![Toxicogenomics in Mechanistic Toxicology 31
nuclear receptors through activation of adenylyl cyclase (29). The conver-
gence of membrane and nuclear estrogen signaling results in the modulation
of transcription and cell function beyond those genes with estrogen response
elements in their promoters (30,31). Nuclear estrogen receptors undergo confor-
mational changes when bound to ligand that reveal binding sites for coactivators
and corepressors that have chromatin remodeling activity (28). The coacti-
vators are targets for modulation by signal transduction cascades themselves
and are shared with other nuclear receptors leading to cross-talk between
pathways (32,33). ER tethering to other transcription factors, such as HIF-1
and Sp1, also allows estrogen signaling to modulate genes lacking an estrogen
response element (34,35). Finally, xenoestrogens and selective estrogen receptor
modifiers (SERMs) each have distinct profiles of ER binding and recruitment
of cofactors leading to diverse transcriptional responses (28). The complexity of
estrogen signaling makes predictive modeling of tissue-specific gene expression
difficult, but a genomics approach linking gene expression to mechanisms of
action is proving useful for evaluating risks in both environmental and pharma-
ceutical toxicology.
The toxicogenomics approach to linking gene expression and mechanism of
action can be thought of in a stepwise manner. First, compounds are admin-
istered and gene expression profiles are collected from the tissue of interest.
For example, the role of estradiol in rat uterus gene expression was studied
by Wu and colleagues (36). Estradiol was found to be necessary and suffi-
cient for inducing the expression of a number of genes. Second, classifi-
cation of estrogen-responsive genes according to annotations in gene databases
using a predefined vocabulary (http://www.geneontology.org) allows specu-
lative association of gene changes with observed phenotypic changes (37). For
example, Naciff and colleagues examined gene expression changes in prepu-
bertal rat uterus after treatment with 17-ethynyl estradiol, an estrogenic drug
with estradiol-like effects (38). They associated phenotypic changes of ethynyl
estradiol–treated uterus (such as edema and hyperemia) with genes known
to effect vascular function (e.g., vascular endothelial growth factor [VEGF]
and cysteine-rich protein 61). Third, these gene-phenotype associations can be
experimentally directly tested. For example, Heryanto and colleagues admin-
istered estradiol in ovariectomized rats and analyzed blood vessel and stromal
cell density in the presence and absence of an antibody to VEGF (39). The
antibody caused increased stromal cell density, which was interpreted as having
blocked uterine edema. Thus, the observation of uterine edema is phenotypi-
cally anchored to VEGF gene expression. Fourth, experimentally proven gene-
phenotype associations can be used for screening future agents for undesirable
toxic effects when the phenotype is below the sensitivity of detection or does
not present itself in the model system used.](https://image.slidesharecdn.com/134-250507171337-4bc3304a/85/Essential-Concepts-in-Toxicogenomics-1st-Edition-Donna-L-Mendrick-Auth-45-320.jpg)
Essential Concepts in Toxicogenomics 1st Edition Donna L. Mendrick (Auth.)
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- 7. M E T H O D S I N M O L E C U L A R B I O L O G YTM Essential Concepts in Toxicogenomics Edited by Donna L. Mendrick Gene Logic Inc, Gaithersburg, MD, USA and William B. Mattes The Critical Path Institute, Rockville, MD, USA
- 8. Editors Donna L. Mendrick William B. Mattes Gene Logic Inc, The Critical Path Institute, Gaithersburg, MD, USA Rockville, MD, USA dmendrick@genelogic.com wmattes@C-Path.org Series Editor John M. Walker Professor Emeritus School of Life Sciences University of Hertfordshire Hatfield Hertfordshire AL10 9AB, UK john.walker543@ntlworld.com ISBN: 978-1-58829-638-2 e-ISBN: 978-1-60327-048-9 Library of Congress Control Number: 2008921701 ©2008 Humana Press, a part of Springer Science+Business Media, LLC All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, 999 Riverview Drive, Suite 208, Totowa, NJ 07512 USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Cover illustration: Chapter 2, Fig. 1. Printed on acid-free paper 9 8 7 6 5 4 3 2 1 springer.com
- 9. Preface The field of toxicogenomics is moving rapidly, so it is impossible at the time of this writing to compile a classic methods textbook. Instead, we chose to identify experts in all aspects of this field and challenged them to write reviews, opinion pieces, and case studies. This book covers the main areas important to the study and use of toxicogenomics. Chapter 1 speaks to the convergence of classic approaches alongside toxicogenomics. Chapter 2 deals with the usefulness of toxicogenomics to identify the mechanism of toxicity. Chapter 3 calls attention to the issues that affect the quality of toxicogenomics experiments, as well as the implications of using microarrays as diagnostic devices. The need for appropriate statistical approaches to genomic data is discussed in Chapter 4, and Chapters 5 and 6 describe the use of genomic data to build toxicogenomic models and provide insights from the approaches of two companies. The important topic of storing the data generated in such experiments and the correct annotation that must accompany such data is considered in Chapter 7. The discussion in Chapter 8 speaks to the use of toxicogenomics to identify species similarities and differences. Chapters 9 and 10 deal with the use of genomics to identify biomarkers within the preclinical and clinical arenas. Biomarkers will only be useful if the community at large accepts them as meaningful. Consortia are important to drive this function, and Chapter 11 discusses current efforts in this area. Last but not least, Chapter 12 presents a perspective on the regulatory implications of toxicogenomic data and some of the hurdles that can be seen in its implication in GLP studies. Although this book tends to focus on pharmaceuticals, the issues facing toxicology are shared by the chemical manufacturers, the tobacco industry, and their regulators. We want to thank our contributors for their generous time and energy in providing their insights. Sadly, we must note the unexpected passing of one of our authors, Dr. Joseph Hackett of the FDA. Joe’s contribution serves as a testimony to his accomplishments in this field, and his insight will be missed in the years to come. Donna L. Mendrick William B. Mattes v
- 10. Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix Color Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi 1 Toxicogenomics and Classic Toxicology: How to Improve Prediction and Mechanistic Understanding of Human Toxicity Donna L. Mendrick . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2 Use of Traditional End Points and Gene Dysregulation to Understand Mechanisms of Toxicity: Toxicogenomics in Mechanistic Toxicology Wayne R. Buck, Jeffrey F. Waring, and Eric A. Blomme . . . . . . . . . . . . 23 3 Quality Control of Microarray Assays for Toxicogenomic and In Vitro Diagnostic Applications Karol L. Thompson and Joseph Hackett. . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 4 Role of Statistics in Toxicogenomics Michael Elashoff . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 5 Predictive Toxicogenomics in Preclinical Discovery Scott A. Barros and Rory B. Martin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 6 In Vivo Predictive Toxicogenomics Mark W. Porter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 7 Bioinformatics: Databasing and Gene Annotation Lyle D. Burgoon and Timothy R. Zacharewski . . . . . . . . . . . . . . . . . . . . . 145 8 Microarray Probe Mapping and Annotation in Cross-Species Comparative Toxicogenomics John N. Calley, William B. Mattes, and Timothy P. Ryan . . . . . . . . . . . 159 9 Toxicogenomics in Biomarker Discovery Marc F. DeCristofaro and Kellye K. Daniels. . . . . . . . . . . . . . . . . . . . . . . . 185 10 From Pharmacogenomics to Translational Biomarkers Donna L. Mendrick . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195 11 Public Consortium Efforts in Toxicogenomics William B. Mattes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221 vii
- 11. viii Contents 12 Applications of Toxicogenomics to Nonclinical Drug Development: Regulatory Science Considerations Frank D. Sistare and Joseph J. DeGeorge . . . . . . . . . . . . . . . . . . . . . . . . . . 239 Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
- 12. Contributors Scott A. Barros • Toxicology, Archemix Corp., Cambridge, Massachusetts Eric A. Blomme • Department of Cellular and Molecular Toxicology, Abbott Laboratories, Abbott Park, Illinois Wayne R. Buck • Department of Cellular and Molecular Toxicology, Abbott Laboratories, Abbott Park, Illinois Lyle D. Burgoon • Department of Biochemistry & Molecular Biology, Michigan State University, East Lansing, Michigan John N. Calley • Department of Integrative Biology, Eli Lilly and Company, Greenfield, Indiana Kellye K. Daniels • Department of Toxicogenomics, Gene Logic Inc., Gaithersburg, Maryland Marc F. DeCristofaro • Biomarker Development, Novartis Pharmaceuticals Corporation, East Hanover, New Jersey Joseph J. DeGeorge • Laboratory Sciences and Investigative Toxicology, Merck & Co Inc, West Point, Pennsylvania Michael Elashoff • Department of BioStatistics, CardioDx, Palo Alto, California Joseph Hackett • Office of Device Evaluation, Center for Devices and Radiological Health, U.S. Food and Drug Administration, Rockville, Maryland Rory B. Martin • Drug Safety and Disposition, Millennium Pharmaceuticals, Cambridge, Massachusetts William B. Mattes • Department of Toxicology, The Critical Path Institute, Rockville, Maryland Donna L. Mendrick • Department of Toxicogenomics, Gene Logic Inc., Gaithersburg, Maryland Mark W. Porter • Department of Toxicogenomics, Gene Logic Inc., Gaithersburg, Maryland Timothy P. Ryan • Department of Integrative Biology, Eli Lilly and Company, Greenfield, Indiana Frank D. Sistare • Laboratory Sciences and Investigative Toxicology, Merck & Co Inc., West Point, Pennsylvania ix
- 13. x Contributors Karol L. Thompson • Division of Applied Pharmacology Research, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, Maryland Jeffrey F. Waring • Department of Cellular and Molecular Toxicology, Abbott Laboratories, Abbott Park, Illinois Timothy R. Zacharewski • Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan
- 14. Color Plates Color plates follow p. 112. Color Plate 1 Identification of genes regulated in the liver of rats after xenobiotic activation of the nuclear receptors PPAR- , aromatic hydrocarbon receptor (AhR), or pregnane X receptor (PXR). (Chapter 2, Fig. 1; see legend and discussion on p. 26.) Color Plate 2 Hierarchical clustering of gene expression profiles of the testes of male Sprague-Dawley rats treated with a single dose of various testicular toxicants and sacrificed 24 h after treatment. (Chapter 2, Fig. 2; see legend and discussion on p. 35.) Color Plate 3 Heatmap of gene expression profiles from the liver of rats treated with Cpd-001 (arrow) and a wide variety of reference compounds including nonhepatotoxicants and hepatotoxi- cants. (Chapter 2, Fig. 4; see legend and discussion on p. 38.) Color Plate 4 Distributions for error estimators based on proteasome data. (Chapter 5, Fig. 2; see legend and discussion on p. 95.) Color Plate 5 Operating characteristics of the baseline in vitro classifier as a function of classification cutpoint. Replicate observations were treated independently. (Chapter 5, Fig. 5; see legend and discussion on p. 104.) Color Plate 6 Operating characteristics of the baseline in vivo classifier as a function of classification cutpoint. (Chapter 5, Fig. 6; see legend on p. 105 and discussion on p. 104.) Color Plate 7 Similarity tree for in vitro compounds. (Chapter 5, Fig. 7; see legend on p. 107 and discussion on p. 106.) Color Plate 8 Model scores for two doses of thioacetamide or vehicle- treated samples at 6-, 24-, and 48-h exposures. (Chapter 6, Fig. 4; see legend and discussion on p. 138.) xi
- 15. 1 Toxicogenomics and Classic Toxicology: How to Improve Prediction and Mechanistic Understanding of Human Toxicity Donna L. Mendrick Summary The field of toxicogenomics has been advancing during the past decade or so since its origin. Most pharmaceutical companies are using it in one or more ways to improve their productivity and supplement their classic toxicology studies. Acceptance of toxicoge- nomics will continue to grow as regulatory concerns are addressed, proof of concept studies are disseminated more fully, and internal case studies show value for the use of this new technology in concert with classic testing. Key Words: hepatocytes; hepatotoxicity; idiosyncratic; phenotypic anchoring; toxicogenomics; toxicology. 1. Introduction The challenges facing the field of toxicology are growing as companies demand more productivity from their drug pipelines. The intent of this chapter is to identify the issues facing classic approaches to nonclinical toxicity testing, the cause of the deficiencies, and ways in which toxicogenomics can improve current in vitro and in vivo testing paradigms. The public at large continues to exert pressure on the pharmaceutical industry to develop new drugs yet are intolerant of safety issues and the high cost of drugs when they reach the market. This is setting up a “perfect storm” with a recognized decrease in productivity in this industry, continual increase in costs of developing new drugs, and rising attrition rates due to nonclinical and clinical safety failures (1–4). From: Methods in Molecular Biology, vol. 460: Essential Concepts in Toxicogenomics Edited by: D. L. Mendrick and W. B. Mattes © Humana Press, Totowa, NJ 1
- 16. 2 Mendrick 2. Classic Toxicology Those in the field of drug and chemical development know of the multitude of compounds to which humans were never exposed either in the clinic or in the environment because of obvious toxicity seen in preclinical species. However, it is well-known that classic testing in animals is not infallible. A study done by a group within the Institutional Life Sciences Institute (ILSI) illustrates the problem (5). Twelve companies contributed data on 150 compounds that have shown toxicity in humans of a significant enough nature to warrant one of four actions: (1) termination, (2) limitation of dosage, (3) need to monitor drug level, or (4) restriction of target population. The group compared the human toxicities with the results of the classic toxicity employed for each drug. They found that only ∼70% of these toxicities could be predicted in classic animal testing even when multiple species, primarily the rat (rodent) and dog (nonrodent), were employed. The dog was better than the rat in predicting human toxicity (63% vs. 43%, respectively), with the success rate varying depending on the human target organ. However, escalating concerns regarding the use of animals in medical research, the amounts of compound required for such large animals, and the cost of such studies prevents this species from being used as the first species or in sufficient numbers to detect subtle toxicities. The exact failure rate due to toxicity and the time of its detection continues to be the subject of study because only by understanding the problem can one begin to propose solutions. Authors tend to report somewhat different findings. The drugs terminated because of human toxicities evaluated in the ILSI study (5) failed most often during Phase II (Fig. 1). Suter et al. at Roche 43 10 0 5 10 15 20 25 30 35 40 45 % Terminated Phase I Phase II Phase III 39 Fig. 1. Data illustrating the termination rate of compounds due to human toxicity during clinical trials. (Data adapted from Olson, H., Betton, G., Robinson, D., Thomas, K., Monro, A., Kolaja, G., et al. 2000. Concordance of the toxicity of pharmaceuticals in humans and in animals. Regul. Toxicol. Pharmacol. 32, 56–67.)
