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From mutagen hunter to genome defender, a change in paradigm
Mutation Research 437 Ž1999. 113–119 www.elsevier.comrlocaterreviewsmr Community address: www.elsevier.comrlocatermutres
From mutagen hunter to genome defender, a change in paradigm Herbert S. Rosenkranz
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Office of the Dean, Graduate School of Public Health, UniÕersity of Pittsburgh, Pittsburgh, PA 15261, USA Dr. Frederick J. de Serres in appreciation for his scientific leadership and his many kindnesses Received 18 November 1998; accepted 28 November 1998
1. Prologue As a ‘‘product’’ of ‘‘Arrowsmith’’ w1x, ‘‘Microbe Hunters’’ w2x and ‘‘Eleven Blue Men’’ w3x, I confess that my entry into the biomedical sciences was the cumulation of dreams of helping mankind by conquering dangerous diseases. Thus, early in my professional career, I enthusiastically enrolled in President Nixon’s ‘‘War on Cancer’’. In fact, I became a ‘‘Mutagen hunter’’ w4,5x. Of course, that safari was based upon the simplistic Žand subsequently shown erroneous. premise, prevalent at the time w6–8x, that since 85% of all human cancers were due to exposure to chemicals in the environment and the work place, if we could identify the culprits, we could eliminate them and hence see a tremendous reduction in cancer incidence! Indeed together with our illustrious honoree, Dr. Frederick J. de Serres, many of us participated in this public health endeavor, in fact we assisted at the birth of ‘‘Genetic Toxicology’’. Of course, over the years our initially naive notions and incorrect premises were revised, corrected and updated. Moreover, the great achievements of molecular biology allowed for great refinements in our concepts and approaches. Oncogenes, suppressor genes, receptors, signal transduction and
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biomarkers joined our lexicon and became objects of our interest. In turn we realized that our initial paradigm that DNA was the primary target of carcinogenesis w9x was no more infallible than the central dogma of molecular biology that DNA was the source of all genetic information w10x. Coupled with this was the realization that 85% of cancers were not the result solely of exposure to chemicals w11x but that factors and modulators such as lifestyle Žcigarette smoking, alcohol consumption., genetic predisposition Že.g., BRCA1 and BRCA2rbreast and ovarian cancers; RB1rretinoblastoma; WT1rWilms’ tumor; APCrcolon, thyroid and stomach carcinomas; etc.. and pathophysiological status Že.g., immunosuppression, hepatitis virus infection. all played pivotal roles. Quickly we realized that cancer prevention by elimination of causative agents was not readily feasible as our ability to recognize the suspect culprits was rudimentary at best and the decision on whether or not to remove them was a societal one as exemplified by the action of the US Congress to exempt saccharin, a suspect carcinogen, from previously enacted restrictions Ži.e., the Delaney Clause of the Food, Drug, and Cosmetic Act.. Recently, this led us to explore other intervention modalities. One of the most appealing of these is cancer chemoprevention by means of ‘‘innocuous’’ chemical food additives w12–17x. Yet, in spite of its attractiveness, recent findings suggest that this approach represents a double-edged sword. There are obvious benefits but also
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newly recognized risks w18–22x which must be considered. However, simultaneously, public health and preventative considerations have led to the acceptance of certain risks, provided they are far outweighed by the benefits Že.g., breast cancer prevention by tamoxifen w23x.. These are, of course, risk management and therefore, they, too, are societal and individual decisions, Yet, because most, if not all, of us have relevant expertises, we might ask what should be our contribution to this developing field? The answer seems obvious to me, we should provide the best possible data and understanding to those charged with making these public health policy decisions. Moreover, our contributions can be most useful in the generation and integration of data related to exposure at the target tissue and the elucidation of mechanisms of potential toxicity. In the past, many of us have deplored what we perceived as the lack of rigor of ‘‘regulatory science’’ w24x. However, we failed to realize that often such situations resulted not from inherently sloppy science but from the legislative or court mandates to regulators to set health criteria by specific deadlines. Thus over two decades ago to protect public health, the US Environmental Protection Agency ŽEPA. set carbon monoxide exposure criteria based upon measurements on only 10 human subjects Žusing methodologies ŽCO-oximetry. subsequently found to be inappropriate for environmental studies. w25x. More recently, to decrease mortality and morbidity attributed to inhalation of particulate material, the EPA w26,27x sought to regulate exposure to 2.5 and 10 mm air particulate matter Ži.e., PM 2.5 and PM 10 .. This was to be done despite a paucity of monitoring data and therefore the absence of Ža. proof of causality and Žb. an understanding of the mechanismŽs. involved w28–30x. One might argue that such regulations in the absence of a thorough mechanistic understanding are unwarranted. Yet, were we to demand mechanistic certainty before taking remedial andror regulatory action in the face of recognized or perceived adverse health effects, we would not have, two decades ago, considered cigarette smoking a hazard until its mechanistic basis was well understood in spite of persuasive epidemiological data. Clearly, as concerned citizens and health professionals we accept the concept of preventative measures in the
