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Mechanistic basis for the development of biomarkers
Toxicology Letters Toxicolog$ Letters 77 (1995) 81-83
Summary
report
Mechanistic basis for the development of biomarkers Canice Nolan European Com,wission. Directorate
General for Science, Research and Development,
Rue de la Loi 200, Brussels, Belgium
Accepted 13 January 1995
This session was comprised of 4 presentations entitled (1) Mech,anisms and biomarkers of genotoxicity. Molecul:ar dosimetry of chemical mutagens (presented by A.A. Van &eland), (2) Non-genotoxic mechanisms of carcinogenesis: development and use of in vitro cell transformation systems (presented by H. Yamasaki), (3) Mechanisms of neurotoxicity: applications to human biomonitoring (presented by L. Manzo) and (4) Early detection of immunotoxicity: from animal studies to human biomonitoring (presented by H. Van Loveren). All 4 presentations were based on research supported by the ENVIRONMENT research programme of the European Commission. A key point, raised by G. Becking in the previous session - that we need mechanistic data to reduce uncertainty in risk assessment for toxic chemicals -was addressed by the 4 speakers. The non-identity of correlation and causation is sometimes forgotten and sometimes disregarded in the selection of parameters for biomonitoring studies, Selection of an inappropriate or pseudobiomarkers can have consequences ranging from wasted time and resources to the arrival at false conclusions and even erroneous decisions in public health policy, e.g. the setting of too high or too low reference values for exposure to toxic chemicals, the mistaken priority setting or inade-
quate allocation of resources. Selection of biomarkers of exposure or effects of toxic chemicals should be based not only on practical criteria such as sample availability and analytical costs but also on a sound understanding of the mechanisms of action, the pharmacokinetics and the pharmacodynamics of the noxious agents being investigated. This is needed to assure conlidence in the results of the studies, particularly if decisions, relating to health or environmental quality, will be based on those results. Our ability to select and to use biomarkers for routine monitoring varies both with the chemical classes and the endpoints under investigation. Whereas highly specific information was presented by A.A. Van Zeeland for some chemical mutagens, and by H. Yamasaki for non-genotoxic carcinogens, we are, as was shown by L. Manzo and H. Van Loveren, obliged to use indirect means to infer neurotoxic and immunotoxic effects respectively. This latter, because of the inaccessibility of the target tissue - brain - for neurotoxic effects and because of our inadequate knowledge of the specific effects of chemicals on the immune system. The presentation of A.A. Van Zeeland concerned molecular dosimetry, i.e. assessing the dose, at the molecular level, of monofunctional alkylating agents. He focused on DNA adduct frequencies in hprt genes of cultured Chinese
0378-4274/95609.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0378-4274(95)103275-P
82
C. Nolan/Toxicology
Hamster Ovary cells and in rat skin fibroblasts. He showed that not all mutations are harmful, that some can be repaired and that in terms of biomonitoring, determination of adduct frequencies is neither a quantitative indicator of exposure nor of effects. Rather frequencies of DNA adducts can be indicative of the types of risks to be expected from exposure to different classes of compounds. These frequencies, or mutation spectra vary strongly with cell type and the nature and extent of mutations is greatly influenced not only by exposure but also by repair processes. Using the examples of 06-alkylguanine and O*-ethylthymine he showed that the former is a good indicator of DNA lesions in the absence of repair but that where cells had the capability to remove alkyl groups from the O6 position then the latter is the better indicator. It is thus only when the genotoxicity profile of a class of chemicals is known in the defined target cells that DNA adduct frequencies can be based as a quantitative biomarker (molecular dosimeter) for genotoxic effects. In his presentation on non-genotoxic carcinogens, H. Yamasaki stressed that mutagenesis was not equivalent to carcinogenesis and that genotoxic and non-genotoxic mechanisms are not mutually exclusive. They could, in many cases, act complementarily and even synergistically. If one assesses the probabilities of each stage in the carcinogenesis pathway occurring in a single cell, then, statistically, cancers should not occur. Furthermore, unlike genotoxin-mediated carcinogenesis, it is not yet possible to find evidence of the non-genotoxic carcinogens at the cancer endpoint even when it is known that they were involved in the carcinogenesis pathway. Indeed many carcinogens do not give positive results in classical carcinogenesis test systems. Dr. Yamasaki proposed that these non-genotoxic carcinogens act indirectly and probably on previously ‘primed’ cells, e.g. through the induction of cell proliferation or through cytosine methylation. He showed examples of cell systems where interaction of genotoxic (initiators) and non-genotoxic (promoters) agents occurred. He also proposed that altered gap junctional intercellular communication was involved in the post-initiation stages of car-
