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Dose-response in the guinea pig maximization test
Symposium 5. Progress in Antidotic Therapy The application of this strategy is illustrated with the widely used nonsteroidal antiinflammatory 2-arylpropionic acids. This is a homogeneous group of drugs; they are safe and effective, but several members of the group have been reported to produce phototoxic and/or photoallergic side-effects. The incidence of these undesired processes is rationalized in the light of the above mechanistic studies.
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DOSE-RESPONSE IN THE GUINEA PIG MAXIMIZATION TEST
Anders Boman 1, S~aren Dyring Jacobsen 2, Signe Klastrup 2, Ove Svendsen * 2, jan E. Wahlberg 1. i Dept. of Occupational
Dermatology, Swedish Institute of Work and Health, S- 171 84 Solna; 2 Dept. of Occupational Dermatology, Scantox, DK-4623 Lille Skensved The standard Guinea Pig Maximization test is among the most sensitive tests for detecting the potential sensitizing capacity of chemicals. Potency evaluation of chemical contact allergens is a quantitative expression of the ability to induce contact allergy based on determination of the relationship between dose and frequency of sensitization in the test population. The guinea pig allergy tests may be developed to give a potency evaluation of the chemicals tested, requiring dose-response determinations. Instead of using one dose group of 20 animals and 10 vehicle treated control animals, these animals could be divided into one control group and five test groups and each group treated with different induction concentrations. Formaldehyde, chloroacetamide, benzocaine, methyl methacrylate and potassium dichromate were included in such a study. For each chemical, the five groups were treated intradermally with concentrations reduced per group with increments of a factor three from the highest concentration that could be applied intradermally. Two of the five groups were treated topically with the highest nonirritating concentration and the three other groups with a 100 times lower concentration. All groups were challenged and rechallenged with the highest non-irritating concentration. A significant dose related sensitization rate was seen after induction with formaldehyde, methyl methacrylate and potassium dichromate. The results with chloroacetamide suggest a strong sensitizing potential. Although no dose-response relationship was obtained with benzocaine, the results suggest that benzocaine was a weak sensitizer. Future studies should attempt to evaluate different dose designs for the intradermal and the topical treatment, and to evaluate if larger increments between concentrations during induction would improve the method. Acknowledgement: Contract 711/18-0002, LST/12 Nordic Council of Ministers
Keywords: guinea pig maximization test; allergens; dose-response
EARLY MOLECULAR EVENTS INDUCED BY TRIBUTYLTIN IN MURINE KERATINOCYTES Emanuela Corsini *, Marina Marinovich, Corrado L. Galli, Barbara Viviani. University of Milan, Institute of Pharmacological
Sciences, Lab. of Toxicology, V~aBalzaretti 9, 20133 Milano The biocidal agent tributyltin (TBT) is used mainly in wood preservation and marine antifouling paints. Occupational exposure of workers to TBT can result in skin and eye irritation, and severe dermatitis has been reported after direct contact with the skin. We have previously demonstrated that TBT induced both intracellular production of IL-lot and its release into culture medium in a murine keratinocyte cell line (HELJ0). We were also able to demonstrate an in vivo role of I L - l a in TBT-induced skin irritation (Toxicol & Appl Pharmacol, in press). Here, we report that TBT induces a direct and dose-related (0.1-2.5 /zM) activation of NF-KB, which peaks at 2 hours and is blocked by PDTC, an antioxidant potent NF-rB
7
inhibitor; by rotenone, an inhibitor of the electron entry from complex I to ubiquinone, and by BAPTA, an intracellular Ca 2+ chelator. NF-~cB activation is preceded in time by an increase of cellular oxidative activity as measured by a fluorimetric assay. This event can be almost completely abrogated by BAPTA, which suggests that intracellular Ca 2+ mobilization could be one of the first event following TBT treatment, leading to an increased cellular oxidative activity. Furthermore, the partial modulation observed after rotenone pretreatment indicates mitochondria as the main intracellular source of reactive oxygen species. To further demonstrate the important role of mitochondria, prolonged treatment with ethidium bromide, an inhibitor of mitochondrial DNA and RNA synthesis, has been used to deplete cells of functional mitochondria. After 5 days of treatment a 50% reduction in MTT conversion is obtained, under this condition a 53% reduction in cellular oxidative activity is observed after 15 minutes of treatment with TBT. The impairment of mitoehondrial metabolism also results in inhibition of NF-rB activation by TBT. These findings indicate that mitochondria are the source of second messanger molecules essential for TBT-induced NF-rB activation and IL-lot production.
Keywords: organotins; keratinocyte; reactive oxygen species; transcription factor; interleukin-lot
S5. Progress in Antidotic Therapy ~'~']
THE EVOLUTION OF ANTIDOTAL THERAPY IN THE LAST DECADES
Jenny Pronczuk de Garbino. World Health Organization,
International Programme on Chemical Safety, Poisoning Prevention & Treatment Unit, Geneva, Switzerland The evolution of antidotal therapy in the last several decades has been marked by: (a) growing international concern on evaluation of its real efficacy, (b) development of a few new antidotes, and (c) innovative approaches in antidote administration. A meeting of the World Federation of Associations of Poisons Centres and Clinical Toxicology and a number of poisons centres, hosted by the IPCS (Geneva, October 1985), recognized the need to define antidotes and evaluate their efficacy. As a ~sult, during the last decade antidotes have become a subject of international evaluation (the results of which are to be presented in another communication). In recent decades, a number of antidotes have been developed and proven to be effective for specific poisoning: Naloxone, N-acetylcisteine, Fab fragments, new chelators, flumazenil, Fab fragment antivenom and 4-methyl pyrazole. Although the clinical benefits in the use of these compounds have been shown, some controversial issues still exist (e.g. doses, routes of administration, opportunity for use). The approach to the use of a number of antidotes has changed in recent years. Some examples are presented, namely in the antidotal management of poisoning by organophosphorous pesticides, cyanide, carbon monoxide, and also of the local effects produced by exposure to bites, stings and to some corrosive agents. Unfortunately no major developments have taken place in the last years concerning (a) the development of antidotes for some extremely hazardous substances, (b) expanding the availability of antidotes, especially to areas of need, or (c) dissemination of up-to-date information concerning the real role of antidotes and their efficacy in the management of poisoned patients.
