- Email: [email protected]
Role of inflammatory cytokines in hepatic and pulmonary toxicity
Symposium 11. Cytokines no-advesre-effect levels, on which authorities may base their regulatory actions, taking uncertainty factors into consideration. In addition to immunotoxicity assessment in animals, possibilities exist for such a type of testing in humans. Depending on the agent, in vivo or in vitro effects of exposure can be studied. However, direct information on endpoints such as resistance to infections or sensitization cannot easily be obtained in volunteer studies or in vitro with human cells. An additional approach to risk assessment is the parallellogram approach, that comprises 4 cornerstones : (I ) the health effect assessed as an endpoint in animals, (2) the quantitative prediction of this endpoint in humans, and the assays in either animals (3) or humans (4) that are used for species comparisons. Examples of how non-treshold mathematical models were used to evaluate dose-response relationships observed in studies of chemicals inducing allergey or direct toxicity to the immune system will be discussed. In addition, it will be discussed how such relationships can be extrapolated to the human situation.
I81 O/L41
WHATARE THE PERSPECTIVES FORREGULATION IN IMMUNOTOXICOLOGY?
K.L. Hastings. Divison of Special Pathogens and Immunologic Drug
Products, Center for Drug Evaluationand Research, US Foodand Drug Administration, Rockville, MD, USA Evaluating the immunotoxic potential of investigational new drugs is a standard component of nonclinical safety assessment, Effects evaluated include the potential for drugs to induce hypersensitivity and/or autoimune reactions or to produce unintended immunosuppression. The Center for Drug Evaluation and Research (COER) is considering approaches for evaluating potential imrnunotoxicity, In particular, two methods are being examined for potential recommendation where indicated: immune cell phenotype determination and the murine local lymph node assay. Issues concerning immunotoxicology testing will be discussed.
811. Cytokines
I811/L1 I
CHEMICAL-INDUCED ACTIVATION OF NUCLEAR TRANSCRIPTION FACTORS AND THEIRREGULATION OF CYTOKINE SECRETION
M.1. Luster. National Institutefor OccupationalSafety and Health,
Morgantown; WestVirginia, USA Non-immune cells from a variety of organs (including the liver, lung and skin) when targeted by toxic agents secrete pro-inflammatory cytokines and chemokines which participate in pathological as well as repair processes. In particular, tumor necrosis factor alpha (TNFa) as well as members of the neutrophil chemoattractant family of chemokines (CXC), including interleukin-S in humans and macrophage inflammatory protein-2 in rodents, play major roles in these toxic responses. This presentation will describe several organ model systems for toxicity including asbestos-induced lung disease as well as carbon tetrachloride and heavy metal-mediated hepatotoxicity and demonstrate cytokine participation. In addition to describing the nature of these responses, the molecular events responsible will be detailed. A common pathway responsible for their induction is chemical-mediated cellular oxidative stress, which causes the activation of the nuclear transcription factors NF-kB, NF-IL-6 and AP-I which, in turn, regulate cytokine gene transcription. Unraveling the complex mechanisms associated with these events will provide novel opportunities for therapeutic intervention.
I811/L21
13
CYTOKINES AND IRRITANT CONTACT DERMATITIS
E. Corsini. InstituteofPharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy Skin irritation is a complex phenomenon that involves resident epidermal cells, fibroblasts of dermis, and endothelial cells as well as invading leukocytes interacting each other under the control of a network of cytokines and lipid mediators. In the last two decades it has become clear that keratinocytes play an important role in the initiation and perpetuation of skin inflammatory and immunological reactions through the release of cytokines and responses to cytokines, While resting keratinocytes produce some cytokines constitutively, a variety of environmental stimuli, such as tumor promoters, ultraviolet light and chemical agents, can induce epidermal keratinocytes to release inflammatory cytokines (IL- I, TNF-a), chemotactic cytokines (IL-S, IP-lO), growth promoting cytokines (IL-6, IL-7, IL-IS, OM-CSF, TOF-a) and cytokines regulating humoral versus cellular immunity (lL-lO, IL-12, IL-IS). In vivo and in vitro data will be presented, concerning both the role of cytokines in xenobiotics induced skin irritation and the early molecular events that follow the treatment with irritant compounds.