Letters 77 (1995) 81-83
cinogenesis and explained how progression of initiated cells to the clonal expansion stage could occur following their isolation. Finally, he presented evidence showing an effect of functional gap junctional genes on growth control in rodent cells and described current efforts to determine why human cells are resistant to chemical transformation. Unlike the presentations of A.A. Van &eland and H. Yamasaki, on the use of mechanistic studies for chemical screening, that of L. Manzo concerned biomonitoring of humans for neurotoxic effects of chemicals and for the measurement of recovery following disease. According to Dr. Manzo, we are as yet about 10 years away from routinely usable biomarkers for human biomonitoring of neurotoxicity due predominantly to the inaccessibility of neural tissues. Nonetheless, using mechanistic studies, Dr. Manzo and his colleagues have identified biochemical and molecular indicators measurable in peripheral tissues as surrogate biomarkers of effects in neural tissues. Their use allows the possibility of assessing complex interactions of chemicals with receptor systems, signal transduction pathways and other regulatory cell functions. Using the examples of lead and organophosphates he showed how their utility in providing an important advance over current biomarkers which are generally limited to the assessment of exposure. H. Van Loveren gave an overview of the immune system and, highlighting its complexity and adaptability, discussed some of the problems involved in the selection and use of biomarkers of exposure (where hardly anything is available, particularly for exposure to chemicals), of early effects (where many non-specific indicators are available but where many confounders exist), of susceptibility (where very little is known and where the roles of stress and socio-economic status need investigation), and of disease. Using the examples of dioxins, furams and PCB, he described some practical examples of the use of immune system biomarkers in routine and accidental exposure situations. He proposed a tiered strategy for the testing of immunotoxic effects of chemicals in human populations. The tier comprised (a) a complete differ-
C. Nolan/Toxicology
ential blood count, (b) measurement of nonspecific immunity, e.g. NK cells, (c) phenotypic analysis of lymphocytes by flow cytometry (surface analysis of ClD3, CD4, CD8 and DCD20), (d) measurement of cellular immunity, (e) antibodymediated immunilty and (f) autoantibodies/inflam-
Letters 77 (1995) 81-83
83
mation. He concluded by stressing the need for more research, not only basic research on the functioning of the immune system but also specific research on its responses to toxic insult by different classes of chemicals.
Summary
report
Mechanistic basis for the development of biomarkers Canice Nolan European Com,wission. Directorate
General for Science, Research and Development,
Rue de la Loi 200, Brussels, Belgium
Accepted 13 January 1995
This session was comprised of 4 presentations entitled (1) Mech,anisms and biomarkers of genotoxicity. Molecul:ar dosimetry of chemical mutagens (presented by A.A. Van &eland), (2) Non-genotoxic mechanisms of carcinogenesis: development and use of in vitro cell transformation systems (presented by H. Yamasaki), (3) Mechanisms of neurotoxicity: applications to human biomonitoring (presented by L. Manzo) and (4) Early detection of immunotoxicity: from animal studies to human biomonitoring (presented by H. Van Loveren). All 4 presentations were based on research supported by the ENVIRONMENT research programme of the European Commission. A key point, raised by G. Becking in the previous session - that we need mechanistic data to reduce uncertainty in risk assessment for toxic chemicals -was addressed by the 4 speakers. The non-identity of correlation and causation is sometimes forgotten and sometimes disregarded in the selection of parameters for biomonitoring studies, Selection of an inappropriate or pseudobiomarkers can have consequences ranging from wasted time and resources to the arrival at false conclusions and even erroneous decisions in public health policy, e.g. the setting of too high or too low reference values for exposure to toxic chemicals, the mistaken priority setting or inade-
quate allocation of resources. Selection of biomarkers of exposure or effects of toxic chemicals should be based not only on practical criteria such as sample availability and analytical costs but also on a sound understanding of the mechanisms of action, the pharmacokinetics and the pharmacodynamics of the noxious agents being investigated. This is needed to assure conlidence in the results of the studies, particularly if decisions, relating to health or environmental quality, will be based on those results. Our ability to select and to use biomarkers for routine monitoring varies both with the chemical classes and the endpoints under investigation. Whereas highly specific information was presented by A.A. Van Zeeland for some chemical mutagens, and by H. Yamasaki for non-genotoxic carcinogens, we are, as was shown by L. Manzo and H. Van Loveren, obliged to use indirect means to infer neurotoxic and immunotoxic effects respectively. This latter, because of the inaccessibility of the target tissue - brain - for neurotoxic effects and because of our inadequate knowledge of the specific effects of chemicals on the immune system. The presentation of A.A. Van Zeeland concerned molecular dosimetry, i.e. assessing the dose, at the molecular level, of monofunctional alkylating agents. He focused on DNA adduct frequencies in hprt genes of cultured Chinese