- 17. Toxicogenomics and Classic Toxicology 3 0 5 10 15 20 25 30 35 40 45 50 Preclinical Phase I Phase II Phase III Registration Clinical Safety Animal Toxicology % Terminated Fig. 2. Failure rate due to animal toxicity and human safety during the devel- opment pipeline. (Data adapted from Suter, L., Babiss, L.E., and Wheeldon, E.B. 2004. Toxicogenomics in predictive toxicology in drug development. Chem. Biol. 11, 161–171.) (6) examined the failure rate of compounds from preclinical to registration and divided safety failures into animal toxicity and human toxicities. Their work found the highest failure rates due to human safety in Phase I and registration (Fig. 2). Note that both studies found a high rate of failure in Phase II or beyond, a costly scenario. Dimasi and colleagues have examined financial models of drug development and have estimated the savings of terminating unsafe compounds earlier within the clinical trial paradigm (7). For example, if 25% of the drugs that will fail in Phase II were discontinued in Phase I, the clinical cost savings alone per approved drug would be $13 million to $38 million dollars. Obviously, the cost savings will be greater if one could prevent such a drug from even entering clinical trials by improving preclinical detection and/or by failing it earlier within the clinical testing phase (e.g., Phase I vs. Phase III). The Food and Drug Administration’s (FDA) Critical Path Initiative (www.fda.gov/oc/initiatives/criticalpath/) quotes one company executive as saying clinical failures due to hepatotoxicity had cost the company more than $200 million per year over the past decade. Clearly, there are many financial incentives to address the issue of safety. 3. Toxicogenomics Many in the field have written excellent opinion pieces and reviews on the use of toxicogenomics in drug discovery and development and in the chemical/agrochemical sectors. Toxicogenomics is used in three areas: predictive applications for compound prioritization, mechanistic analyses for compounds with observed toxicity, and biomarker identification for future
- 18. 4 Mendrick screens or to develop biomarkers useful in preclinical and/or clinical studies. Though impractical to list all of the relevant publications, a few excellent articles on toxicogenomics are provided (2,3,6,8–15). 3.1. Study Designs As with all scientific endeavors, to answer the questions being posed it is important to have an optimal study design. Genomics tends to be somewhat expensive, so understandably some try to downsize the experimental setting. Unfortunately, that may prevent hypothesis generation or evaluation of a preex- isting theory. As an example, if one is trying to form a hypothesis as to the mechanism of injury induced by a compound, sampling tissue only at the time of such damage may prevent evaluation of the underlying events that started the pathologic processes. Similarly, sampling only one time point will inhibit the fullest evaluation of the dynamic processes of injury and repair. In classic toxicology testing, one would not claim with certainty that a compound is not hepatotoxic if one saw no elevation of serum alanine aminotransferase (ALT) or histologic change in the liver in a snapshot incorporating only one time point and dose level. Likewise, one does not pool blood from all animals and perform clinical pathology on such. Unfortunately, some have approached toxicogenomics in this manner (using restrictive study designs and pooled RNA samples) and then felt betrayed by the lack of information. This does not mean to suggest that all toxicogenomic studies must be all-encompassing as long as the investigator understands beforehand the limits of his or her chosen design. One approach might be to collect samples from multiple time points and doses and triage the gene expression profiling to determine the most important study groups within that experimental setting. Establishment of the appropriate dose is important as well. Classic toxicology endeavors use dose escalation until one sees a phenotypic adverse event such as changes in classic clinical pathology, histology, body weight, and so forth. An anchoring of the dose used for toxicogenomic studies also must be employed and contextual effects of such doses understood. Doses that severely affect body weight likely induce great stress upon the animal, and this must be taken into account if this phenotypic anchor is followed. A recent paper by Shioda et al. studied effects of xenoestrogens in cell culture and explored the relationship of doses to transcriptional profiles (16). This work suggests that doses chosen for equiv- alent cellular responses highlight the differences between compounds while those selected based on the compounds’ action on a particular gene reveal mostly similarities between the compounds. Additional work remains to be done to determine if this conclusion can be extrapolated to other compound types and in vivo environments, but, at a minimum, this report reinforces the
- 19. Toxicogenomics and Classic Toxicology 5 need to understand the chosen study design and fully explain criteria used for dose selection. 3.2. Genomic Approaches Can Clarify Basic Husbandry Issues Genomics enables detection of toxicity parameters as well as differences in animal husbandry. In many cases, the study design may call for food restriction or animals may be accidentally deprived of food. Genomic analysis can detect such events as shown in Fig. 3 and Fig. 4. Studies in Gene Logic’s (Gaithersburg, MD) ToxExpress® database were used for the analysis. In Fig. 3, the data from the probe sets (∼8800) on the Affymetrix Rat Genome U34A GeneChip® microarray (Santa Clara, CA) were subjected to a principal components analysis (PCA). Such a test illustrates underlying differences in the data in a multidimensional picture. For ease in viewing, two-dimensional graphical representations are provided. In Fig. 3, the data from all probe sets were used, and, even with the accompanying noise when so many parameters are measured, one can see differentiation of the groups particularly if one combines the x and y axes, accounting for 39% of the gene expression variability. PCA Mapping (39.3%) 41 31 22 13 4 –5 –14 PC #2 14.3% PC #1 25% –80 –64 –49 –34 –19 –5 10 25 40 55 70 –23 –32 –41 –51 Treatment Fasted Fed Fig. 3. A PCA using all genes on the Rat Genome U34A microarray illustrates the differentiation on a genomic basis between rats fasted for 24 h versus those rats that had food ad libitum. Use of all genes on the array is accompanied by noise and yet one obtains reasonable separation using the x axis and better discrimination if one employs both x and y axes. Such findings illustrate the ability of gene expression to provide insight into animal husbandry.
- 20. 6 Mendrick PCA Mapping (81.5%) PC #2 5.6% PC #1 75.9% –16 –10 –8.6 –7.2 –5.8 –4.4 –3 –1.6 –0.2 1.2 2.6 4 –12.9 –9.8 –6.7 –3.6 –0.5 2.6 5.7 8.8 11.9 15 Effect of Fasting Fasted Fed Fig. 4. An analysis filter was applied to identify differentially regulated genes. The filter has a cutoff as follows: fold-change ≥1.8 with t-test p-value .05 and ≥90% present in reference or experimental group with mean avg. diff. 40. This resulted in 281 genes identified as dysregulated between fed and fasted rats. Almost all of the variation is captured in PC #1 (75.9%) and the groups are more clearly separated than seen when all genes were used as shown in Fig. 3. When genes that were differentially regulated among the fed and fasted animals were chosen, the gene list was reduced from 5000 to 281. The results in Fig. 4 demonstrate a complete discrimination of these rats with the x axis accounting for 76% of the variability. Genomics can be used to discriminate strain and gender as well. In the former case, female rats of Sprague-Dawley (SD) or Wistar origin were compared. Although evaluation using all genes discriminated these strains (data not shown), selection of differentially regulated genes resulted in a clearer separation as shown in Fig. 5. Because both strains are albino, one could envision using a genomics approach should there be a potential mix-up of strains in the animal room. What is likely less surprising is the ability to categorize gender based on gene expression findings. Although it is usually easy to identify the gender of rats by physical examination alone, one could envision the use of a genomic approach to study the feminization of male rats under drug treatment or vice versa. As shown in Fig. 6, a PCA employing all genes discriminates between genders although the first two axes capture only ∼23% of the variation suggesting there
- 21. Toxicogenomics and Classic Toxicology 7 PCA Mapping (69.9%) PC #2 6.72% PC #1 63.2% –9 –5.3 –4.36 –3.42 –2.48 –1.54 –0.6 0.34 1.28 2.22 3.16 4.1 –7.3 –5.6 –3.9 –2.2 –0.5 1.2 2.9 4.6 6.3 8 SD Effect of Strain Wistar Fig. 5. A PCA was performed with 83 genes that were differentially expressed between normal female Sprague-Dawley (SD) or Wistar rats. Such strains are clearly discriminated using a genomic approach. PCA Mapping (24.3%) PC #2 7.44% PC #1 16.9% –90 –54 –44 –34 –24 –14 –5 4 14 24 34 44 –72 –55 –38 –21 –5 12 29 46 63 80 Gender Female Male Fig. 6. A PCA was performed with all genes from male and female rat livers. There is an overall separation particularly if one combines the variability shown along the x and y axes.
- 22. 8 Mendrick PCA Mapping (68.9%) PC #2 5.3% PC #1 63.6% –14 –5 –3.9 –2.8 –1.7 –0.6 0.5 1.6 2.7 3.8 4.9 6 –11.3 –8.6 –5.9 –3.2 –0.5 2.2 4.9 7.6 10.3 13 Effect of Gender Female Male Fig. 7. A PCA was performed with 175 genes that were differentially expressed between the genders. In this case, the majority of difference was captured in the first axis resulting in a very clear separation of the genders. are many subtle effects, a point not likely to be argued by many humans. Using genes differentially regulated, however, found that 175 genes can account for more than 63% of variation in the first axis alone as shown in Fig. 7. In the three cases described above (food deprivation, strain, and gender), differentially regulated genes were identified that enabled clear categorization between the groups. However, the reverse is also true. Removal of genes that identify such differences from the analysis can enable other differences to become more apparent. Such has been accomplished with the predictive models built as part of our ToxExpress program specifically to avoid such confounding variables and enable such models to work well when female or male rats are used of various strains and independent of fasting. 3.3. Toxicogenomics Can Augment Understanding of Classic Toxicology Findings Toxicogenomics can add value to classic testing when one animal appears to have an aberrant finding. It is recognized that preclinical testing of drugs is a complicated process and mistakes can happen. If a rat did not exhibit any signs of toxicology unlike its cohorts, it would be informative to differentiate dosing errors from true differences in toxicity responses. One could monitor
- 23. Toxicogenomics and Classic Toxicology 9 the blood to determine if the drug was detectable, but that would depend on its clearance. Alternatively, one could explore gene expression as a subtle detection method. Figure 8 illustrates how similarity in gene expression can identify the treatment given. If nine rats received carbon tetrachloride and the tenth rat was left untreated, the gene expression of the latter would appear similar to that of untreated rats as shown in the upper left corner in this mock illustration. As seen with these examples, molecular approaches can be more sensitive in terms of discriminating animal husbandry, strain differences, and so forth, than parameters monitored in classic toxicity testing. Microarrays monitor tens of thousands of events (expression levels of genes and Expressed Sequence Tags (ESTs)), whereas classic toxicity testing has been estimated to measure approximately 100 parameters (17). From a strictly statistical approach, one can envision that more knowledge would lead to improved decision making although the challenge of removing the noise when monitoring so many events is real. Arrays provide information on the changes in individual genes, and from this one can then hypothesize on the effects on biological pathways, and PCA Mapping (36.5%) PC #2 12.5% PC #1 24.1% –55 –70 –57 –45 –33 –21 –10 2 14 26 38 50 –41 –27 –13 0 14 28 42 56 70 CCI4 24 hrs CornOil 24 hrs Untreated Fig. 8. A PCA illustrates the greatest source of difference at the gene expression level between the rats. The first component (PC #1) accounts for 24% of the variability, and rats treated with carbon tetrachloride are clearly delineated from those receiving vehicle or left untreated with the exception of the one rat in the upper left. The second component, PC #2, accounts for 12.5% of the variability and discriminates even untreated from vehicle-treated rats further illustrating the sensitivity of this approach.