I811/L31
GLIAL FUNCTION AND MODULATION OF CNS IMMUNE RESPONSES
M. Aschner, DepartmentofPhysiology and Pharmacology, Wake Forest University School ofMedicine, Winston-Salem, NC, USA Because it is shielded by the skull bones, the blood-brain barrier (BBB), the cerebrospinal fluid and the meningeal membranes , the central nervous system (CNS) was regarded for decades as an "immunologically privileged" organ. However, with recent evidence that in response to invasion by microorganisms, the CNS can mount its own defense by resident cells, such as the microglia and astrocytes, this long-standing view that the CNS is isolated from the effects of the immune system has been rethought and revised. It has been shown that both resident astrocytes and microglia can secrete numerous cytokines, and, therefore, modulate lymphoid-mononuclear cell function, establishing an integrative communication pathway between immune cells of the CNS and those of the peripheral immune system. While clearly implicated in the initiation, maintenance, and suppression of immune responses, cytokines produced by astrocytes and microglia, as well as the responses of these cells to cytokines produced elsewhere can propagate eNS damage, suggesting their potential involvement in neurodegenerative disorders. The objective of this presentation is to address the role played by microglial and astrocytic cells in the initiation, modulation, and suppression of immune responses, as well as their potential involvement in propagating CNS damage. Topics of discussion will include (1) the function of astrocytes vs. microglia as the antigen presenting cells (APes), (2) the role afforded by astrocyte-derived cytokines, and astrocytic responses to cytokines secreted elsewhere, in mediating and sustaining immune responses, and (3) experimental evidence that astroglial impairment by pathogens may contribute to the etiology of neurodegenerative disorders.
I811/L41 AND ROLE OF INFLAMMATORY CYTOKINES IN HEPATIC PULMONARY TOXICITY D.L. Laskin *, D.E. Heck, J.D. Laskin. Environmental and Occupational Health Scienceslnst. Rutgers Univ./UMDNJ-RW Johnson Med. Sch., Piscataway; NJ, USA Exposure of humans and experimental animals to inhaled irritants such as ozone induces an acute inflammatory response and lung injury. We hypothesize that macrophage-derived inflammatory cytokines and cytotoxic mediators contribute to the pathogenic process.
14
Symposium i2 . Humanand Environmental Risk Assessmentof Existing Chemicals
Treatment of rats with ozone (2 ppm, 3 h) results in damage to the alveolar epithelium and increased protein in lung lavage fluid. This is associated with an increase in the number of macrophages in the lung. We found that these cells are activated to release the proinflammatory cytokines tumor necrosis factor-a (TNFa) and interleukin- l which have been implicated in tissue injury. Following ozone inhalation , alveolar macrophages as well as Type II cells also produce increased amounts of the cytotoxic mediator, nitric oxide. This response is time dependent and correlated with expression of inducible nitric oxide synthase (NOS II) protein and mRNA. Inhibition of macrophages with gadolinium chloride abrogates ozone induced inflammation , mediator production and tissue injury. These data, together with the findings that the toxicity of ozone is reduced in knockout mice lacking TNFa or NOSII, demonstrate that macrophages and mediators they release contribute to irritant induced lung injury. Ozone inhalation also caused alterations in the liver, including increased nitric oxide production and protein synthesis suggesting that ozone induces an acute phase response. We speculate that this is mediated by cytokines such as TNFa produced by alveolar macrophages. In this regard we noted increased expression of TNFa in both lung and liver tissue . These data suggest that cytokines produced locally by macrophages following toxicant exposure may exert pathophysiologic effects outside the target organ . (Nllf ES04738, GM34310 and Burroughs Wellcome Fund) .
