of trifluoroacetylated-protein adducts in Kupffer cells was investigated to learn how the immune system might come in contact with the proteins. Guinea pigs were exposed to 1.0% halothane, 40% oxygen for 4 h. Kupffer cells were isolated on days 1 through 9 post-exposure, by liver perfusion and purification by elutriation. Using gel electrophoresis and Western blotting techniques, it has been demonstrated that Kupffer cells obtained from halothane-treated guinea pigs, do carry trifluoroacetyl-protein adducts as recognized by an anti-trifluoroacetyl-rabbit serum albumin antibody. Apparent molecular weights of polypeptides bound by trifluoroacetyl were of a wide range, 25-152 kDa. Bands were most prominent in the larger Kupffer cells with more appearing at lower molecular weights. Trifluoroacetyl-protein adducts were not detected in lung, spleen, lymph node or peripheral blood macrophages. This work suggests a role for Kupffer cells in the presentation of altered proteins in the liver to cells of the immune system.

Demonstration of a cellular immune response in halothane-exposed guinea pigs.

Halothane hepatitis is considered to be a result of an idiosyncratic autoimmune reaction brought about by the formation of neoantigens that have been generated by covalent binding of halothane biotransformation intermediates. The guinea pig is being examined as an animal model to investigate an immune-mediated mechanism for halothane hepatotoxicity. Male Hartley guinea pigs were exposed to 1% halothane/40% oxygen for 4 hr, three times with 40-day intervals. Kupffer cells and splenocytes were isolated from animals on various days after each halothane exposure. Splenocytes were cocultured in a lymphocyte transformation test with various concentrations of TFA(trifluoroacetylated)-antigens for 7 days and proliferation was measured by 3H-thymidine incorporation. In a second experiment, Kupffer cells were cocultured with autologous as well as allogeneic splenocytes with or without concanavalin A to determine whole cell sensitization and accessory function by Kupffer cells from treated animals. A 4-fold increase in splenocyte proliferation occurred in response to TFA-guinea pig albumin. No significant increase in proliferation could be detected with TFA-lysine or guinea pig albumin. A 14-fold increase in splenocyte proliferation also occurred in response to Kupffer cells from halothane-exposed animals. Autologous splenocytes demonstrated more of a response from treated versus control animals, indicating possible involvement of major histocompatibility complex II antigens. These results indicate recognition of TFA-antigens and Kupffer cells as antigen-presenting cells in halothane-exposed guinea pigs. This study provides good evidence that a cellular immune response is involved in the guinea pig after halothane exposure.

The effect of carbamazepine and its reactive metabolite, 9-acridine carboxaldehyde, on immune cell function in vitro.

Carbamazepine, a widely used anticonvulsant, is associated with a wide range of adverse reactions including agranulocytosis, aplastic anemia and drug-induced lupus. It has also been reported to alter immune function in a variety of ways. We had previously demonstrated that carbamazepine is oxidized by activated neutrophils to several metabolites and this leads to covalent binding of the drug to the cells. It appears that the major metabolite responsible for this binding is 9-acridine carboxyaldehyde. In this study the effects on leukocyte function of carbamazepine and its leukocyte-generated metabolites were compared. Incubation of lymphocytes with 100 microM 9-acridine carboxaldehyde resulted in 40% cell death while carbamazepine at this concentration had no effect on viability. The effect on the immune cell function was investigated using the autologous mixed lymphocyte reaction (AMLR), allogeneic mixed lymphocyte reaction (MLR), lymphocyte transformation test (LTT) and mitogenesis assays. Alteration of immune cell function by the reactive metabolite, 9-acridine carboxyaldehyde, was demonstrated by an increased proliferation at low concentrations (0.08-1.0 microM) and inhibition at high concentrations (20-100 microM) in the allogeneic MLRs. Carbamazepine had no measurable effect. 9-Acridine appears to have more of an effect on B-cells since this augmentation-suppression phenomenon was also observed in mitogenesis assays with Staphylococcus aureus, a B-cell mitogen, in contrast to mostly inhibition observed in the mitogenesis assay with phytohemagglutinin, a T-cell mitogen. Again, carbamazepine had no measurable effects at comparable concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Covalent binding of carbamazepine oxidative metabolites to neutrophils.

Carbamazepine therapy is associated with several types of idiosyncratic drug reactions, including hematological disorders. In previous studies, we found that carbamazepine was metabolized by the myeloperoxidase/H2O2 system of activated neutrophils, and covalent binding of the drug to neutrophils was observed. Several metabolites were identified, including 9-acridine carboxaldehyde. Iminostilbene, a minor hepatic metabolite of carbamazepine, was metabolized to a much greater extent than carbamazepine to similar metabolites, including 9-acridine carboxaldehyde. In the present study, the covalent binding of iminostilbene to activated neutrophils was also found to be 10-fold greater than that of carbamazepine. In addition, the binding of 9-acridine carboxaldehyde to neutrophils was 100-fold that of carbamazepine and did not require activation of the neutrophils. This suggests that this aldehyde is the reactive intermediate responsible for much of the binding. To understand possible mechanisms of covalent binding, we investigated the reaction of 9-acridine carboxaldehyde with nucleophiles and found that a reaction occurs with primary amines, such as n-butylamine and N-alpha-acetyllysine, with the formation of an imine. Sodium cyanoborohydride was used to reduce the imine to a stable secondary amine. This suggests a possible mechanism for 9-acridine carboxaldehyde binding to neutrophils that could involve physiological reducing systems in place of the borohydride. 9-Acridine carboxaldehyde may be responsible for some of the adverse reactions associated with carbamazepine, especially those that involve bone marrow.

Carbamazepine metabolism to a reactive intermediate by the myeloperoxidase system of activated neutrophils.

Carbamazepine is an anticonvulsant which is associated with a significant incidence of hypersensitivity reactions including agranulocytosis. We have postulated that many drug hypersensitivity reactions, especially agranulocytosis and lupus, are due to reactive metabolites generated by the myeloperoxidase (MPO) (EC 1.11.1.7) system of neutrophils and monocytes. This led to a study of the metabolism and covalent binding of carbamazepine with MPO/H2O2/Cl- and neutrophils. Metabolism and covalent binding were observed in both systems and the same pathway appeared to be involved; however, the metabolism observed with the MPO system was approximately 500-fold greater than that observed with neutrophils. The metabolites identified were an intermediate aldehyde, 9-acridine carboxaldehyde, acridine, acridone, choloroacridone, and dichloroacridone. We postulate that the first intermediate in the metabolism of carbamazepine is a carbonium ion formed by reaction of hypochlorous acid (HOCl) with the 10,11 double bond. Although we have no direct proof for the proposed carbonium ion, it provides the most likely mechanism for the observed ring contraction. Iminostilbene, a known metabolite of carbamazepine, was also metabolized by a similar pathway leading to ring contraction; however, the rate was much faster and the first step may involve N-chlorination and a nitrenium ion intermediate. These data confirm that carbamazepine is metabolized to reactive intermediates by activated leukocytes. Such metabolites could be responsible for some of the adverse reactions associated with carbamazepine, especially reactions such as agranulocytosis and lupus which involve leukocytes.