- 24. 10 Mendrick so forth, a level of understanding not provided in classic approaches. One could use the analogy of the sensitivity of electron versus light microscopy. Disease processes such as minimal change nephropathy induce severe functional alterations (proteinuria) in the human kidney yet are extremely difficult to visualize with light microscopy. However, the morphologic changes associated with this proteinuria are clearly seen in such diseased kidneys if one uses electron microscopy, a more sensitive technique. At the ultrastructural level, a flattening of visceral epithelial cells upon the glomerular basement membrane is clearly visible, and such a process is known to be associated with proteinuria in preclinical species and in humans. Several recent papers highlight the improved sensitivity of a genomics approach with classic examples of compounds that produce adverse events in rats. Heinloth and her colleagues at the National Institute of Environmental Health and Safety (NIEHS) have examined the dose response relationship of rat liver to acetaminophen (18). They studied multiple doses in the rat using two (they called these “subtoxic”) doses that failed to cause changes monitored with classic approaches. However, when ultrastructural methods were employed, some toxicity-associated mitochondrial changes were observed at one of these “subtoxic” doses. Evaluation of gene expression changes found, as a consequence of acetaminophen toxicity, a loss in cellular energy even at such “subtoxic” doses. As the dose was increased, so was the magnitude of changes observed, and this was accompanied by alterations in associated genes. They concluded that gene expression profiling may provide more sensitivity for detecting adverse effects in the absence of the occurrence of overt toxicity. Another recent paper explored a time savings approach. Nie and his colleagues at Johnson and Johnson Pharmaceutical Research and Development examined changes in gene expression in the rat liver at 24 h after exposure to nongenotoxic carcinogens and control compounds (19). They identified six genes that predicted 89% of nongenotoxic carcinogens after 1 day of exposure instead of the 2 years of chronic dosing normally required for such cellular changes to be clear. They also mined the gene expression results to find biologically relevant genes for a more mechanistic approach to understanding nongenotoxic carcinogenicity. Together the work by Heinloth et al. and Nie et al. highlight the sensitivity of a genomics approach to detect compounds that will elicit classic adverse events in rats either at higher doses or exposure times. Toxicogenomics can be employed to identify species-specific changes providing support for safety claims in humans. Peroxisome proliferator activated receptor alpha (PPAR-) agonists have been widely studied as they have been found to be clinically useful as hypolipidemic drugs yet induce hepatic tumors in rats. It is now appreciated that such an effect has little safety
- 25. Toxicogenomics and Classic Toxicology 11 risk to humans and that genes can be identified in rat liver and hepatocytes that enable discrimination of this mechanism. As suggested by Peter Lord and his colleagues at Johnson and Johnson, this could be incorporated into the safety evaluation of novel compounds by employing expression profiling of rat liver or hepatocytes exposed to the new drug. Similarities between this novel compound and known PPAR- agonists could provide a safety claim for the former’s species-specific effects (15). In some cases, differences in species responses to drugs may rely on dissim- ilarities in drug metabolism enzymes. A study was performed using data in Gene Logic’s ToxExpress database. Expression levels of genes involved in glutathione metabolism were compared among normal tissues and genders of the rat, mouse, and canine. As expected, expression levels varied among genes and tissues. Little differences were seen among male and female animals but large effects were seen among species. If differential gene expression does translate to enzymatic activities differences between species, it would be expected to impact drug responses. Such information could provide guidance into species selection for toxicity testing (20). Chapters later in this book discuss species differences in more detail. 3.4. Use of Toxicogenomics to Detect Idiosyncratic Compounds A well-accepted definition of the term idiosyncratic, particularly on a compound by compound basis, is hard to find as individuals use different criteria. Some depend on incidence alone, some on the inflammatory response induced, some on a lack of dose response relationship, and so forth. However, universal to most users’ definitions is the lack of classic changes observed in preclinical species. To complete the analogy started above in discussing the sensitivity differences between light and electron microscopy, one could suppose that idiosyncratic compounds elicit some effects at the gene and protein level in rats but these events do not lead to changes severe enough to induce classic phenotypic changes in this commonly used testing species. The failure to cause overt injury in the rat might be due to (1) the inability of this species to metabolize the drug into the toxic metabolite responsible for human injury, (2) quantitative differences whereby rats generate the toxic metabolite but at lower levels than seen in humans, (3) a superior ability of the rat to detoxify toxins, and (4) the failure of rats to respond in an immune-mediated manner to adduct formation of intrinsic cell surface proteins. Many if not all compounds today are commonly screened for metabolism differences between species and their development discontinued if such metabolic specific responses are seen. Even so, the postmarketing occurrence of drug hepatotoxicity is a leading cause of regulatory actions and further erodes the pharmaceutical pipeline. The field of
- 26. 12 Mendrick drug development needs new methods to identify such drugs earlier in discovery and development that can inform compound selection, position drugs to areas of unmet clinical need, and influence preclinical and clinical trial designs in a search for earlier biomarkers of liver injury induced by such a compound. Several groups including those from Millennium (Chapter 5 and Ref. 21), AstraZeneca (22), and Gene Logic (Chapter 6) have reported success in (a) training predictive models using all genes and ESTs being monitored on the array platform and (b) classifying the compounds based on their potential to induce toxicity in humans including idiosyncratic compounds. The result is sets of genes and ESTs that may not be easily translated into mecha- nistic understanding on their own yet show robust predictive ability. Such an approach has enabled the prediction of idiosyncratic hepatotoxicants from rats and rat hepatocytes exposed to such agents. Figure 9 illustrates the predictive ability of models built at Gene Logic to identify idiosyncratic compounds as potential human hepatotoxicants using gene expression data obtained from in vivo and in vitro exposure to prototypical compounds. The classification of individual compounds as idiosyncratic tends to be controversial, so to remove any internal bias, the statistics were compiled using marketed compounds classified as idiosyncratic by Kaplowitz (23). Kaplowitz subclassifies idiosyn- cratic compounds as allergic or nonallergic. As can be seen in Fig. 9, toxicoge- nomic predictions performed from in vivo rat exposure are equally accurate regardless of whether or not the idiosyncratic compound induces an allergic responses in the human liver, using Kaplowitz’s classification. In contrast, the model built from rat hepatocyte data does somewhat better with compounds that 92% 71% 92% 88% 92% 78% 0 20 40 60 80 100 In Vivo Exposure in Rats In Vitro Exposure to Primary Rat Hepatocytes 92% 71% 92% 88% 92% 78% 0 20 40 60 80 100 % Predicted as Hepatotoxic in Humans Allergic Non-allergic Allergic Non-allergic Fig. 9. Compounds described as idiosyncratic by Kaplowitz (23) were studied. He discriminates between drugs that induce allergic-type reactions from those that do not, and the results of the predictive toxicogenomic modeling are shown here.
- 27. Toxicogenomics and Classic Toxicology 13 cause nonallergic responses in humans. It should be remembered that idiosyn- cratic compounds do not elicit classic phenotypic changes in rats and other nonclinical species, yet the majority are detected using a predictive toxicoge- nomics approach in rat liver and primary rat hepatocytes. How is it possible for genomics to predict something in the rat that is not seen at a classic phenotypic level? 3.4.1. Case Studies of Idiosyncratic Hepatotoxicants For most of the idiosyncratic drugs on the market today, little is known about their ability to induce hepatotoxicity in humans while avoiding classic detection in preclinical species. Felbamate is an exception, and it has been researched extensively. It has been reported that its metabolism is quantitatively different between species and this is believed to be responsible for the failure of classic testing to detect its potential to induce human hepatotoxicity. Researchers have reported that felbamate is metabolized to a reactive aldehyde (atropaldehyde) and that rats produce far less of this toxin than do humans (24). However, because rats do produce some level of this toxic metabolite, it raises the possi- bility that a technology more sensitive than classic testing (e.g., histopathology) may detect subtle signs of toxicity in rat. To test this hypothesis, an in vivo study done at Gene Logic employed 45 rats in groups of five per dose and time point. Animals were dosed daily by oral gavage with vehicle, low-dose or high- dose felbamate. Groups were sacrificed at 6 h and 24 h after one treatment and 14 days after daily dosing. Histology of the livers was normal. One of the high- dose–treated rats sacrificed after 14 days of exposure exhibited an ALT level above the normal reference range, and the entire group had a minor but statis- tically significant elevation above the control group. The overall conclusion of the board-certified veterinary pathologist was that a test article effect was unclear. Because this drug failed to demonstrate hepatotoxicity in the classic testing employed for registration, this result was not surprising. However, it does raise the issue of whether individual rats are responding in a classic sense to idiosyncratic compounds, and these results are not being interpreted correctly as suggested by retrospective analysis of some idiosyncratic compounds by Alden and colleagues at Millennium Pharmaceuticals (25). To explore the potential effects of felbamate on gene expression, livers were collected from all rats in this study, processed using Affymetrix GeneChip microarrays, and the data passed through the predictive toxicogenomics models built at Gene Logic. The results of the models, illustrated in Fig. 10, were correct in identifying felbamate as a potential human hepatotoxicant, a potential hepatitis-inducing agent in humans (26,27), and an agent that will cause liver enlargement in rats
- 28. 14 Mendrick Hepatitis (human); liver enlargement (rat) E.g., nefazodone, an idiosyncratic compound YES Model Results Pathology/Mechanistic Models Compound Assessment General Model Toxicogenomic Models Questions Addressed Might it induce human hepatotoxicity? What type of injury may it induce in humans and/or rats What well-known compounds does it resemble? Fig. 10. Predictive toxicogenomic models were built at Gene Logic and validated internally and with customer-provided blinded data. The results of the genomic data obtained from felbamate-treated rat liver are shown here. (28). The gene expression data from such treated rats showed some similarity to other idiosyncratic drugs such as nefazodone. Another interesting idiosyncratic drug is nefazodone. This oral antidepressant was approved in 1994 in the United States, but postmarket cases of idiosyncratic adverse reactions were reported that resulted in 20 deaths. A black box warning was added to the label in 2001 and the drug withdrawn in 2004 (23,29–34). The only hepatic effect reported in rats was a dose-related (50, 150, 300 mg kg−1 day−1 ) increase in liver weight reported in a 13-week rat study (SBA NDA 20-152). Several mechanisms for its toxicity have been proposed. The first hypothesis regarding its toxicity is a generation of reactive metabolites via bioactivation, which are capable of covalently modifying CYP3A4, a drug- metabolizing enzyme it is metabolized by and inhibits (35–37). The second theory is that nefazodone compromises biliary elimination, resulting in an increase in drug and bile acids retained in the liver over time (34,38). These authors have found a transient increase in serum bile acids in rats suggesting that rats are responding to this drug inappropriately but can recover from its toxicity prior to developing classic phenotypic changes. To investigate whether gene expression changes might be able to detect nefazodone as a potential human hepatotoxicant in the absence of classic changes in the rat, a study was performed using doses of 0, 50, and 500 mg kg−1 day−1 given by oral gavage. Note that the high dose is only 67% higher than what the manufacturer used in its 13-week rat study in which liver enlargement was reported. Groups of rats were sacrificed 6 h and 24 h
- 29. Toxicogenomics and Classic Toxicology 15 after the first dose and 7 days after daily dosing. Clinical pathology and liver histopathology revealed no clear evidence of liver toxicity as was expected of this idiosyncratic compound. The results from toxicogenomic modeling were similar to that of felbamate, namely that it is a potential human hepatotox- icant that can cause hepatitis in humans and liver enlargement in rats and has similarity to idiosyncratic compounds such as felbamate. Kellye Daniels, Director of Toxicogenomics at Gene Logic, examined the gene expression data in more detail and found evidence for a transitory effect on Phase I and II drug-metabolizing enzymes, oxidative stress response genes, protein-repair associated genes, and a solute carrier. Taken together, this suggests the cells are metabolizing the conjugates for removal, proteins are damaged, and the cell is trying to remove them alongside a compensatory induction of a solute carrier to export them. These changes may reflect the reason rats do not develop obvious hepatotoxicity. It is hypothesized that idiosyncratic compounds have a similar phenotypic pattern at the gene level to compounds known to induce hepatotoxicity in rats and that enables predictive models to detect the former as well as the latter. The effect of compounds on the expression of superoxide dismutase 2 (Sod2), shown in Fig. 11, will serve as an example. This gene encodes a protein that catalyzes the breakdown of superoxide into hydrogen peroxide and water in the mitochondria and is therefore a central regulator of reactive oxygen species (ROS) levels (39). The expression of this gene is upregulated by compounds such as acetaminophen and lipopolysaccharide, known to induce Cross-Compound Comparison of Sod2 Expression Fold Change (24 hr exposure) *p 0.05 * * * * Rosiglitazone Diclofenac Acetaminophen Lipopolysaccharide Nefazodone –2 –1 0 1 2 3 4 5 6 * * * * * * * * Fig. 11. The expression level of superoxide dismutase 2 (Sod2) in toxicant-treated liver tissue compared with vehicle controls is illustrated. Both the fold change and statistical results are shown.