absence of complete scientific certainty. On the other hand, cancer chemopreventative strategies whereby healthy individuals, albeit they may be at risk for a specific disease Že.g., breast cancer., are treated prophylactically for prolonged periods, possibly decades or a lifetime, with specific agents, require a much greater level of certainty of the absence of harmful effects. Thus, the failed b-carotene prevention trials to reduce lung cancer were undoubtedly well meant and possibly even biologically plausible w31–33x. Yet they were undertaken in the absence of sufficient knowledge of the mechanism of the expected chemopreventative effect and without consideration of the possible toxicity of this agent w34–37x. Conceivably the situation could be repeated with resveratrol which is being currently considered for such use w38x. Now, how does one get into a disease prevention mode? It involves a rather ‘‘simple’’ phenotypic change. Most of us are already engaged in experimental approaches that can contribute to the hazard identification and risk assessment processes. It basically requires a readjustment in our perspective. Having advocated such an approach, I would like to illustrate a simple application using the structure–activity relational ŽSAR. methodologies that form the basis of our own research w39x. The
Fig. 1. Structure of indole-3-carbinol.
H.S. Rosenkranzr Mutation Research 437 (1999) 113–119
question that we address here is the determination of whether indole-3-carbinol ŽI3C, Fig. 1., a substance present in many vegetables, is a carcinogenrtumor promoter or a dietary cancer preventer? Because more is known about I3C than, for example, resveratrol, it offers a good case study. 2. Background The possible chemopreventative usefulness of I3C is controversial. This derives from the finding that in some feeding regimens w19,40–43x I3C has been reported to act as a tumor promoter rather than as a preventer. Still given the potential usefulness of I3C in cancer prevention, it is worthy of further investigation w17,19,40x. Accordingly we determined the possible carcinogenic and anticarcinogenic properties of I3C using a number of pertinent predictive knowledge-based SAR models. The advantage of the SAR approach is that it distills the experimentally determined biological activities of several thousand chemicals and identifies structural features significantly associated with activity. Subsequently, when the potential of a yet untested chemical Že.g., I3C. is investigated, the expert system determines whether it contains any of the substructures significantly associated with a particular biological activity of interest w44,45x. The presence of such structural determinants is then translated into a probability and a level or spectrum of activity. 3. Results First we addressed the potential of I3C to act as a rodent carcinogen based upon a variety SAR models Žmouse, rat, rodent, trans-species. derived from cancer bioassays carried out under the aegis of the US National Toxicology Program w46x or listed in the Carcinogenic Potency Data Base w44,45x. We also determined the potential of I3C to induce hepatic preneoplastic glutathione-S-transferase positive foci w47x. In none of these was I3C predicted to have a carcinogenic potential. Based upon a previously derived relationship w48x, this suggests that the probability that I3C is a rodent carcinogen is less than 1%. This finding assumes even greater significance in light of the estimate that ; 50% of all chemicals
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tested will give a carcinogenic response in rodents if the test protocol includes the MTD w49x. Moreover, based upon the same battery of SAR models of rodent carcinogenicity, 33.5% of randomly selected chemicals are predicted to be rodent carcinogens. Because most recognized human carcinogens are also mutagenicrgenotoxic w50–53x, we examined the genotoxic potential of I3C. We found this chemical to be devoid of ‘‘structural alerts’’ for DNA-reactivity w54,55x, it is predicted to be neither a Salmonella mutagen w56,57x, nor an inducer of error-prone ŽSOS. DNA repair w58,59x, nor of unscheduled DNA synthesis w60x. Thus, I3C is unlikely to be either a mutagen or a genotoxicant. Inhibition of gap-junctional intercellular communication has been taken as an attribute of tumor promoters w61–65x. I3C also lacks that potential