SummaryImmune surveillance against tumors usually depends on T cell recognition of tumor antigens presented by major histocompatibility complex (MHC) molecules, whereas MHC class I tumors may be controlled by natural killer (NK) cells. Perforin-dependent cytotoxicity is a major effector function ofCD8 + MHC class I-restricted T cells and of NK cells. Here, we used perforin-deficient C57BL/6 (PKO) mice to study involvement of perforin and Fas ligand in tumor surveillance in vivo.We induced tumors in PKO and normal C57BL/6 mice by (a) injection of different syngeneic tumor cell lines of different tissue origin in naive and primed mice; (b) administration of the chemical carcinogens methylcholanthrene (MCA) or 12-O-tetradecanoylphorbol-13-acetare (TPA) plus 7,12-dimethylbenzanthracene (DMBA), or (c) by injection of acutely oncogenic Moloney sarcoma virus. The first set of models analyzes the defense against a tumor load given at once, whereas the last two sets give information on immune defense against tumors at the very moment of their generation. Most of the tumor cell lines tested were eliminated 10-100-fold better by C57BL/6 mice in an unprimed situation; after priming, the differences were more pronounced. Lymphoma cells transfected with Fas were controlled 10-fold better by PKO and C57BL/6 mice when compared to untransfected control cells, indicating some role for FasL in tumor control. MCA-induced tumors arose more rapidly and with a higher incidence in PKO mice compared to C57BL/6 or CDS-deficient mice. DMBA+TPA-induced skin papillomas arose with similar high incidence and comparable kinetics in both mouse strains. C57BL/6 and PKO mice have a similar incidence ofMoloney murine sarcoma and leukemia virus-induced sarcomas, but tumors are larger and regression is retarded in PKO mice.Thus, perform-dependent cytotoxicity is not only a crucial mechanism of both cytotoxic T lymphocyte-and NK-dependent resistance to injected tumor cell lines, but also operates during viral and chemical carcinogenesis in vivo. Experiments addressing the role of Fasdependent cytotoxlcity by studying resistance to tumor cell lines that were stably transfected with Fas neither provided evidence for a major role of Fas nor excluded a minor contribution of Fas in tumor surveillance.
Dose-response curves for the first interaction of a chemical with a biochemical target molecule are usually monotonic; i.e., they increase or decrease over the entire dose range. However, for reactions of a complex biological system to a toxicant, nonmonotonic (biphasic) dose-effect relationships can be observed, showing a decrease at low dose followed by an increase at high dose, or vice versa. We present four examples to demonstrate that nonmonotonic dose-response relationships can result from superimposition of monotonic dose responses of component biological reactions. Examples include (i) a membrane-receptor model with receptor subtypes of different ligand affinity and opposing downstream effects (adenosine receptors A1 vs. A2), (ii) androgen receptor-mediated gene expression driven by homodimers, but not mixed-ligand dimers, (iii) repair of background DNA damage by enzymatic activity induced by adducts formed by a xenobiotic, (iv) rate of mutation as a consequence of DNA damage times rate of cell division, the latter being modulated by cell-cycle delay at low-level DNA damage, and cell-cycle acceleration due to regenerative hyperplasia at cytotoxic dose levels. Quantitative analyses based on biological models are shown, and factors that affect the degree of nonmonotonicity are identified. It is noted that threshold-type dose-response curves could in fact be nonmonotonic. Our analysis should promote a scientific discussion of biphasic dose responses and the concept termed "hormesis," and of default procedures for low-dose extrapolation in toxicological risk assessment.
Furan has been found in a number of heated food items and is carcinogenic in the liver of rats and mice. Estimates of human exposure on the basis of concentrations measured in food are not reliable because of the volatility of furan. A biomarker approach is therefore indicated. We searched for metabolites excreted in the urine of male Fischer 344 rats treated by oral gavage with 40 mg of furan per kg of body weight. A control group received the vehicle oil only. Urine collected over two 24-h periods both before and after treatment was analyzed by a column-switching LC-MS/MS method. Data were acquired by a full scan survey scan in combination with information dependent acquisition of fragmentation spectra by the use of a linear ion trap. Areas of 449 peaks were extracted from the chromatograms and used for principal component analysis (PCA). The first principal component fully separated the samples of treated rats from the controls in the first post-treatment sampling period. Thirteen potential biomarkers selected from the corresponding loadings plot were reanalyzed using specific transitions in the MRM mode. Seven peaks that increased significantly upon treatment were further investigated as biomarkers of exposure. MS/MS information indicated conjugation with glutathione on the basis of the characteristic neutral loss of 129 for mercapturates. Adducts with the side chain amino group of lysine were characterized by a neutral loss of 171 for N-acetyl- l-lysine. Analysis of products of in vitro incubations of the reactive furan metabolite cis-2-butene-1,4-dial with the respective amino acid derivatives supported five structures, including a new 3-methylthio-pyrrole metabolite probably formed by beta-lyase reaction on a glutathione conjugate, followed by methylation of the thiol group. Our results demonstrate the potential of comprehensive mass spectrometric analysis of urine combined with multivariate analyses for metabolic profiling in search of biomarkers of exposure.
SummaryMitotane [1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2-dichloroethane, (o,p'-DDD)] is the only drug approved for the treatment for adrenocortical carcinoma (ACC) and has also been used for various forms of glucocorticoid excess. Through still largely unknown mechanisms, mitotane inhibits adrenal steroid synthesis and adrenocortical cell proliferation. Mitotane increases hepatic metabolism of cortisol, and an increased replacement dose of glucocorticoids is standard of care during mitotane treatment. Recently, sunitinib, a multityrosine kinase inhibitor (TKI), has been found to be rapidly metabolized by CYP3A4 during mitotane treatment, indicating clinically relevant drug interactions with mitotane. We here summarize the current evidence concerning mitotane-induced changes in hepatic monooxygenase expression, list drugs potentially affected by mitotane-related CYP3A4 induction and suggest alternatives. For example, using standard doses of macrolide antibiotics is unlikely to reach sufficient plasma levels, making fluoroquinolones in many cases a superior choice. Similarly, statins such as simvastatin are metabolized by CYP3A4, whereas others like pravastatin are not. Importantly, in the past, several clinical trials using cytotoxic drugs but also targeted therapies in ACC yielded disappointing results. This lack of antineoplastic activity may be explained in part by insufficient drug exposure owing to enhanced drug metabolism induced by mitotane. Thus, induction of CYP3A4 by mitotane needs to be considered in the design of future clinical trials in ACC.
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