- 30. 16 Mendrick hepatotoxicity in multiple species yet acting by differing mechanisms. Two idiosyncratic compounds, diclofenac and nefazodone, also elevate the level of this gene, whereas a drug not associated with hepatotoxicity, rosiglitazone, has no significant effect. This illustrates the commonality of gene expression among many compounds that cause human hepatotoxicity whether or not they induce similar toxicity in rats, the species examined here. 4. Use of Toxicogenomics in an In Vitro Approach Because earlier identification of a compound destined to fail in the market- place would save significant resources, it is important to examine what can be done in terms of both cell culture (in vitro) and animal (in vivo) testing. Although cells in a culture environment do not maintain their in situ morphology, their homeostatic function and potentially the full complement of drug-metabolizing genes, the ease of use, and relative savings in terms of compound and time continues to induce researchers to exploit in vitro systems. Companies tend to focus on cell culture approaches to rank compounds on the basis of potential toxicity during lead optimization but only if suffi- cient throughput and cost constraints can be met (3,40). Because the in vitro environment does not represent all of the complex processes at play upon in vivo exposure, during lead prioritization some prefer to focus on short-term in vivo approaches instead (41). How can toxicogenomics add value to classic in vitro approaches (e.g., cytotoxicity) and in vivo studies? First, it is helpful to identify what is needed in these stages. Donna Dambach and her colleagues at Bristol-Myers Squibb were working with a tiered testing strategy using immortalized human hepatocyte cell lines and primary rat and human hepatocytes (40). They report good success in using their five human hepatocyte cell lines in a cytotoxicity assay wherein compounds with an IC50 value of 50 μM are deemed to be of increased risk and may be subjected to further testing. Although this assay only detects necrosis-causing compounds, they find it valuable because it has been reported that 50% of all drug-related hepatotoxicity is due to necrosis. Because genomic approaches can monitor thousands of genes at one time, it can enlarge our understanding of the various cellular responses to an agent that can lead to both predictive models based on a statistical approach and to better mechanistic understanding. Barros and Martin (Chapter 5 and Ref. 21) detail a successful approach for using gene expression data from primary rat hepatocytes exposed to hepatotoxicants and safe compounds to generate a predictive toxicogenomic model of hepatotoxicity that can be used as a screen to rank compounds on the basis of their potential to induce hepatotoxicity. Similarly, Hultin-Rosenberg and colleagues at AstraZeneca report success in
- 31. Toxicogenomics and Classic Toxicology 17 using such approaches (22). These authors used several statistical methods and found little gene overlap between them. They concluded that the ability to “map biological pathways onto predictive sets will be problematic” as genes chosen with statistical approaches may or may not respond in similar ways to other members of the same pathway. Jeff Waring and his colleagues at Abbott have reported the use of toxicogenomics to identify mechanisms of toxicity using primary rat and human hepatocytes (42,43), and Sawada et al. identified a set of genes in HepG2 cells that can be used in an in vitro screen to identify compounds that cause phospholipidosis (44). Thus, teams of researchers are exploring new ways to improve accuracy of toxicity assessment using a genomics approach that may be used with in vitro or in vivo exposure to build predictive models and to enhance mechanistic understanding. 5. Regulatory Issues The FDA has been proactive in the use of genomics and in March 2005 released a guidance paper on the use of pharmacogenomics (including toxicogenomics) in drug development. This document and recent presenta- tions and papers by FDA staff can be found on their dedicated Web site (www.fda.gov/cder/genomics). Chapter 3 and Chapter 12 discuss in more detail issues and regulatory implications of genomic data. Some investi- gators have expressed concerns about the generalized use of toxicogenomics until factors such as quality control standards are in place, sufficient proof of concept studies emerge, and the concern about overinterpre- tation or misinterpretation of genomic data has been addressed (8,11,45). Hopefully, some of these concerns have been alleviated by the recent spate of publications arising from the work of the MicroArray Quality Control (MAQC) project. (The articles can be obtained at the MAQC Web site www.fda.gov/nctr/science/centers/toxicoinformatics/maqc/.) This consortium reported good reproducibility of gene expression measurements between and within platforms using multiple platforms and test sites (46,47) and found analytical consistency across platforms when evaluating specific toxicogenomic studies (48). The second phase of this work began in September 2006 and will provide a venue for individuals from government agencies (FDA, EPA, etc.), pharmaceutical companies, biotechnology companies, and platform providers to discuss differing approaches for the identification of biomarkers for preclinical and clinical use. Whereas some believe that the field of toxicogenomics is not “ready for prime time,” others embrace its usefulness today in predictive, mechanistic, and biomarker applications particularly in the investigative and compound ranking aspects of drug development (2,9,12). Besides the MAQC efforts, other public efforts are involved in standardizing genomic controls,
- 32. 18 Mendrick analysis tools, protocols, and identifying minimal genomic data submissions requirements. These include the External RNA Controls Consortium (ERCC) and the HL7/CDISC/I3C Pharmacogenomics Data Standards Committee. Even if the community can find common ground on data-mining approaches applied to genomic data, there remains a concern as to biomarker qualification particularly as a designation of valid confers mandatory submission of such data with the drug application. Although many use the term validation, Dr. Woodcock (Deputy Commissioner for Operations and Chief Operating Officer, FDA) suggested that qualification may be a better term as the former means many things to different people and tends to remind people of the physical test and not the underlying biological truth (Woodcock J, Presentation at the FDA, DIA, PhRMA, BIO, PWG Workshop on Application and validation of genomic biomarkers for use in drug development and regulatory submissions. 2005. http://www.fda.gov/cder/genomics/presentations.htm). Some uneasiness exists in the pharmaceutical industry related to how biomarkers will be validated (i.e., qualified) and how they will learn of this as it does then elicit reporting requirements. As a beginning, the FDA recently published examples of valid genomic biomarkers and how they are affecting drug labels (“Table of Valid Genomic Biomarkers in the Context of Approved Drug Labels” found at www.fda.gov/cder/genomics/genomic biomarkers table.htm.). However, because no method exists today for the qualification of biomarkers, Goodsaid and Frueh in the Office of Clinical Pharmacology (CDER/FDA) have taken the initiative to propose a process (49). There is an ongoing collabo- ration between the FDA and Novartis to identify a process map and examine biomarkers of renal injury in the rat with the goal of extending this work into man in a consortium to fully validate the biomarkers (Maurer, G, Presen- tation at the FDA, DIA, PhRMA, BIO, PWG Workshop on Application and validation of genomic biomarkers for use in drug development and regulatory submissions. 2005. http://www.fda.gov/cder/genomics/presentations.htm). A consortium approach will further widespread acceptance of methods to qualify biomarkers as well as the biomarkers themselves. Chapter 11 will address public consortia in more detail. 6. Conclusion Toxicogenomics is posed to augment and improve the safety testing of compounds but not replace classic testing as some originally proposed. If applied appropriately with correct study designs, genomics can have widespread uses in toxicology. It can identify toxicity at doses of compounds that do not cause overt toxicity as shown by Heinloth and her colleagues at NIEHS
- 33. Toxicogenomics and Classic Toxicology 19 (18), speed detection of nongenotoxicity as reported by Peter Lord and co- workers at Johnson and Johnson (19), detect rat-specific effects such as those induced by PPAR- agonists (15), and identify idiosyncratic compounds prior to human exposure using predictive models built on exposed primary rat hepatocytes or rat liver as reported by Hultin-Rosenberg and colleagues at AstraZeneca (22) and Martin et al. at Millennium Pharmaceuticals (21). More and more companies are using this new technology in such predictive appli- cations, mechanistic evaluations, and/or in biomarker discovery. The use of toxicogenomics and the balance between the three areas tend to be based on the number of compounds being evaluated among other issues. However, compli- cating the adoption of toxicogenomics is the interdisciplinary aspect of the field wherein critical analysis and understanding by people from many backgrounds including toxicologists, chemists, bioinformaticians, biostatisticians, patholo- gists, and regulators is needed. It is beneficial to have such individuals together in discussions of toxicogenomics so each can contribute their expertise to a facet of the field. Unfortunately, such groups rarely communicate effectively in large organizations although many pharmaceutical companies are beginning to realign teams of researchers through discovery and development to better meet the new demands of successful drug development. Hopefully, new technologies including toxicogenomics can be used to improve the prediction of drug toxicity prior to human exposure to provide better guidance in protecting human safety. References 1. Kola, I. and Landis, J. (2004) Can the pharmaceutical industry reduce attrition rates? Nat. Rev. Drug Discov. 3, 711–715. 2. Mayne, J.T., Ku, W.W., and Kennedy, S.P. (2006) Informed toxicity assessment in drug discovery: systems-based toxicology. Curr. Opin. Drug Discov. Dev. 9, 75–83. 3. Barros, S.A. (2005) The importance of applying toxicogenomics to increase the efficiency of drug discovery. Pharmacogenomics 6, 547–550. 4. DiMasi, J.A., Hansen, R.W., and Grabowski, H.G. (2003) The price of innovation: new estimates of drug development costs. J. Health Econ. 22, 151–185. 5. Olson, H., Betton, G., Robinson, D., Thomas, K., Monro, A., Kolaja, G., et al. (2000) Concordance of the toxicity of pharmaceuticals in humans and in animals. Regul. Toxicol. Pharmacol. 32, 56–67. 6. Suter, L., Babiss, L.E., and Wheeldon, E.B. (2004) Toxicogenomics in predictive toxicology in drug development. Chem. Biol. 11, 161–171. 7. DiMasi, J.A. (2002) The value of improving the productivity of the drug development process: faster times and better decisions. Pharmacoeconomics 20 (Suppl 3), 1–10. 8. Boverhof, D.R. and Zacharewski, T.R. (2006) Toxicogenomics in risk assessment: applications and needs. Toxicol. Sci. 89, 352–360.
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- 37. 2 Use of Traditional End Points and Gene Dysregulation to Understand Mechanisms of Toxicity: Toxicogenomics in Mechanistic Toxicology Wayne R. Buck, Jeffrey F. Waring, and Eric A. Blomme Summary Microarray technologies can be used to generate massive amounts of gene expression information as an initial step to decipher the molecular mechanisms of toxicologic changes. Identifying genes whose expression is associated with specific toxic end points is an initial step in predicting, characterizing, and understanding toxicity. Analysis of gene function and the chronology of gene expression changes represent additional methods to generate hypotheses of the mechanisms of toxicity. Follow-up experiments are typically required to confirm or refute hypotheses derived from toxicogenomic data. Under- standing the mechanism of toxicity for a compound is a critical step in forming a rational plan for developing counterscreens for toxicity and for increasing productivity of research and development while decreasing the risk of late-stage failure in pharmaceutical development. Key Words: gene expression; mechanism of toxicity; microarray; phenotype. 1. Introduction A study published in 2002 demonstrated a robust, statistically significant link between wine drinking and healthy diet, nonsmoking, and higher household income (1). Yet, no one would presume that starting to drink wine would cause someone to suddenly choose a healthier lifestyle or get a pay increase! Whereas the distinction between cause and effect is clear in this simple example, analysis of cause and effect in the practice of toxicogenomics is less obvious. Statistical analysis of a large set of microarray gene expression profiles derived from From: Methods in Molecular Biology, vol. 460: Essential Concepts in Toxicogenomics Edited by: D. L. Mendrick and W. B. Mattes © Humana Press, Totowa, NJ 23
- 38. 24 Buck et al. treatments with particular toxicants will essentially always produce a signature set of genes that can distinguish treatment from control groups. However, the predictive or diagnostic value of such a signature, as it applies to the practical toxicologic evaluation of compounds, requires validation as described in previous chapters. Nonetheless, gene expression signatures that discriminate based on epiphenomena instead of mechanisms of toxicity may fail to recognize toxic changes caused by new chemical series. Therefore, establishing gene expression signatures that are causally linked to a specific toxic process, so- called phenotypic anchoring, can increase one’s confidence in the value of such signatures. Signatures can also be developed that will detect the activation of mechanistically important pathways in test animal species that are known to be associated with potential toxicity in humans but have no recognized phenotypic outcomes in test animals. Toxicogenomic data are also extremely valuable to formulate new hypotheses regarding the mechanisms responsible for undesirable phenotypic changes. The extraordinary wealth of information on cell and tissue transcriptomes gained through microarrays can be combined with annotation libraries to group expression data into functionally related pathways, a process called pathway analysis (2). Data collection at multiple time points can further imply cause- effect relationships between early and later gene expression. Investigation into mechanisms of toxicity is particularly helpful when a toxic phenotype, such as hepatocellular hypertrophy, can be caused by a number of distinct molecular mechanisms. In pharmaceutical discovery and development, efficient assays can then be designed to screen backup compounds against activation of the relevant pathway to select less toxic alternatives. In this chapter, we will first discuss the situation where a single phenotype (liver hypertrophy) is associated with a number of distinct gene expression profiles, each of which has unique implications in safety assessment. Second, we will cover the utility of a cardiotoxic agent to pinpoint mechanisms of toxicity in a tissue having a limited range of morphologic and functional responses to injury, such as the heart. Third, the value of toxicogenomics in distinguishing beneficial, toxic, and incidental mechanisms of estrogens on the uterus will be explored as it relates to environmental and pharmaceutical toxicology. Fourth, we will highlight the advantage of using toxicogenomics to screen environ- mental compounds for testicular toxicity and the challenge of determining a mechanism of action in this complex tissue. Finally, we will discuss the value of toxicogenomics in discovering the activation of phenotypically inapparent cellular stress pathways as a predisposing mechanism for the development of idiosyncratic drug reactions and the ability to test for these changes in animal models.