as well w66x. The availability of an SAR model of inhibitors of rodent carcinogenesis Žw67x, unpublished results. enabled us to test I3C for this potential as well. In fact I3C contains a biophore associated with this anticarcinogenic potential. Its presence might explain the reported anticarcinogenicity of I3C. According to the weight of evidence criteria used herein, based upon the SAR models considered, no carcinogenic risk associated with I3C was uncovered while a cancer chemopreventative potential was identified. While these findings in and of themselves are not definitive, they support previously reported findings suggestive of a beneficial effect. Thus, techniques developed by us to identify toxic potentials Ži.e., carcinogenicity and mutagenicity. were used to document the probable safety of I3C. 4. Conclusions The approach taken herein is still rudimentary and the tentative conclusions can only be used as adjuncts to other experimental and population-based data. Still, it has several philosophical implications. Heretofore, our discipline Žgenetic and molecular toxicology. has been concerned primarily with the identification of toxicants Že.g., mutagens, carcinogens.. However, as a result of the changed public perception, prevention, because of its cause-effectiveness, is being recognized as a major component of the health care system. That realignment has shifted the paradigm from the clinician’s emphasis on the
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individual patient to population-based analyses of the effectiveness of preventive interventions and health enhancement. The experimental approaches that are needed are certainly not new to many of us. In fact the application of biomarkers of exposure and of early disease manifestation have been part of our armamentarium for more than a decade. The new unfolding health care panorama gives us the unique opportunity to apply our expertise to aspects of disease prevention. Thus, instead of recognizing toxicants, we can apply our skills to identifying, with assurance, non-toxicants. This we could accomplish simply by shifting our emphasis from tests with high specificity to tests with high sensitivity w68x and from an emphasis on biomarkers of DNA damage to biomarkers of early disease manifestation Že.g., inactivation of suppressor genes. and how they are modulated by presumably non-toxic chemopreventative agents w69,70x. Moreover, because unlike, for example, cancer chemotherapy, which is limited in duration, cancer chemoprevention may require a lifetime of diet supplementation for persons at risk Že.g., individuals with specific breast cancer genes., intervention trials will need to be designed with lifetime epidemiological and biomarker follow-up studies. It is my opinion that acquiring andror re-engineering the tools for this undertaking must be done now. In the not too distant future the results of the ‘‘Environmental Genome’’ project w71x may enable us to identify individuals genetically at increased risk of developing diseases of environmental and occupational etiology. Heretofore, the potential for social harm of such identifications have inhibited their further development and application. However the availability of prophylactic regimens has changed the picture. Now, these subpopulations may be the likely target of intervention studies of the type advocated herein. I think we are equipped professionally as well as philosophically to meet this new challenge. Just as our honoree ŽDr. Frederick J. de Serres. had the vision to use the elegant Neurospora crassa system to identify and understand the induction of inherited changes and to encourage others to make similar leaps of faith in their research careers to prevent diseases caused by environmental mutagens and carcinogens. So too, current and new practitioners of our discipline should be encouraged to confront the challenge, and pursue the, as yet,
uncompleted task. In so doing they will have the satisfaction of being part of a noble tradition w2,3x dedicated to the improvement of the quality and duration of human life.
5. Epilogue The pioneering generation represented by our honoree, building upon the monumental work of earlier microbiologists, epidemiologists and physicians, has made enormous strides in our understanding and prevention of genetic and molecular processes that contribute to acute and chronic diseases. On the other hand, due to changes in lifestyles, migrations and global climate changes, we now witness the re-emergence of old scourges Že.g., tuberculosis, cholera. and the emergence of new ones Že.g., HIV, Hanta and Ebola viruses.. Yet the techniques, tools, knowledge and concepts legated to us by Dr. de Serres and his peers, enable us to face these new challenges. Perhaps the progress from alchemy to the ‘‘magic bullet’’, and from ‘‘microbe hunter’’ to ‘‘mutagen hunter’’ will ultimately lead us to the ‘‘genome defender’’. It is my sincerest wish that Dr. de Serres and his family will continue to witness for many more years the fruits of the profession that he was instrumental in founding and guiding to maturity.