IS11/LSI
CARBONMONOXIDE AND NITRICOXIDE CONTROL INTERLEUKIN-1 PRODUcnON AND RELEASEIN RAT BRAIN
P. Navarra *, P. Preziosi. institute of Pharmacology, Catholic
University MedicalSchool, Rome, italy Inflammatory cytokines, interleukin-I (IL- I) in particular, are associated to various pathologic events in the central nervous system (CNS), ranging from the central signs of acute phase response (fever, anorexia., sleepiness, neuroendocrine changes) to neurodegenerative disorders such as Alzheimer disease. Apart from systemic IL-I, which is able to signal to the CNS through various mechanisms , IL-l is also synthesized within the brain. Here, most of the cytokine is produced in glial cells upon induction by immune-inflammatory stimuli. Besides, a less defined preformed pool seems to exist, which is probably neuronal in nature. We have characterized the release of immunoreactive (ir)-IL-l from rat hypothalarnic explants, At least part of IL-I released appears to come from the above mentioned pre-formed pool, since release can be modified after as early as 20 minutes of treatment with appropriate stimuli. Indeed, release is modulated by high KCI concentrations, neurotransmitters and neuropeptides . Moreover, a new class of neural messengers, the gaseous neurotransminers nitric oxide (NO) and carbon monoxide (CO) were able to modulate ir-IL-I secretion. In fact, increases in the generation of NO and CO were associated with increased and decreased IL-l secretion from the hypothalamus, respectively.
to the Directive (791831IEEC) provided a notification system for substances being placed on the EU market for the first time, thus making a distinction between the so-called ' new' substances and those 'existing ' substances, already on the EU market in 1981. The legislation was made under Article lOOA of the Treaty of Rome, aimed at maintaining the functioning of the internal market Risk assessment first entered community legislation in 1992 with the seventh amendment to the Directive (921321EEC), requiring risk assessment be carried out for new chemicals . In order to also provide the legal framework for a systemic evaluation of the existing chemical substances, Council Regulation (EEC) 793193 was adopted. The Regulation requires that the evaluation be conducted in four steps; data collection, priority setting, risk assessment, and, where necessary, risk reduction. The principles for carrying out a risk assessment were laid down in Commission Directive 93/671EEC for new substances and Council Regulation 1488/94 for existing substances. Further detailed guidance on how to perform the risk assessments are given in a published Technical Guidance Document. The protection of three human populations are prescribed , these being workers, consumers, and man exposed indirectly via the environment, and for each, coverage of the following endpoint s is stipulated; acute toxicity, irritation/corrosivity, sensitisation, general systemic effects, mutagenicity, carcinogenicity and reproductive effects. Decisions on the level of risk for each population are made, where possible, on the basis of the margins of safety, these being a comparison of the estimated exposure levels with those levels producing adverse effects. Where quantitative data are unavailable, an assessment of the likelihood of the risk is required .
IS121L2!
SCIENCE-BASED CRITERIA, RULESAND METHODS OF RISK ASSESSMENT
CJ. van Leeuwen. RIVM, The Netherlands Because of international trade and long-range transport , risk management of chemicals is an international challenge. To protect human health and the environment risk assessment procedures have been developed, based on information on use patterns and emissions as well as standard physicochemical and (eco)toxicological information. The generic risk assessment methodologies developed in the European Union for new and existing chemicals are a prerequisite for successful risk management. The development of the so-called European Union System for the Evaluation of Substances (EUSES) comprises many science-policy issues. The development of risk assessment guidance and the EUSES model required time as well as a well-organised and transparent discussion between academia, industries and regulatory authorities. Once available, such a tool can be improved and adapted on the bases of scientific and regulatory developments . The framework of EUSES and the experiences over the past few years will be presented. From the risk assessments carried out so far it can be concluded that it is not the (eco)toxicology that poses the greatest uncertainties in risk assessment but the exposure of chemicals, i.e, the use patterns and releases.