- 39. Toxicogenomics in Mechanistic Toxicology 25 2. Liver Hypertrophy: One Phenotype with Many Mechanisms Gene expression profiles allow for a global, detailed evaluation of the molecular events that precede and accompany toxicity and therefore are extremely useful to understand the molecular mechanisms underlying various histopathologic or clinical chemistry changes. This is best illustrated with liver enlargement, a relatively frequent observation in rodents treated with xenobiotic agents. There are a variety of mechanisms by which drug-induced hepatomegaly can occur, including increases in smooth endoplasmic reticulum contents or in cytochrome P450 monooxygenase activities, peroxisome proliferation, hyper- trophy of mitochondria, or hepatocellular proliferation (3). In some cases, these changes are adaptive and are not considered of toxicologic significance (4). In other cases, these changes are rodent specific and are not considered relevant to humans, such as peroxisome proliferation (5). In contrast, enzyme induction responses can sometimes be associated with liver injury, as evidenced by increased alanine aminotransferase (ALT) levels and hepatocellular apoptosis and necrosis (6,7). Finally, many compounds that induce liver hypertrophy fall into the category of nongenotoxic carcinogens (NGTCs). NGTCs are agents that do not cause a direct effect on DNA (i.e., nongenotoxic) yet test positive in long-term rodent carcinogenicity studies. Whereas the relevance of these findings to humans is not always clear, a positive result in this bioassay will result in a tremendous amount of time and resources allocated to identifying the mechanism and determining its relevance to humans (8). Thus, in the case of liver hypertrophy, toxicogenomics represents a useful addition to rapidly under- stand the underlying mechanism, which helps in interpreting and positioning liver findings. In one of the pioneering toxicogenomic studies, Hamadeh et al. used gene expression profiling to distinguish different classes of enzyme inducers (9). In this study, rats were treated with three peroxisome proliferators (clofibrate, Wyeth 14,643, and gemfibrozil) and an enzyme inducer (phenobarbital). Pheno- barbital is known to induce a variety of drug-metabolizing enzymes, including cytochrome P450 2B (CYP2B), 2C, and 3A (10). Rats treated for a single day with all compounds showed no histopathologic changes, whereas drug- related microscopic hepatocellular hypertrophy was observed in the livers of all animals after 2 weeks of treatment. Gene expression analysis conducted on the livers from the 24-h–treated rats revealed distinct gene expression patterns induced by the peroxisome proliferators compared with phenobarbital. Further refinement of the gene expression analysis identified a set of 22 genes that could accurately classify blinded liver hypertrophy–inducing compounds as being either compounds that induce peroxisome proliferation or phenobarbital-like compounds (9).
- 40. 26 Buck et al. In our studies, we have used gene expression analysis to identify the mechanism underlying liver hypertrophy induced by an experimental anti- inflammatory agent, A-277249 (6). In this study, treatment of rats for 3 days by A-277249 resulted in a twofold increase in liver weights, coupled with hepato- cellular hypertrophy. A toxicogenomic analysis on the livers from treated rats revealed that A-277749 induced gene expression changes highly similar to those induced by the aromatic hydrocarbon receptor agonists 3-methylcholanthrene and Aroclor 1254, including increases in CYP1A1, CYP1B1, and glutathione-S- transferase (Fig. 1 and Color Plate 1). In addition, the gene expression analysis suggested that the induction of these enzymes might be driven by the targeted pharmacological activities of A-277249, making it unlikely that compounds in this class could be identified that would not result in liver hypertrophy. These studies allowed for a rapid termination of a program with little probability of success. Toxicogenomics has also been applied to demonstrate that fumonisin mycotoxins cause liver hypertrophy and rodent liver carcinogenicity independent of the peroxisome proliferator activated receptor alpha (PPAR-) pathway (11). In this study, wild type and PPAR- knockout mice were treated with fumonisin or Wy-14,463, a prototypical PPAR- agonist. The gene expression analysis showed that PPAR- was necessary for the regulation of Fig. 1. Identification of genes regulated in the liver of rats after xenobiotic activation of the nuclear receptors PPAR-, aromatic hydrocarbon receptor (AhR), or pregnane X receptor (PXR). The heatmap shows the genes significantly regulated in liver by several prototypical inducers of the three nuclear receptors. Genes shown in light gray are either up- or down-regulated relative to vehicle-treated control, and genes shown in black are unchanged. These data were extracted from the Iconix DrugMatrix database. (see Color Plate 1).
- 41. Toxicogenomics in Mechanistic Toxicology 27 lipid metabolism genes associated with liver hypertrophy in Wy-14,463–treated mice, whereas treatment with fumonisin resulted in similar expression changes for the lipid metabolism genes in both wild type and PPAR- knockout mice. These results confirmed that mouse liver carcinogenicity induced by fumonisin was independent of PPAR-, thus suggesting that the toxicity induced by fumonisin is not necessarily species-specific in contrast with what is seen with PPAR- agonists. A number of studies have attempted to apply toxicogenomics in order to identify compounds associated with liver hypertrophy that are likely to test positive in long-term rodent carcinogenicity studies. Identifying these compounds early would result in significant savings in time and resources and would allow for the prioritization of compounds unlikely to test positive in long-term carcinogenicity studies. Fielden et al. (12) used gene expression data from rats treated for 5 days with more than 100 structurally and mecha- nistically diverse nongenotoxic hepatocarcinogens and non-hepatocarcinogens. From these data, the authors identified a gene expression signature consisting of 37 genes that could classify hepatocarcinogens and non-hepatocarcinogens with 86% and 81% sensitivity and specificity, respectively. In addition, by comparing the gene expression data with a reference database, this signature can provide an understanding of potential modes of action for hepatic tumorigenicity such as regenerative proliferation, proliferation associated with xenobiotic receptor activation, peroxisome proliferation, and steroid hormone–mediated mechanisms. A similar approach was taken by Nie et al. (13). In this study, male rats were treated for 1 day with 52 compounds, 24 of which were NGTCs and 28 were non-carcinogens. Microarray analysis on the livers from treated rats revealed a set of six genes that could distinguish NGTCs from non-carcinogenic compounds. Further work is under way to verify the robustness of these signa- tures across different laboratories and gene expression platforms. Nonetheless, these early proof-of-concept studies suggest that gene expression analysis repre- sents a valid approach toward distinguishing NGTCs from non-carcinogens using short-term rodent studies. Overall, the toxicologic relevance of rodent liver hypertrophy to humans is variable and highly dependent on the underlying mechanism. Without an understanding of the mechanism, potentially safe and efficacious compounds may be deprioritized for further development. Thus, the application of gene expression analysis toward identifying the mechanism underlying drug-induced liver hypertrophy represents a clear example of how toxicogenomics can complement traditional toxicologic end points and bring value as a safety evaluation tool.
- 42. 28 Buck et al. 3. Cardiotoxicity: Use of a Tool Compound to Dissect the Mechanism of Toxicity The heart is a relatively common target organ of toxicity for pharmaceu- tical, natural, or industrial agents. For pharmaceutical agents, hepatic toxicity is probably the most common toxicity identified, but cardiac toxicity is sufficiently prevalent to warrant interest by toxicologists. For the toxicologist in devel- opment, pharmaceutical agents can be withdrawn from the market because of cardiovascular toxicity or development compounds can be terminated because of the discovery of unanticipated cardiac toxic changes in chronic toxicology studies. For the discovery toxicologist, the challenge may be in selecting devel- opment candidates that will not induce development-limiting cardiovascular toxicology. Current systems to monitor functional deficits of the cardiovascular system are robust enough to ensure the safety of patients involved in clinical trials or prescribed agents with potential cardiac effects. In contrast, there is a lack of robust biomarkers to detect, assess, and monitor the progression of cardiac structural damage or altered cellular homeostasis (14). Therefore, there is a significant interest in avoiding compounds that may affect myocardiocyte homeostasis or cause structural damage. Histologic changes induced by cardiac toxicants are relatively limited in nature and usually are characterized by myocardiocyte degeneration, apoptosis, or necrosis. In addition, cardiac toxicity may be identified in subacute to chronic studies, at a time when the toxicity is well advanced and when it is difficult to formulate hypotheses regarding mechanisms. Ultrastructural evaluation may allow for an increased definition of the morphologic changes. For instance, mitochondrial swelling in degenerating cells is considered a good indication that mitochondria represent a primary target organelle of toxicity. However, for the most part, morphologic changes observed by histopathology or electron microscopy reveal no real clue regarding mechanism of cardiac toxicity. Gene expression profiling represents a unique approach to rapidly generate a vast amount of molecular data relevant to the toxic event and to use these data to formulate hypotheses that can then be confirmed or refuted in follow-up experiments. This concept will be illustrated in this section using literature examples but also a specific case example that we have encountered in our internal discovery programs. Doxorubicin represents the ideal tool compound to interrogate cardiac toxicity, and consequently doxorubicin has been extensively used in the liter- ature for biochemical studies of cardiac toxicity. Doxorubicin is an anthra- cycline antibiotic with broad-spectrum chemotherapeutic activity. The use of this agent is however limited by its cardiotoxicity, which may occur months to years after treatment (15). The toxicity also occurs in animals, providing excellent preclinical models to understand the mechanism of cardiotoxicity of
- 43. Toxicogenomics in Mechanistic Toxicology 29 the anthracycline antibiotics. One of the proposed mechanisms of cardiotoxicity involves the formation of reactive oxygen species (ROS) via redox cycling of the semiquinone radical intermediate (16,17). ROS formation results in damage to cellular macromolecules, such as DNA, proteins, or lipids (17). In particular, cardiac mitochondria are considered the principal early organelle of toxicity due to damage by the ROS or disturbances in mitochondrial homeostasis (16). The cardiotoxicity of doxorubicin is difficult to detect after a single dose in the rat with serum chemistry and light microscopy (18). In contrast, large numbers of relevant gene expression changes can be detected in the heart shortly after treatment with doxorubicin, and these early transcriptomic effects are useful to unravel the mechanism of toxicity. In various internal gene profiling studies, we have demonstrated that doxorubicin affects biological pathways related to mitochondrial function and calcium regulation, thereby demonstrating that gene expression profiles represent an ideal tool for hypothesis gener- ation. Interestingly, these transcriptomic changes indicative of mitochondrial dysfunction occurred well before decreased ATP production, which can be shown using isolated mitochondria from the heart of doxorubicin-treated rats (19,20). This reinforces the concept that gene expression changes most relevant to the mechanism of toxicity can occur well before actual organelle dysfunction. This property is very useful and can represent a very effective approach to select exploratory compounds without cardiac toxicity liabilities. This is best illustrated by a recent study that our discovery organization published (21). Our team was interested in developing inhibitors of acetyl-coA carboxylase (ACC) as potential therapeutic agents for hyperlipidemia. Evaluation of the early series revealed neurologic and cardiovascular liabilities in preclinical models, precluding further evaluation of this chemotype. This prompted us to investigate the mechanism of toxicity of this series, in an effort to develop a counterscreen that could be used in the selection of backup compounds. Briefly, male Sprague-Dawley rats were treated daily with toxic doses of the exploratory compounds for 3 days. Doxorubicin was used as a positive control in this study. As expected, no evidence of cardiotoxicity was evident after this short-term dosing period when evaluating histopathologic sections and serum chemistry panels. In contrast, the gene expression profiles induced in the heart by these compounds were very similar to those induced by doxorubicin, confirming their cardiotoxic liability and also suggesting similar mechanisms of toxicity. DrugMatrix is a commercial database that contains gene expression profiles of multiple organs of rats treated with a wide variety of pharmaceutical and toxicologic agents (22). When these gene expression changes were compared with those present in the DrugMatrix commercial database, the expression profiles from the ACC inhibitors had strong correlations with a number of reference expression profiles, especially profiles induced by cardiotoxicants or