0378-4274/95609.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0378-4274(95)103275-P
82
C. Nolan/Toxicology
Hamster Ovary cells and in rat skin fibroblasts. He showed that not all mutations are harmful, that some can be repaired and that in terms of biomonitoring, determination of adduct frequencies is neither a quantitative indicator of exposure nor of effects. Rather frequencies of DNA adducts can be indicative of the types of risks to be expected from exposure to different classes of compounds. These frequencies, or mutation spectra vary strongly with cell type and the nature and extent of mutations is greatly influenced not only by exposure but also by repair processes. Using the examples of 06-alkylguanine and O*-ethylthymine he showed that the former is a good indicator of DNA lesions in the absence of repair but that where cells had the capability to remove alkyl groups from the O6 position then the latter is the better indicator. It is thus only when the genotoxicity profile of a class of chemicals is known in the defined target cells that DNA adduct frequencies can be based as a quantitative biomarker (molecular dosimeter) for genotoxic effects. In his presentation on non-genotoxic carcinogens, H. Yamasaki stressed that mutagenesis was not equivalent to carcinogenesis and that genotoxic and non-genotoxic mechanisms are not mutually exclusive. They could, in many cases, act complementarily and even synergistically. If one assesses the probabilities of each stage in the carcinogenesis pathway occurring in a single cell, then, statistically, cancers should not occur. Furthermore, unlike genotoxin-mediated carcinogenesis, it is not yet possible to find evidence of the non-genotoxic carcinogens at the cancer endpoint even when it is known that they were involved in the carcinogenesis pathway. Indeed many carcinogens do not give positive results in classical carcinogenesis test systems. Dr. Yamasaki proposed that these non-genotoxic carcinogens act indirectly and probably on previously ‘primed’ cells, e.g. through the induction of cell proliferation or through cytosine methylation. He showed examples of cell systems where interaction of genotoxic (initiators) and non-genotoxic (promoters) agents occurred. He also proposed that altered gap junctional intercellular communication was involved in the post-initiation stages of car-
Letters 77 (1995) 81-83
cinogenesis and explained how progression of initiated cells to the clonal expansion stage could occur following their isolation. Finally, he presented evidence showing an effect of functional gap junctional genes on growth control in rodent cells and described current efforts to determine why human cells are resistant to chemical transformation. Unlike the presentations of A.A. Van &eland and H. Yamasaki, on the use of mechanistic studies for chemical screening, that of L. Manzo concerned biomonitoring of humans for neurotoxic effects of chemicals and for the measurement of recovery following disease. According to Dr. Manzo, we are as yet about 10 years away from routinely usable biomarkers for human biomonitoring of neurotoxicity due predominantly to the inaccessibility of neural tissues. Nonetheless, using mechanistic studies, Dr. Manzo and his colleagues have identified biochemical and molecular indicators measurable in peripheral tissues as surrogate biomarkers of effects in neural tissues. Their use allows the possibility of assessing complex interactions of chemicals with receptor systems, signal transduction pathways and other regulatory cell functions. Using the examples of lead and organophosphates he showed how their utility in providing an important advance over current biomarkers which are generally limited to the assessment of exposure. H. Van Loveren gave an overview of the immune system and, highlighting its complexity and adaptability, discussed some of the problems involved in the selection and use of biomarkers of exposure (where hardly anything is available, particularly for exposure to chemicals), of early effects (where many non-specific indicators are available but where many confounders exist), of susceptibility (where very little is known and where the roles of stress and socio-economic status need investigation), and of disease. Using the examples of dioxins, furams and PCB, he described some practical examples of the use of immune system biomarkers in routine and accidental exposure situations. He proposed a tiered strategy for the testing of immunotoxic effects of chemicals in human populations. The tier comprised (a) a complete differ-
C. Nolan/Toxicology
ential blood count, (b) measurement of nonspecific immunity, e.g. NK cells, (c) phenotypic analysis of lymphocytes by flow cytometry (surface analysis of ClD3, CD4, CD8 and DCD20), (d) measurement of cellular immunity, (e) antibodymediated immunilty and (f) autoantibodies/inflam-
Letters 77 (1995) 81-83
83
mation. He concluded by stressing the need for more research, not only basic research on the functioning of the immune system but also specific research on its responses to toxic insult by different classes of chemicals.