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From mutagen hunter to genome defender, a change in paradigm Herbert S. Rosenkranz
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Office of the Dean, Graduate School of Public Health, UniÕersity of Pittsburgh, Pittsburgh, PA 15261, USA Dr. Frederick J. de Serres in appreciation for his scientific leadership and his many kindnesses Received 18 November 1998; accepted 28 November 1998
1. Prologue As a ‘‘product’’ of ‘‘Arrowsmith’’ w1x, ‘‘Microbe Hunters’’ w2x and ‘‘Eleven Blue Men’’ w3x, I confess that my entry into the biomedical sciences was the cumulation of dreams of helping mankind by conquering dangerous diseases. Thus, early in my professional career, I enthusiastically enrolled in President Nixon’s ‘‘War on Cancer’’. In fact, I became a ‘‘Mutagen hunter’’ w4,5x. Of course, that safari was based upon the simplistic Žand subsequently shown erroneous. premise, prevalent at the time w6–8x, that since 85% of all human cancers were due to exposure to chemicals in the environment and the work place, if we could identify the culprits, we could eliminate them and hence see a tremendous reduction in cancer incidence! Indeed together with our illustrious honoree, Dr. Frederick J. de Serres, many of us participated in this public health endeavor, in fact we assisted at the birth of ‘‘Genetic Toxicology’’. Of course, over the years our initially naive notions and incorrect premises were revised, corrected and updated. Moreover, the great achievements of molecular biology allowed for great refinements in our concepts and approaches. Oncogenes, suppressor genes, receptors, signal transduction and
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biomarkers joined our lexicon and became objects of our interest. In turn we realized that our initial paradigm that DNA was the primary target of carcinogenesis w9x was no more infallible than the central dogma of molecular biology that DNA was the source of all genetic information w10x. Coupled with this was the realization that 85% of cancers were not the result solely of exposure to chemicals w11x but that factors and modulators such as lifestyle Žcigarette smoking, alcohol consumption., genetic predisposition Že.g., BRCA1 and BRCA2rbreast and ovarian cancers; RB1rretinoblastoma; WT1rWilms’ tumor; APCrcolon, thyroid and stomach carcinomas; etc.. and pathophysiological status Že.g., immunosuppression, hepatitis virus infection. all played pivotal roles. Quickly we realized that cancer prevention by elimination of causative agents was not readily feasible as our ability to recognize the suspect culprits was rudimentary at best and the decision on whether or not to remove them was a societal one as exemplified by the action of the US Congress to exempt saccharin, a suspect carcinogen, from previously enacted restrictions Ži.e., the Delaney Clause of the Food, Drug, and Cosmetic Act.. Recently, this led us to explore other intervention modalities. One of the most appealing of these is cancer chemoprevention by means of ‘‘innocuous’’ chemical food additives w12–17x. Yet, in spite of its attractiveness, recent findings suggest that this approach represents a double-edged sword. There are obvious benefits but also
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newly recognized risks w18–22x which must be considered. However, simultaneously, public health and preventative considerations have led to the acceptance of certain risks, provided they are far outweighed by the benefits Že.g., breast cancer prevention by tamoxifen w23x.. These are, of course, risk management and therefore, they, too, are societal and individual decisions, Yet, because most, if not all, of us have relevant expertises, we might ask what should be our contribution to this developing field? The answer seems obvious to me, we should provide the best possible data and understanding to those charged with making these public health policy decisions. Moreover, our contributions can be most useful in the generation and integration of data related to exposure at the target tissue and the elucidation of mechanisms of potential toxicity. In the past, many of us have deplored what we perceived as the lack of rigor of ‘‘regulatory science’’ w24x. However, we failed to realize that often such situations resulted not from inherently sloppy science but from the legislative or court mandates to regulators to set health criteria by specific deadlines. Thus over two decades ago to protect public health, the US Environmental Protection Agency ŽEPA. set carbon monoxide exposure criteria based upon measurements on only 10 human subjects Žusing methodologies ŽCO-oximetry. subsequently found to be inappropriate for environmental studies. w25x. More recently, to decrease mortality and morbidity attributed to inhalation of particulate material, the EPA w26,27x sought to regulate exposure to 2.5 and 10 mm air particulate matter Ži.e., PM 2.5 and PM 10 .. This was to be done despite a paucity of monitoring data and therefore the absence of Ža. proof of causality and Žb. an understanding of the mechanismŽs. involved w28–30x. One might argue that such regulations in the absence of a thorough mechanistic understanding are unwarranted. Yet, were we to demand mechanistic certainty before taking remedial andror regulatory action in the face of recognized or perceived adverse health effects, we would not have, two decades ago, considered cigarette smoking a hazard until its mechanistic basis was well understood in spite of persuasive epidemiological data. Clearly, as concerned citizens and health professionals we accept the concept of preventative measures in the