- 44. 30 Buck et al. cardiotonic agents, such as cyclosporin A, haloperidol, or norepinephrine, again suggesting cardiotoxic liability. Finally, when the gene expression changes were analyzed in the context of biological pathways, the mitochondrial oxidative phosphorylation pathway was mostly affected. Overall, these results indicated that this series of compounds had cardiotoxic liability due to a mechanism very similar to that of doxorubicin. This rapid mechanistic understanding allowed us to rapidly select safer chemotypes for this program by ensuring the lack of similar transcriptomic effects in the hearts of treated rats. 4. Estrogenic Activity: Complex Mechanisms Arising from Similarly Acting Compounds Compounds with hormone-mimetic effects have the potential to influence fetal and prepubertal development, fertility, and mammary and reproductive carcinogenesis. The significance for carcinogenesis in high-dose screening studies depends, in part, on the particular hormone pathways that are activated or inhibited (23). The contribution of toxicogenomics for understanding the mechanisms of action and toxicity for individual estrogenic compounds screened in the rat uterotropic assay is discussed after a brief review of the complexities of estrogen signaling. Estrogen effects are mediated by nuclear-mediated and membrane-mediated mechanisms. Nuclear-mediated estrogen effects employ the estrogen receptor alpha (ER-) and, recently discovered in 1996, the estrogen receptor beta (ER-) (24). ER- and ER- are encoded by separate genes with unique devel- opmental and tissue distributions and each undergoes alternative splicing to form multiple isoforms (25,26). In the presence of estradiol, the nuclear estrogen receptors undergo dimerization and activate transcription by binding to the estrogen response element. ER- recruitment of transcription initiation factors is less efficient than that of ER-, and these two estrogen receptors tend to have opposing effects within a particular cell (26). The ER- isoform ER- 46 has a truncated N-terminus and is unable to bind to the estrogen response element, but when palmitoylated, it can associate with the Shc adapter protein and the insulin-like growth factor I receptor (IGF-IR) to transduce signals from the plasma membrane upon binding to estrogen (27). Membrane-mediated estrogen effects are responsible for fast signaling events that have been described in vascular endothelium, neurons, and the myometrium. Direct phosphorylation of estrogen receptors by mitogen-activated protein kinase (MAPK) and growth factor receptors alters ER binding to coactivators and corepressors causing altered function in nuclear-mediated pathways (28). Additionally, an orphan G-protein–coupled receptor, GPR30, is estrogen responsive independent of
- 45. Toxicogenomics in Mechanistic Toxicology 31 nuclear receptors through activation of adenylyl cyclase (29). The conver- gence of membrane and nuclear estrogen signaling results in the modulation of transcription and cell function beyond those genes with estrogen response elements in their promoters (30,31). Nuclear estrogen receptors undergo confor- mational changes when bound to ligand that reveal binding sites for coactivators and corepressors that have chromatin remodeling activity (28). The coacti- vators are targets for modulation by signal transduction cascades themselves and are shared with other nuclear receptors leading to cross-talk between pathways (32,33). ER tethering to other transcription factors, such as HIF-1 and Sp1, also allows estrogen signaling to modulate genes lacking an estrogen response element (34,35). Finally, xenoestrogens and selective estrogen receptor modifiers (SERMs) each have distinct profiles of ER binding and recruitment of cofactors leading to diverse transcriptional responses (28). The complexity of estrogen signaling makes predictive modeling of tissue-specific gene expression difficult, but a genomics approach linking gene expression to mechanisms of action is proving useful for evaluating risks in both environmental and pharma- ceutical toxicology. The toxicogenomics approach to linking gene expression and mechanism of action can be thought of in a stepwise manner. First, compounds are admin- istered and gene expression profiles are collected from the tissue of interest. For example, the role of estradiol in rat uterus gene expression was studied by Wu and colleagues (36). Estradiol was found to be necessary and suffi- cient for inducing the expression of a number of genes. Second, classifi- cation of estrogen-responsive genes according to annotations in gene databases using a predefined vocabulary (http://www.geneontology.org) allows specu- lative association of gene changes with observed phenotypic changes (37). For example, Naciff and colleagues examined gene expression changes in prepu- bertal rat uterus after treatment with 17-ethynyl estradiol, an estrogenic drug with estradiol-like effects (38). They associated phenotypic changes of ethynyl estradiol–treated uterus (such as edema and hyperemia) with genes known to effect vascular function (e.g., vascular endothelial growth factor [VEGF] and cysteine-rich protein 61). Third, these gene-phenotype associations can be experimentally directly tested. For example, Heryanto and colleagues admin- istered estradiol in ovariectomized rats and analyzed blood vessel and stromal cell density in the presence and absence of an antibody to VEGF (39). The antibody caused increased stromal cell density, which was interpreted as having blocked uterine edema. Thus, the observation of uterine edema is phenotypi- cally anchored to VEGF gene expression. Fourth, experimentally proven gene- phenotype associations can be used for screening future agents for undesirable toxic effects when the phenotype is below the sensitivity of detection or does not present itself in the model system used.
- 46. 32 Buck et al. Toxicogenomics can enhance the sensitivity of screening assays for pharma- cological effect. For example, phytoestrogens are present in laboratory animal feed containing soy, but it is unclear whether or not they interfere with the uterotropic assay for screening potential endocrine disruptors for estrogenic effects. Naciff and colleagues (40) found gene expression analysis to be more sensitive to estrogenic compound administration than phenotypic markers (i.e., wet uterus weight). Therefore, they performed gene expression analysis on prepubertal rats fed a phytoestrogen-containing diet compared with rats treated with a phenotypically subthreshold dose of ethynyl estradiol (41). None of the animals had detectable changes in uterine wet weight. Gene expression was altered on the phytoestrogen-containing diet, but there were no genes in common between the subthreshold estrogen control and the phytoestrogen diet. The conclusion was that phytoestrogen-containing feed does not interfere with uterotropic screening assays. As a second example, the objective of a pharmaceutical project was to develop a new SERM with significantly reduced incidence of pelvic organ prolapse, which is a side effect of estrogen therapy, while preserving the beneficial antiproliferative effects in breast and uterine tissue and osteoprotective effects. Unfortunately, the rat uterotropic assay does not distinguish SERMs that predispose to pelvic organ prolapse. Helvering and colleagues have associated increased matrix metalloproteinase 2 (MMP2) gene expression with a greater risk of prolapse in humans and then translated this finding to comparative microarray analysis of rat uterine gene expression using multiple SERMs (42). If MMP2 is proven to be involved in the mechanisms leading to pelvic organ prolapse, it could be exploited as a reliable indicator against which to screen compounds in laboratory animal species for the relative risk of this complication in humans. 5. Testicular Toxicity: Defining Toxic Mechanisms in a Complex Tissue Compared with studies in the liver or kidney, there have been few published studies using gene expression profiling to investigate mechanisms of testicular toxicity. Yet, testicular toxicity is not uncommon during the development of pharmaceutical agents and can represent a challenge, as it may not be detected morphologically until chronic studies are conducted, but also because early histopathologic changes can be very subtle and can easily be missed unless more sophisticated techniques, such as tubular staging, are used (43). Current biomarkers of toxicity (such as serum follicular stimulating hormone or semen analysis) are not robust enough to detect early changes both in preclinical studies and in clinical trials. A recent working group sponsored by the Institutional Life Sciences Institute (ILSI) Health and Environmental Sciences Institute
- 47. Toxicogenomics in Mechanistic Toxicology 33 (HESI) has tried to address this issue through an evaluation of additional biomarkers, such as Inhibin B (44). Data released do not suggest that plasma inhibin B represents a marker sufficiently sensitive to detect modest testicular dysfunction in rats. Because sensitive biomarkers are currently lacking, gene expression profiling represents a potentially useful approach to develop an improved understanding of the various mechanisms of testicular toxicity. This improved understanding could ultimately be used to discover new sensitive markers of testicular injury. This is of particular interest to the pharmaceutical industry, but especially in the field of environmental toxicology, as a significant number of pesticides and other environmental toxicants have known testicular effects. Investigating the mechanism of testicular toxicity can be quite challenging, given the complexity of the testis as a tissue, but also the complexity of the regulatory mechanisms for testicular function. The testis is composed of several different cell types characterized by unique functional and morphologic features but having close paracrine interactions and complex cellular interdependence. In vitro models have been used to circumvent this complexity, but ultimately these models do not reflect the paracrine interactions occurring in the tissue. Furthermore, cell lines of Sertoli or Leydig cell origin have lost major functional characteristics, and primary cultures of testicular cells require significant resources of time and can only be used for short-term experiments. For instance, Leydig cell cultures are frequently used to interrogate specific mechanisms of toxicity. Cultures are prepared with Percoll gradient centrifugation methods but are usually not pure, and preparations only yield a limited number of cells that need to be used shortly after preparations. These primary cultures are, however, extremely useful to confirm or refute hypotheses. For instance, adult Leydig cells were used to further understand the mechanism by which Aroclor 1254, a polychlorinated biphenyl, disrupts gonadal function (45). By reverse transcription PCR, the authors demonstrated treatment-induced downregulation of the transcripts of various enzymes involved in steroidogenesis. These results coupled with a demonstration of decreased basal and luteinizing hormone (LH)- stimulated testosterone and estradiol production confirmed that Leydig cells represent a primary target cell for Aroclor 1254 and that the mechanism of toxicity was partly related to an effect of steroidogenesis. A similar approach will be illustrated in a subsequent example. Several studies have used gene expression profiling to investigate the molecular basis of testicular toxicity (46–51). For instance, testicular gene expression profiles were generated using a custom nylon DNA array and evaluated after exposure of mice to bromochloroacetic acid, a known testicular toxicant. Transcript changes were detected involving genes with known functions in fertility, such as Hsp70-2 and SP22, as well as genes encoding