512. Human and Environmental Risk Assessment of Existing Chemicals
IS121LS! CONSUMER EXPOSURE ESTIMATION IN RISK
IS12/L1 I LEGAL BACKGROUND AND GENERALPRINCIPLES
A. Wilson. UK
OF RISK ASSESSMENT
B.G. Hansen. European Commission, European Chemicals Bureau,
JRC 1-21020, Ispra (VA), Italy The first pieces of chemical control legislation were aimed at hazard identification. Council Directive 67/5481EEC was introduced to provide uniform EU-wide rules for the classification, packaging and labelling of dangerou s chemicals . The sixth amendment
ASSESSMENT
Abstract not received at time of publication .
I81 O/L41
WHATARE THE PERSPECTIVES FORREGULATION IN IMMUNOTOXICOLOGY?
K.L. Hastings. Divison of Special Pathogens and Immunologic Drug
Products, Center for Drug Evaluationand Research, US Foodand Drug Administration, Rockville, MD, USA Evaluating the immunotoxic potential of investigational new drugs is a standard component of nonclinical safety assessment, Effects evaluated include the potential for drugs to induce hypersensitivity and/or autoimune reactions or to produce unintended immunosuppression. The Center for Drug Evaluation and Research (COER) is considering approaches for evaluating potential imrnunotoxicity, In particular, two methods are being examined for potential recommendation where indicated: immune cell phenotype determination and the murine local lymph node assay. Issues concerning immunotoxicology testing will be discussed.
811. Cytokines
I811/L1 I
CHEMICAL-INDUCED ACTIVATION OF NUCLEAR TRANSCRIPTION FACTORS AND THEIRREGULATION OF CYTOKINE SECRETION
M.1. Luster. National Institutefor OccupationalSafety and Health,
Morgantown; WestVirginia, USA Non-immune cells from a variety of organs (including the liver, lung and skin) when targeted by toxic agents secrete pro-inflammatory cytokines and chemokines which participate in pathological as well as repair processes. In particular, tumor necrosis factor alpha (TNFa) as well as members of the neutrophil chemoattractant family of chemokines (CXC), including interleukin-S in humans and macrophage inflammatory protein-2 in rodents, play major roles in these toxic responses. This presentation will describe several organ model systems for toxicity including asbestos-induced lung disease as well as carbon tetrachloride and heavy metal-mediated hepatotoxicity and demonstrate cytokine participation. In addition to describing the nature of these responses, the molecular events responsible will be detailed. A common pathway responsible for their induction is chemical-mediated cellular oxidative stress, which causes the activation of the nuclear transcription factors NF-kB, NF-IL-6 and AP-I which, in turn, regulate cytokine gene transcription. Unraveling the complex mechanisms associated with these events will provide novel opportunities for therapeutic intervention.
I811/L21
13
CYTOKINES AND IRRITANT CONTACT DERMATITIS
E. Corsini. InstituteofPharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy Skin irritation is a complex phenomenon that involves resident epidermal cells, fibroblasts of dermis, and endothelial cells as well as invading leukocytes interacting each other under the control of a network of cytokines and lipid mediators. In the last two decades it has become clear that keratinocytes play an important role in the initiation and perpetuation of skin inflammatory and immunological reactions through the release of cytokines and responses to cytokines, While resting keratinocytes produce some cytokines constitutively, a variety of environmental stimuli, such as tumor promoters, ultraviolet light and chemical agents, can induce epidermal keratinocytes to release inflammatory cytokines (IL- I, TNF-a), chemotactic cytokines (IL-S, IP-lO), growth promoting cytokines (IL-6, IL-7, IL-IS, OM-CSF, TOF-a) and cytokines regulating humoral versus cellular immunity (lL-lO, IL-12, IL-IS). In vivo and in vitro data will be presented, concerning both the role of cytokines in xenobiotics induced skin irritation and the early molecular events that follow the treatment with irritant compounds.