absence of complete scientific certainty. On the other hand, cancer chemopreventative strategies whereby healthy individuals, albeit they may be at risk for a specific disease Že.g., breast cancer., are treated prophylactically for prolonged periods, possibly decades or a lifetime, with specific agents, require a much greater level of certainty of the absence of harmful effects. Thus, the failed b-carotene prevention trials to reduce lung cancer were undoubtedly well meant and possibly even biologically plausible w31–33x. Yet they were undertaken in the absence of sufficient knowledge of the mechanism of the expected chemopreventative effect and without consideration of the possible toxicity of this agent w34–37x. Conceivably the situation could be repeated with resveratrol which is being currently considered for such use w38x. Now, how does one get into a disease prevention mode? It involves a rather ‘‘simple’’ phenotypic change. Most of us are already engaged in experimental approaches that can contribute to the hazard identification and risk assessment processes. It basically requires a readjustment in our perspective. Having advocated such an approach, I would like to illustrate a simple application using the structure–activity relational ŽSAR. methodologies that form the basis of our own research w39x. The
Fig. 1. Structure of indole-3-carbinol.
H.S. Rosenkranzr Mutation Research 437 (1999) 113–119
question that we address here is the determination of whether indole-3-carbinol ŽI3C, Fig. 1., a substance present in many vegetables, is a carcinogenrtumor promoter or a dietary cancer preventer? Because more is known about I3C than, for example, resveratrol, it offers a good case study. 2. Background The possible chemopreventative usefulness of I3C is controversial. This derives from the finding that in some feeding regimens w19,40–43x I3C has been reported to act as a tumor promoter rather than as a preventer. Still given the potential usefulness of I3C in cancer prevention, it is worthy of further investigation w17,19,40x. Accordingly we determined the possible carcinogenic and anticarcinogenic properties of I3C using a number of pertinent predictive knowledge-based SAR models. The advantage of the SAR approach is that it distills the experimentally determined biological activities of several thousand chemicals and identifies structural features significantly associated with activity. Subsequently, when the potential of a yet untested chemical Že.g., I3C. is investigated, the expert system determines whether it contains any of the substructures significantly associated with a particular biological activity of interest w44,45x. The presence of such structural determinants is then translated into a probability and a level or spectrum of activity. 3. Results First we addressed the potential of I3C to act as a rodent carcinogen based upon a variety SAR models Žmouse, rat, rodent, trans-species. derived from cancer bioassays carried out under the aegis of the US National Toxicology Program w46x or listed in the Carcinogenic Potency Data Base w44,45x. We also determined the potential of I3C to induce hepatic preneoplastic glutathione-S-transferase positive foci w47x. In none of these was I3C predicted to have a carcinogenic potential. Based upon a previously derived relationship w48x, this suggests that the probability that I3C is a rodent carcinogen is less than 1%. This finding assumes even greater significance in light of the estimate that ; 50% of all chemicals
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tested will give a carcinogenic response in rodents if the test protocol includes the MTD w49x. Moreover, based upon the same battery of SAR models of rodent carcinogenicity, 33.5% of randomly selected chemicals are predicted to be rodent carcinogens. Because most recognized human carcinogens are also mutagenicrgenotoxic w50–53x, we examined the genotoxic potential of I3C. We found this chemical to be devoid of ‘‘structural alerts’’ for DNA-reactivity w54,55x, it is predicted to be neither a Salmonella mutagen w56,57x, nor an inducer of error-prone ŽSOS. DNA repair w58,59x, nor of unscheduled DNA synthesis w60x. Thus, I3C is unlikely to be either a mutagen or a genotoxicant. Inhibition of gap-junctional intercellular communication has been taken as an attribute of tumor promoters w61–65x. I3C also lacks that potential as well w66x. The availability of an SAR model of inhibitors of rodent carcinogenesis Žw67x, unpublished