- 48. 34 Buck et al. proteins involved in cell communication, adhesion, and signaling, supporting the hypothesis that the toxicologic effect was the result of disruption of cellular interactions between Sertoli cells and spermatids (49,52). Several studies have investigated using genomics technologies to identify the mechanisms of testicular toxicity associated with exposure to di(n-butyl) phthalate (DBP) (50,53). DBP is a plasticizer widely used in products such as food wraps, blood bags, and cosmetics (54). Because of its wide use, levels of DBP metabolites are detectable in human urine (55). DBP is a male reproductive toxicant, and the developing testis is a primary target through a suspected antiandrogenic effect (56). Using fetal rat testes exposed in utero to DBP, Shultz et al. evaluated global changes in gene expression and demonstrated reduced expression of several steroidogenic enzymes, thereby providing a molecular mechanism for the antiandrogenic effect of this agent (50). These findings are nicely supported by and explained at the molecular level by other investigations showing that DPB impairs cholesterol transport and steroidogenesis in the fetal rat testis (57). Likewise, a recent study investigated the mechanisms of toxicity associated with the triazole fungicides (51). It is known that these agents can modulate in mammalian species many cytochrome mixed function oxidase (CYP) enzymes that are involved in the metabolism of xenobiotics and endogenous molecules. Testicular toxicity associated with these agents is typically not detected until chronic or reproductive studies are conducted. Using custom oligonucleotide microarrays, the authors confirmed that these agents altered the expression of many CYPs and affected the sterol and steroid metabolic pathways in the testis. Although these studies are preliminary, they do suggest several plausible mechanisms of toxicity that need to be further interrogated with specifically designed follow-up experiments. In the pharmaceutical industry, the primary objective of gene expression profiling studies in the testis is to develop approaches that would allow one to weed out potential testicular toxicants at an early stage in the candidate selection process in order to avoid compound termination at an advanced stage of development. A recent study used this approach (58). Using four prototypical testicular toxicants, this study evaluated whether gene expression profiling could facilitate the identification of compounds with testicular toxicity liabilities. In this study, gene expression profiles were generated 6 h after dosing. Not surprisingly, histopathologic changes were absent, except for one agent. However, the microarray analysis identified several differentially expressed genes that were consistent with the known action of the toxicants. Our laboratory has also evaluated this approach. However, in contrast with the above studies, our gene expression profiles were generated 1 and 4 days after treatment with the prototypical toxicants. As expected, these treatments did not result in significant histopathologic changes, although all toxicants used in the
- 49. Other documents randomly have different content
- 50. eläinparka oli kuollut. Silmät olivat pullistuneet päästä, kieli riippui ulkona ja turpa oli vaahdossa. Otin sen syliini, koetin tulen edessä herätellä sitä eloon — mahdotonta. Suruni lemmikkini kadottamisesta oli sitä suurempi, koska en voinut olla vapaa omantunnon tuskista. Minun täytyi syyttää itseäni sen kuolemaan syypääksi, sillä en voinut muuta otaksua kun että se kuoli kauhusta. Mutta kuinka hämmästyinkään nähdessäni, että sillä oli niska poikki, sillä kun lähemmin tarkastin, huomasin sen kaulanikamien vääntyneen irti selkärangasta. Eiköhän se ollut tapahtunut pimeässä ja samallaisen käden kautta kuin minunkin oli? Eiköhän inhimilliset voimat sittenkin olleet koko ajan vaikuttaneet tapahtumiin tässä huoneessa? Monta syytä näytti olevan tähän otaksumiseen. Todistaa en sitä voi, voin ainoastaan yksinkertaisesti kertoa mitä omilla silmilläni näin. Lukija voi itse tehdä omat johtopäätöksensä. Päivän nousuun saakka ei mitään enää tapahtunut. Ensimäisen auringonsäteen esiin pilkistäessä jätin kummitustalon. Ennenkuin lähdin, kävin kerran vielä siinä pienessä, tyhjässä huoneessa, josta ei ole uloskäytävää, ja jossa olin palvelijani kera vankina. En voinut olla ajattelematta, että sillä voimalla, joka kaiken kauhun oli saanut aikaan, oli sijansa juuri tuossa pienessä huoneessa. Vaikkakin menin sinne nyt selvällä päivällä ja aurinko kirkkaasti loisti likaisten ikkunaruutujen lävitse, valtasi minut jälleen yön kauhu. En voinut pakottaa itseäni olemaan siellä enempää kuin noin puoli minuuttia. Menin portaita alas ja taasen kuulin edelläni askeleita; ja kun suljin oven, olin kuulevinani hiljaista naurua. Menin kotiin. Otaksuin sieltä löytäväni karkuripalvelijani, mutta hän ei ollut sinne palannut. Seuraavinakaan päivinä en hänestä mitään kuullut, kunnes vihdoin sain häneltä Liverpoolista seuraavan kirjeen: Kunnioitettavin herra, pyydän nöyrimmin teiltä anteeksi, vaikka
- 51. tuskin voin toivoa että pidätte minua sen arvoisena, olkoonpa että tekin — mistä taivas kuitenkin lie teitä varjellut — näitte sen mitä minä näin. Minusta tuntuu että vuosia kuluu, ennenkuin pääsen entiselleni. En enää kelpaa palvelijaksi, siitä ei ole epäilemistäkään, sentähden matkustan lankoni luo Melbourneen. Huomenna laiva lähtee. Mahdollisesti rauhoitun jälleen tällä pitkällä matkalla. Kymmenenkin kertaa päivässä hätkähdän ja joka jäseneni vapisee; minusta tuntuu niinkuin tuo olisi yhä takanani. Pyydän teitä nöyrimmin, arvoisa herra, lähettämään kaikki sinne jääneet tavarani ja loput palkastani äidilleni Walworthiin. — Johan tietää hänen osoitteensa. Kirje loppui monilla anteeksi pyynnöillä sekä yksityiskohtaisilla selityksillä asioista, jotka koskivat hänen palvelustaan. Varmaankin lienee monelle tämä pako Australiaan todistuksena siitä, että tämä mies oli tavalla tai toisella petollisessa yhteydessä yön tapahtumien kanssa. En väitä näitä otaksumisia vastaan, mutta luulen kuitenkin, että ne ovat suurimmalle osalle helpoimpana selityksenä näihin yliluonnollisiin asioihin. Illalla palasin jälleen kummituspaikalle hakemaan tavaroitani ja koiraparkani ruumista. Ei minua silloin mikään häirinnyt, eikä mitään outoa sattunut, paitsi että portaita noustessani ja laskeutuessani kuulin jälleen askeleiden sipsuttavan edelläni. Lähdettyäni talosta menin herra J:n luo ja tapasin hänet kotoa. Annoin avaimen hänelle takaisin, vakuuttaen että uteliaisuuteni oli täydellisesti tyydytetty. Rupesin juuri kertomaan hänelle, mitä oli tapahtunut, kun hän keskeytti minut ja sanoi kohteliaasti, että hän oli kadottanut kaiken mielenkiinnon salaisuuteen, jonka perille ei kuitenkaan koskaan pääsisi. Siitä huolimatta päätin ainakin kertoa
- 52. kirjeistä, jotka olin lukenut, sekä niitten kummallisesta katoamisesta ja kysyin häneltä, luuliko hän niitten olevan osoitettuja äsken kuolleelle talonhoitajattarelle; eiköhän hänen elämästään löytäisi vastausta niihin hämäriin otaksumiin, joihin kirjeissä viitattiin? J. näytti hämmästyneeltä ja hetkisen mietittyään sanoi hän: En tiedä paljoa tuon vaimon entisistä elämänvaiheista, ainoastaan sen, että hän on ollut perheeni tuttu. Kuitenkin herätätte muistooni jotain hänelle epäedullista. Tahdon ottaa asiasta selkoa ja aikanaan kertoa teille tulokset. Mutta jos hän olisikin johonkin rikokseen syypää ja me uskoisimme siihen, että olennon, joka on täällä maan päällä ollut jonkun selvittämättömän rikoksen tekijänä tai uhrina, täytyisi palata ja rauhattomana haamuna käyskennellä sillä paikalla jossa rikos on tapahtunut, niin täytyy minun vastata tähän, että talossa kuului kolinaa ja näkyi outoja näkyjä jo ennenkuin tämä vanhus siellä kuoli. Te hymyilette, mitä tahdotte sanoa? — Luulen että jos pääsemme asiasta selville, huomaamme kyllä inhimillisten olentojen vaikutusta näissä tapahtumissa. — Mitä? Te pidätte siis koko kummittelua ainoastaan petoksena? Ja mistä syystä? — En petoksena sanan täydessä merkityksessä — enempää kuin sitäkään, jos voisin, esimerkiksi syvään uneen vaipuneena, josta ette minua voisi herättää, vastata kysymyksiin selvyydellä, joka hereillä ollessani olisi minulle mahdotonta. Jos voisin sanoa teille, paljonko rahaa on kukkarossanne, tai lukea ajatuksenne, niin en pitäisi sitä petoksena enempää kuin yliluonnollisenakaan. Olen silloin tietämättäni eläinmagnetismin vaikutuksen alaisena, jota joku kaukana oleva, minulle ennen tuntematon henkilö, minua kohtaan suuntaa. Löytynee kai eläinsähkölle sukua oleva, jopa
- 53. voimakkaampikin voima; — ennen muinoin sitä sanottiin magiaksi, salaisvoimaksi tai -opiksi. Mahdollisesti sellainen voima on myös vainajissa, nimittäin niin, että sen vaikutus ulottuu ainoastaan joihinkin määrättyihin ajatuksiin ja muistoihin, eikä siihen osaan, jota oikeastaan sanomme sieluksi, sillä se on kuoleman jälkeen kaikesta maallisesta irti. Se voima ulottunee, kuten sanottu ainoastaan siihen osaan, joka on maallinen ja synnillinen, ja siten aistiemme käsitettävissä. Tämä on kuitenkin vanhentunut teoria, jota en voi arvostella. Sittenkään en mitenkään usko niitä voimia, jotka tässä toimivat, yliluonnollisiksi. Sallitteko minun esimerkillä selittää teille, mitä tarkoitan. Paracelsus pitää seuraavaa koetta helppona ja myöhemmät kirjailijat uskovat siihen myöskin. Kukka kuolee, te poltatte sen. Olkoon kukka millainen tahansa, tuhkana ei sitä miksikään tunne. Kuitenkin voi kemiallisen prosessin avulla kukan tuhkasta saada värispektrumin, joka hyvin muistuttaa tuota elävää kukkaa. Samoinhan voi olla elävien ihmistenkin laita. Sielu on poissa, kuten tuoksu, kukan sielu. Kuitenkin voi synnyttää väri-ilmiön, jota taikauskoiset voivat pitää poismenneen sieluna. Se ei ole muuta kuin kuolleen ruumiin kuva. Siten on myös selvitettävissä se, että kaikkein parhaistakin kummitusjutuista aina säännöllisesti puuttuu — kaikki sielullinen, ylevä henki. Kummitukset esiintyvät enimmiten vähäpätöisten tai aivan olemattomien syiden vuoksi, puhuvat harvoin ja silloinkin tuskin mitään, joka kohoaisi jokapäiväisten ajatusten tasoa ylemmäs. Amerikalaisilla spiritisteillä on kokonaisia nidoksia suorasanaista ja runomittaista kirjallisuutta, joka muka olisi suurten kuolleiden kuten Shakespeare'n, Baco'n, ja luoja tiesi keiden sanelemaa. Vaikka tarkastelisimme parhaita tällaisia vainajien ilmoituksia, eivät ne ole hituistakaan parempia, kuin mitä tavallisin lahjakas kasvatuksen saanut kuolevainen voi kuvitella. Ne ovat paljoa huonompia kuin mitä Baco, Shakespeare ja Plato sanoivat ja
- 54. kirjoittivat elossa ollessaan. Silmiinpistävää on myös, etteivät ne sisällä ainoatakaan uutta ajatusta. — Minä puolestani pidän kaikkia näitä ilmiöitä ainoastaan ajatuksina, jotka jollain tuntemattomalla tavalla kulkevat kuolevaisen aivoista toiseen. Olkoonpa, että pöydät itsestään liikkuisivat, pirulliset haamut näyttäytyisivät salaperäisen sädekehän ympäröiminä tai ruumiittomat kädet sukeltaisivat esiin ja tarttuisivat kiinni näkyviin esineihin, tai että joku hämärä, outo, kuten minä näin, saisi veremme jähmettymään. Uskon varmasti, että kaikki ainoastaan olisi vierasten aivojen sähkölankojen kautta tapahtunutta vaikutusta aivoihini. Löytyy luonnollisia sähkövoimasia ruumiita ja nämä voivat aikaansaada magneettisia ihmeitä; toisissa on taas jonkunmoista luonnollista fluidumia, sähköä, jos niin tahdotte, ja nekin voivat tehdä sähköihmeitä. Molemmat eroavat normaali-tieteestä ja ovat sille aivan tarkoituksettomia, tuloksettomia ja arvottomia. Ne eivät vie mihinkään korkeaan päämäärään, ja sentähden ei maailma niistä välitä eikä todellinen tiede ole tätä voimaa ihmisessä viljellyt. Uskon varmasti, että kaikella, mitä kuulin ja näin, on lihasta ja verestä oleva olento, kuten minäkin, kaukaisena aikaansaajana ja täydellisesti tietämättömänä seurauksista. Luulen, että tästä syystä ei kaksi henkilöä, kuten kerroitte, ole samaa kokenut. Jos koko kummittelu johtuisi tavallisesta petoksesta, olisi koneisto niin järjestetty, että vain pienet poikkeukset tavallisesta olisivat mahdollisia. Jos ne olisivat yliluonnollisia, luojan sallimuksella toimivia voimia, olisi niillä varmasti joku päämäärä. Nämä ilmiöt eivät kuulu kumpaankaan luokkaan. Päinvastoin olen vakuutettu siitä, että ne lähtevät aivoista, jotka ovat kaukana meistä, ja että nämä aivot eivät tarkoituksella aiheuta sitä, mitä tapahtuu, vaan että nämä tapahtumat vain kuvastavat niiden harhailevia kirjavia, vähän vaihtelevia puolinaisia ajatuksia. Lyhyesti,
- 55. se mitä on tapahtunut, ei ole ollut mitään muuta kuin tuollaisten aivojen todellistuneita unia, aivojen, jotka todella voivat osittain ruumiillistua. Näillä aivoilla lienee suunnaton voima, niin että se voi panna liikkeelle pahansuopia ja hävittäviä välikappaleita, sillä sellainen voima oli selvästi koirani tappanut. Todennäköisesti olisi se tappanut minutkin, jos olisin joutunut samallaisen pelon valtaan kuin koira, elleivät järkeni ja henkiset voimani olisi antaneet minulle vastustusvoimaa. — Sekö on tappanut koiranne? Sehän on kauheata! — Todellakin, on hyvin outoa, ettei mitään eläimiä koskaan ole saatu viihtymään tässä talossa, ei edes kissaakaan; ei ole siellä näkynyt rottia eikä hiiriäkään. Eläimet varmaan tuntevat vaistollaan heille kuolettavien voimien vaikutusta. Tässä kohden on ihmisen ymmärrys vähemmän luotettava. Mutta kylliksi tästä. Ymmärrättekö teoriani? — Kyllä, ainakin osapuilleen, ja hyväksyn mielelläni minkä hyvänsä järkevään päin olevan selityksen mieluummin kuin uskon tässä haamujen ja tonttujen peliään pitävän — sillä nehän kuuluvat lastenkamariin. Mutta mitä ihmeessä teen minä talolle? — Sanon teille, miten teidän on tähän asiaan ryhtyminen. Se otaksuma että tuo pieni kalustamaton huone makuuhuoneen vieressä on koko kummittelun lähtöpaikka, näyttää minusta epäämättömän varmalta. Aivan tosissani annan siis teille sen neuvon, että revitte muurit ja hävitätte koko huoneen. Huomasin nimittäin, että se on rakennettu muista huoneista ulkonevasti erikseen takapihalle ja että sen siis taloa vahingoittamatta voi hävittää.