I811/L31
GLIAL FUNCTION AND MODULATION OF CNS IMMUNE RESPONSES
M. Aschner, DepartmentofPhysiology and Pharmacology, Wake Forest University School ofMedicine, Winston-Salem, NC, USA Because it is shielded by the skull bones, the blood-brain barrier (BBB), the cerebrospinal fluid and the meningeal membranes , the central nervous system (CNS) was regarded for decades as an "immunologically privileged" organ. However, with recent evidence that in response to invasion by microorganisms, the CNS can mount its own defense by resident cells, such as the microglia and astrocytes, this long-standing view that the CNS is isolated from the effects of the immune system has been rethought and revised. It has been shown that both resident astrocytes and microglia can secrete numerous cytokines, and, therefore, modulate lymphoid-mononuclear cell function, establishing an integrative communication pathway between immune cells of the CNS and those of the peripheral immune system. While clearly implicated in the initiation, maintenance, and suppression of immune responses, cytokines produced by astrocytes and microglia, as well as the responses of these cells to cytokines produced elsewhere can propagate eNS damage, suggesting their potential involvement in neurodegenerative disorders. The objective of this presentation is to address the role played by microglial and astrocytic cells in the initiation, modulation, and suppression of immune responses, as well as their potential involvement in propagating CNS damage. Topics of discussion will include (1) the function of astrocytes vs. microglia as the antigen presenting cells (APes), (2) the role afforded by astrocyte-derived cytokines, and astrocytic responses to cytokines secreted elsewhere, in mediating and sustaining immune responses, and (3) experimental evidence that astroglial impairment by pathogens may contribute to the etiology of neurodegenerative disorders.
I811/L41 AND ROLE OF INFLAMMATORY CYTOKINES IN HEPATIC PULMONARY TOXICITY D.L. Laskin *, D.E. Heck, J.D. Laskin. Environmental and Occupational Health Scienceslnst. Rutgers Univ./UMDNJ-RW Johnson Med. Sch., Piscataway; NJ, USA Exposure of humans and experimental animals to inhaled irritants such as ozone induces an acute inflammatory response and lung injury. We hypothesize that macrophage-derived inflammatory cytokines and cytotoxic mediators contribute to the pathogenic process.
14
Symposium i2 . Humanand Environmental Risk Assessmentof Existing Chemicals
Treatment of rats with ozone (2 ppm, 3 h) results in damage to the alveolar epithelium and increased protein in lung lavage fluid. This is associated with an increase in the number of macrophages in the lung. We found that these cells are activated to release the proinflammatory cytokines tumor necrosis factor-a (TNFa) and interleukin- l which have been implicated in tissue injury. Following ozone inhalation , alveolar macrophages as well as Type II cells also produce increased amounts of the cytotoxic mediator, nitric oxide. This response is time dependent and correlated with expression of inducible nitric oxide synthase (NOS II) protein and mRNA. Inhibition of macrophages with gadolinium chloride abrogates ozone induced inflammation , mediator production and tissue injury. These data, together with the findings that the toxicity of ozone is reduced in knockout mice lacking TNFa or NOSII, demonstrate that macrophages and mediators they release contribute to irritant induced lung injury. Ozone inhalation also caused alterations in the liver, including increased nitric oxide production and protein synthesis suggesting that ozone induces an acute phase response. We speculate that this is mediated by cytokines such as TNFa produced by alveolar macrophages. In this regard we noted increased expression of TNFa in both lung and liver tissue . These data suggest that cytokines produced locally by macrophages following toxicant exposure may exert pathophysiologic effects outside the target organ . (Nllf ES04738, GM34310 and Burroughs Wellcome Fund) .