results. enabled us to test I3C for this potential as well. In fact I3C contains a biophore associated with this anticarcinogenic potential. Its presence might explain the reported anticarcinogenicity of I3C. According to the weight of evidence criteria used herein, based upon the SAR models considered, no carcinogenic risk associated with I3C was uncovered while a cancer chemopreventative potential was identified. While these findings in and of themselves are not definitive, they support previously reported findings suggestive of a beneficial effect. Thus, techniques developed by us to identify toxic potentials Ži.e., carcinogenicity and mutagenicity. were used to document the probable safety of I3C. 4. Conclusions The approach taken herein is still rudimentary and the tentative conclusions can only be used as adjuncts to other experimental and population-based data. Still, it has several philosophical implications. Heretofore, our discipline Žgenetic and molecular toxicology. has been concerned primarily with the identification of toxicants Že.g., mutagens, carcinogens.. However, as a result of the changed public perception, prevention, because of its cause-effectiveness, is being recognized as a major component of the health care system. That realignment has shifted the paradigm from the clinician’s emphasis on the
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individual patient to population-based analyses of the effectiveness of preventive interventions and health enhancement. The experimental approaches that are needed are certainly not new to many of us. In fact the application of biomarkers of exposure and of early disease manifestation have been part of our armamentarium for more than a decade. The new unfolding health care panorama gives us the unique opportunity to apply our expertise to aspects of disease prevention. Thus, instead of recognizing toxicants, we can apply our skills to identifying, with assurance, non-toxicants. This we could accomplish simply by shifting our emphasis from tests with high specificity to tests with high sensitivity w68x and from an emphasis on biomarkers of DNA damage to biomarkers of early disease manifestation Že.g., inactivation of suppressor genes. and how they are modulated by presumably non-toxic chemopreventative agents w69,70x. Moreover, because unlike, for example, cancer chemotherapy, which is limited in duration, cancer chemoprevention may require a lifetime of diet supplementation for persons at risk Že.g., individuals with specific breast cancer genes., intervention trials will need to be designed with lifetime epidemiological and biomarker follow-up studies. It is my opinion that acquiring andror re-engineering the tools for this undertaking must be done now. In the not too distant future the results of the ‘‘Environmental Genome’’ project w71x may enable us to identify individuals genetically at increased risk of developing diseases of environmental and occupational etiology. Heretofore, the potential for social harm of such identifications have inhibited their further development and application. However the availability of prophylactic regimens has changed the picture. Now, these subpopulations may be the likely target of intervention studies of the type advocated herein. I think we are equipped professionally as well as philosophically to meet this new challenge. Just as our honoree ŽDr. Frederick J. de Serres. had the vision to use the elegant Neurospora crassa system to identify and understand the induction of inherited changes and to encourage others to make similar leaps of faith in their research careers to prevent diseases caused by environmental mutagens and carcinogens. So too, current and new practitioners of our discipline should be encouraged to confront the challenge, and pursue the, as yet,
uncompleted task. In so doing they will have the satisfaction of being part of a noble tradition w2,3x dedicated to the improvement of the quality and duration of human life.
5. Epilogue The pioneering generation represented by our honoree, building upon the monumental work of earlier microbiologists, epidemiologists and physicians, has made enormous strides in our understanding and prevention of genetic and molecular processes that contribute to acute and chronic diseases. On the other hand, due to changes in lifestyles, migrations and global climate changes, we now witness the re-emergence of old scourges Že.g., tuberculosis, cholera. and the emergence of new ones Že.g., HIV, Hanta and Ebola viruses.. Yet the techniques, tools, knowledge and concepts legated to us by Dr. de Serres and his peers, enable us to face these new challenges. Perhaps the progress from alchemy to the ‘‘magic bullet’’, and from ‘‘microbe hunter’’ to ‘‘mutagen hunter’’ will ultimately lead us to the ‘‘genome defender’’. It is my sincerest wish that Dr. de Serres and his family will continue to witness for many more years the fruits of the profession that he was instrumental in founding and guiding to maturity.
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