- 56. — Luuletteko todellakin, että jos sen tekisin, nuo sähkövirrat sillä häviäisivät? — Koettakaa. Olen niin varmasti vakuutettu siitä, etten erehdy, että mielihyvällä otan osalleni puolet kustannuksista, eritoten jos vielä uskotte minulle työn johdon. — Se ei tule kysymykseenkään, kustannukset maksan itse, kaikesta tulette saamaan tarkat tiedot. Noin kymmenen päivää sen jälkeen sain herra J:ltä kirjeen. Hän oli itse käynyt talossa sekä löytänyt nuo kaksi kirjettä siitä samasta paikasta, mistä minä olin ne ottanut. Ne olivat herättäneet hänessä samat epäilykset kuin minussakin, ja hän oli koettanut mitä huolellisimmin ottaa selkoa tuosta naisesta, jolle ne olivat osoitetut. Näytti siltä, että tämä nainen oli mennyt naimisiin vasten vanhempainsa tahtoa, vieläpä erään sangen epäilyttävän amerikalaisen kanssa — häntä pidettiin yleensä merirosvona. Nainen itse oli kunniallisen kauppiaan tytär ja ammatiltaan opettajatar. Hänellä oli hyvissä varoissa elävä veli, joka oli leski ja jolla oli 6- vuotias lapsi. Vuosi avioliiton jälkeen löydettiin tämän veljen ruumis Thames-virrasta, aivan Lontoonsillan luota. Hänen kaulassansa näkyi väkivallan merkkejä, mutta ei niitä pidetty kylliksi riittävinä muuttamaan ruumiinkatsastuksen tulosta: tapaturmaisesti hukkunut. — Amerikalainen ja hänen vaimonsa ottivat lapsen kasvattaakseen kuten kuollut velikin oli viimeisenä tahtonaan ilmoittanut; testamentissa oli samalla määrätty, että jos lapsi kuolee, perii omaisuuden sisar. Lapsi kuoli noin kuusi kuukautta myöhemmin; huhuttiin että sen kasvatusta oli laiminlyöty ja sitä pidelty pahoin. Sanoivatpa naapurit kuulleensa sen öisin huutavankin. Lääkäri, joka tutki lapsen ruumiin, todensi, että se oli kuollut riittämättömästä
- 57. ravinnosta; ruumiissa saattoi huomata myös mustankeltaisia mustelmia. Näytti siltä kuin olisi pienokainen koettanut paeta eräänä talviyönä, hiipinyt takapihalle ja yrittänyt kiivetä muurin yli; nääntyneenä oli se kuitenkin pudonnut pihalle, josta se aamulla oli kuolevana löydetty. Joskin siis saattoi epäillä harjoitetun kamalaa julmuutta, niin murhaa ei kuitenkaan voitu sanoa tapahtuneen. Täti ja hänen miehensä koettivat asiaa peitellä — kertomalla lapsen aivan erikoisesta vastustushalusta ja kovapäisyydestä, sanoen häntä jopa tylsämieliseksikin. Miten hyvänsä, tuo nainen peri nyt pienen orvon kuoleman jälkeen veljensä omaisuuden. Mutta ennenkuin heidän avioliittonsa ensimäinenkään vuosi oli kulunut katosi mies äkkiä Englannista, enää koskaan takasin palaamatta. Hän vuokrasi risteilijän, joka teki kaksi vuotta myöhemmin Atlannilla haaksirikon. Leski jäi tosin hyviin varoihin, mutta hänelle tapahtui kaikellaisia onnettomuuksia. Eräs pankki teki vararikon, hyvin sijoitettu pääoma oli menetetty; hän osti erään liikkeen, mutta menetti siinä lopunkin omaisuudestaan; hoiti taloudenhoitajattaren toimia, mennen kuitenkin yhä enemmän alaspäin, kunnes lopulta päätyi siivoojattareksi. Hän ei pysynyt missään, vaikkakaan häntä vastaan ei ollut mitään erikoisempaa huomauttamista; päinvastoin, jokainen tunnusti hänen kykeneväisyytensä, rehellisyytensä ja hiljaisen käytöstapansa. Mutta mikään ei hänelle näyttänyt olevan siunaukseksi. Niin vaipui hän köyhäintalon hoidokiksi, josta tilasta herra J. hänet vapautti pannen hänet juuri sen talon hoitajaksi, jonka vuokraajana nainen itse oli kerran ollut. Herra J. lisäsi vielä kirjeessään olleensa hetkisen yksin siinä huoneessa, jonka olin tahtonut hävitettäväksi, mutta ei nähneensä eikä kuullensa siellä mitään; hänet oli kuitenkin vallannut sellainen
- 58. selittämätön kauhu, että hän mielihyvällä suostui ehdotukseni mukaan huoneen hävittämään. Hän oli jo tilannut työmiehiä ja aikoi alottaa työn heti kun se vain minulle sopisi. Menin kummitustaloon tuohon salaperäiseen huoneeseen; särimme puusisustuksen ja revimme lattian auki. Lattialankkujen alla, tomun ja lian peitossa, oli miehen mentävä luukku. Se oli huolellisesti suljettu rautaisilla kiskoilla ja säpeillä. Aukaistuamme sen, laskeuduimme luukusta maanalaiseen huoneeseen, jonka olemassa oloa ei kukaan ollut aavistanut. Siinä oli ikkuna ja tulisija, mutta olivat molemmat nähtävästi jo aikoja sitten umpeen muuratut. Tulen valossa tutkimme huoneen. Sen huonekalut olivat kaikki 18:nnen vuosisadan mallia ja oli niitä kolme tuolia, tamminen pöytä ja nojatuoli. Seinällä oli vaatekaappi, josta löysimme puoleksi lahonneita miesten vaatteita, nekin edellisen vuosisadan mallia, ja nähtävästi korkeassa asemassa olevalle miehelle kuuluvia. Ne olivat koristetut kalliilla terässoljilla ja napeilla. Paitsi sitä oli kaapissa miekka sekä liivit, jotka muinoin olivat olleet kultaompeleilla koristetut, mutta nyt jo mustuneet ja kosteuden turmelemat. Vieläkin löysimme sieltä viisi kultarahaa, muutamia hopearahoja sekä norsunluusta tehdyn pääsylipun erääseen huvipaikkaan, jonka ilot ovat ammoin haihtuneet. Mutta päälöytömme oli kuitenkin eräänlainen seinään kiinnitetty tulenkestävä kaappi, jonka lukon avaaminen tiirikalla oli sangen vaikeaa. Tässä säiliössä oli kolme hyllyä ja kaksi laatikkoa. Edellisillä oli hyvässä järjestyksessä lasipulloja. Niissä oli värittömiä, haihtuvia nesteitä, jotka eivät tuntuneet myrkyllisiltä, joissakuissa oli fosforia ja salmiakkia. Vielä oli kaapissa muutamia sangen omituisia
- 59. lasiputkia, pieni, toisesta päästään terävä rautatanko, suuri lohkare vuorikristallia ja toinen samallainen merenvahaa, sekä vielä hyvin voimakas magneetti. Eräässä lokerossa oli kultakehyksinen pienoismuotokuva, jonka värit olivat säilyneet ihmeellisen kirkkaina, katsoen siihen, että se oli niin kauan ollut sellaisessa paikassa. Kuva esitti keski-ikäistä miestä. Kasvot olivat sangen merkilliset, niin ettei niitä hevillä unohda. Jos voisi muuttaa käärmeen ihmiseksi, jossa ihmishaahmosta huolimatta olisi säilynyt vanha käärmeen-ilme, niin olisi tuo näiden kasvojen paras kuva. Otsan leveys ja mataluus, pään terävä muoto, pitkien, raollaan olevien kamalien silmien murhaava katse, joka vivahti vihreään ja säkenöi kuin smaragdi, ja tähän vielä lisäksi jonkunmoinen armoton levollisuus, voittamattoman voiman merkki — siinä kuva, joka painui mieleen. Koneellisesti käänsin muotokuvaa kädessäni. Sen takapuolella oli viisikulmio ja sen keskellä pienet tikapuut, joiden kolmannella astimella oli vuosiluku 1765. Tutkin tarkemmin tuota vuosilukua ja löysinkin siitä ponnahtimen, jota painamalla kuvan takaosa avautui kuin kansi. Sisäpuolelle oli kaiverrettu: Sinulle Mariana! Ole uskollinen elämässä ja kuolemassa sinun —, tässä oli nimi, jota en huoli mainita. Olin sen kuullut lapsuudessani vanhoilta ihmisiltä, jotka kertoivat hänestä kuin jostakin häikäisevästä taikurista, joka aikoinaan oli herättänyt suurta huomiota. Lopuksi täytyi hänen paeta syytettynä rakastajattarensa ja kilpakosijansa murhasta. Herra J:lle en kertonut asiasta mitään, ja annoin hänelle, vaikkakin vastenmielisesti, tuon pienoismuotokuvan. Tämän rautaisen kaapin ensimäisen laatikon olimme saaneet helposti auki, mutta sitä vaikeampi oli aukaista toista laatikkoa. Se ei ollut lukossa, mutta ei sitä siitä huolimatta tahtonut mitenkään saada auki. Vihdoin saimme sen kuitenkin taltalla väännetyksi auki, jolloin eteemme ilmestyi
- 60. aivan merkillinen laitos. Pienellä ohuella levyllä oli lasinen kuppi täynnä kirkasta ainetta; kupin päällä oli jonkunmoinen kompassi, jonka neula liikkui nopeasti ympäriinsä ja jossa oli tavallisten ilmansuuntamerkkien sijassa seitsemän merkillistä kirjainta, niiden näköisiä, joita tähtien tutkijat käyttävät planeettien merkkeinä. Aivan erikoinen, ei väkevä eikä vallan vastenmielinenkään, haju lemusi tästä laatikosta. Olipa hajun syy mikä hyvänsä, se teki meihin väkevän fyysillisen vaikutuksen, jonka tunsimme kaikki, jopa apunamme olevat työmiehetkin. Pistelevä tunne levisi yli koko ruumiin, sormenpäistä hiuksenjuuriin saakka. Päästäkseni tuota levyä tutkimaan otin sen päällä olevan lasikupin pois. Äkkiä pyörähti silloin kompassi tavattomalla kiivaudella, ja tunsin koko ruumiissani iskun, joka pudotti kupin kädestäni lattiaan. Kuppi meni rikki, neste virtasi lattialle ja kompassi sinkosi nurkkaan. Samassa silmänräpäyksessä tuntuivat seinät vapisevan, ikäänkuin jättiläinen olisi niitä järkyttänyt. Nuo molemmat työmiehet säikähtivät niin, että juoksivat tikapuita ylös huoneesta; kun ne kuitenkin näkivät ettei mitään enää tapahtunut, palasivat he takasin. Sillävälin olin katsellut tuota levyä. Se oli jonkunmoinen kirja, sidottu punaiseen nahkaan, varustettu hopeisilla haoilla, ja sisälsi vain yhden pergamenttilehden; tälle oli munkki-latinalla kirjoitettu seuraavat sanat: Kaikki, jotka näiden muurien sisällä saan valtaani, olipa se tuntevaa tai elotonta, elävää tai kuollutta, tahdon minä hävittää — niinkuin neula liikkuu, niin työskentelee tahtoni! Kirottu olkoon tämä talo ja rauhattomat sen asukkaat! Muuta emme löytäneet: Herra J. poltti levyn kirouksineen ja hävitti tuon maanalaisen sekä sen päällä olevan huoneen perustuksia myöten.
- 61. Sen jälkeen asui hän talossa kokeeksi kuukauden ja todellakin kodikkaampaa taloa ei Lontoossa lie ollut. Myöhemmin vuokrasi hän sen, eivätkä vuokralaiset ole mitään valittaneet.
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