IS11/LSI
CARBONMONOXIDE AND NITRICOXIDE CONTROL INTERLEUKIN-1 PRODUcnON AND RELEASEIN RAT BRAIN
P. Navarra *, P. Preziosi. institute of Pharmacology, Catholic
University MedicalSchool, Rome, italy Inflammatory cytokines, interleukin-I (IL- I) in particular, are associated to various pathologic events in the central nervous system (CNS), ranging from the central signs of acute phase response (fever, anorexia., sleepiness, neuroendocrine changes) to neurodegenerative disorders such as Alzheimer disease. Apart from systemic IL-I, which is able to signal to the CNS through various mechanisms , IL-l is also synthesized within the brain. Here, most of the cytokine is produced in glial cells upon induction by immune-inflammatory stimuli. Besides, a less defined preformed pool seems to exist, which is probably neuronal in nature. We have characterized the release of immunoreactive (ir)-IL-l from rat hypothalarnic explants, At least part of IL-I released appears to come from the above mentioned pre-formed pool, since release can be modified after as early as 20 minutes of treatment with appropriate stimuli. Indeed, release is modulated by high KCI concentrations, neurotransmitters and neuropeptides . Moreover, a new class of neural messengers, the gaseous neurotransminers nitric oxide (NO) and carbon monoxide (CO) were able to modulate ir-IL-I secretion. In fact, increases in the generation of NO and CO were associated with increased and decreased IL-l secretion from the hypothalamus, respectively.
to the Directive (791831IEEC) provided a notification system for substances being placed on the EU market for the first time, thus making a distinction between the so-called ' new' substances and those 'existing ' substances, already on the EU market in 1981. The legislation was made under Article lOOA of the Treaty of Rome, aimed at maintaining the functioning of the internal market Risk assessment first entered community legislation in 1992 with the seventh amendment to the Directive (921321EEC), requiring risk assessment be carried out for new chemicals . In order to also provide the legal framework for a systemic evaluation of the existing chemical substances, Council Regulation (EEC) 793193 was adopted. The Regulation requires that the evaluation be conducted in four steps; data collection, priority setting, risk assessment, and, where necessary, risk reduction. The principles for carrying out a risk assessment were laid down in Commission Directive 93/671EEC for new substances and Council Regulation 1488/94 for existing substances. Further detailed guidance on how to perform the risk assessments are given in a published Technical Guidance Document. The protection of three human populations are prescribed , these being workers, consumers, and man exposed indirectly via the environment, and for each, coverage of the following endpoint s is stipulated; acute toxicity, irritation/corrosivity, sensitisation, general systemic effects, mutagenicity, carcinogenicity and reproductive effects. Decisions on the level of risk for each population are made, where possible, on the basis of the margins of safety, these being a comparison of the estimated exposure levels with those levels producing adverse effects. Where quantitative data are unavailable, an assessment of the likelihood of the risk is required .
IS121L2!
SCIENCE-BASED CRITERIA, RULESAND METHODS OF RISK ASSESSMENT
CJ. van Leeuwen. RIVM, The Netherlands Because of international trade and long-range transport , risk management of chemicals is an international challenge. To protect human health and the environment risk assessment procedures have been developed, based on information on use patterns and emissions as well as standard physicochemical and (eco)toxicological information. The generic risk assessment methodologies developed in the European Union for new and existing chemicals are a prerequisite for successful risk management. The development of the so-called European Union System for the Evaluation of Substances (EUSES) comprises many science-policy issues. The development of risk assessment guidance and the EUSES model required time as well as a well-organised and transparent discussion between academia, industries and regulatory authorities. Once available, such a tool can be improved and adapted on the bases of scientific and regulatory developments . The framework of EUSES and the experiences over the past few years will be presented. From the risk assessments carried out so far it can be concluded that it is not the (eco)toxicology that poses the greatest uncertainties in risk assessment but the exposure of chemicals, i.e, the use patterns and releases.
512. Human and Environmental Risk Assessment of Existing Chemicals
IS121LS! CONSUMER EXPOSURE ESTIMATION IN RISK
IS12/L1 I LEGAL BACKGROUND AND GENERALPRINCIPLES
A. Wilson. UK
OF RISK ASSESSMENT
B.G. Hansen. European Commission, European Chemicals Bureau,
JRC 1-21020, Ispra (VA), Italy The first pieces of chemical control legislation were aimed at hazard identification. Council Directive 67/5481EEC was introduced to provide uniform EU-wide rules for the classification, packaging and labelling of dangerou s chemicals . The sixth amendment
ASSESSMENT
Abstract not received at time of publication .