Keywords: antidote; treatment; poisoning
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DOSE-RESPONSE IN THE GUINEA PIG MAXIMIZATION TEST
Anders Boman 1, S~aren Dyring Jacobsen 2, Signe Klastrup 2, Ove Svendsen * 2, jan E. Wahlberg 1. i Dept. of Occupational
Dermatology, Swedish Institute of Work and Health, S- 171 84 Solna; 2 Dept. of Occupational Dermatology, Scantox, DK-4623 Lille Skensved The standard Guinea Pig Maximization test is among the most sensitive tests for detecting the potential sensitizing capacity of chemicals. Potency evaluation of chemical contact allergens is a quantitative expression of the ability to induce contact allergy based on determination of the relationship between dose and frequency of sensitization in the test population. The guinea pig allergy tests may be developed to give a potency evaluation of the chemicals tested, requiring dose-response determinations. Instead of using one dose group of 20 animals and 10 vehicle treated control animals, these animals could be divided into one control group and five test groups and each group treated with different induction concentrations. Formaldehyde, chloroacetamide, benzocaine, methyl methacrylate and potassium dichromate were included in such a study. For each chemical, the five groups were treated intradermally with concentrations reduced per group with increments of a factor three from the highest concentration that could be applied intradermally. Two of the five groups were treated topically with the highest nonirritating concentration and the three other groups with a 100 times lower concentration. All groups were challenged and rechallenged with the highest non-irritating concentration. A significant dose related sensitization rate was seen after induction with formaldehyde, methyl methacrylate and potassium dichromate. The results with chloroacetamide suggest a strong sensitizing potential. Although no dose-response relationship was obtained with benzocaine, the results suggest that benzocaine was a weak sensitizer. Future studies should attempt to evaluate different dose designs for the intradermal and the topical treatment, and to evaluate if larger increments between concentrations during induction would improve the method. Acknowledgement: Contract 711/18-0002, LST/12 Nordic Council of Ministers
Keywords: guinea pig maximization test; allergens; dose-response
EARLY MOLECULAR EVENTS INDUCED BY TRIBUTYLTIN IN MURINE KERATINOCYTES Emanuela Corsini *, Marina Marinovich, Corrado L. Galli, Barbara Viviani. University of Milan, Institute of Pharmacological
Sciences, Lab. of Toxicology, V~aBalzaretti 9, 20133 Milano The biocidal agent tributyltin (TBT) is used mainly in wood preservation and marine antifouling paints. Occupational exposure of workers to TBT can result in skin and eye irritation, and severe dermatitis has been reported after direct contact with the skin. We have previously demonstrated that TBT induced both intracellular production of IL-lot and its release into culture medium in a murine keratinocyte cell line (HELJ0). We were also able to demonstrate an in vivo role of I L - l a in TBT-induced skin irritation (Toxicol & Appl Pharmacol, in press). Here, we report that TBT induces a direct and dose-related (0.1-2.5 /zM) activation of NF-KB, which peaks at 2 hours and is blocked by PDTC, an antioxidant potent NF-rB
7
inhibitor; by rotenone, an inhibitor of the electron entry from complex I to ubiquinone, and by BAPTA, an intracellular Ca 2+ chelator. NF-~cB activation is preceded in time by an increase of cellular oxidative activity as measured by a fluorimetric assay. This event can be almost completely abrogated by BAPTA, which suggests that intracellular Ca 2+ mobilization could be one of the first event following TBT treatment, leading to an increased cellular oxidative activity. Furthermore, the partial modulation observed after rotenone pretreatment indicates mitochondria as the main intracellular source of reactive oxygen species. To further demonstrate the important role of mitochondria, prolonged treatment with ethidium bromide, an inhibitor of mitochondrial DNA and RNA synthesis, has been used to deplete cells of functional mitochondria. After 5 days of treatment a 50% reduction in MTT conversion is obtained, under this condition a 53% reduction in cellular oxidative activity is observed after 15 minutes of treatment with TBT. The impairment of mitoehondrial metabolism also results in inhibition of NF-rB activation by TBT. These findings indicate that mitochondria are the source of second messanger molecules essential for TBT-induced NF-rB activation and IL-lot production.
Keywords: organotins; keratinocyte; reactive oxygen species; transcription factor; interleukin-lot
S5. Progress in Antidotic Therapy ~'~']
THE EVOLUTION OF ANTIDOTAL THERAPY IN THE LAST DECADES
Jenny Pronczuk de Garbino. World Health Organization,
International Programme on Chemical Safety, Poisoning Prevention & Treatment Unit, Geneva, Switzerland The evolution of antidotal therapy in the last several decades has been marked by: (a) growing international concern on evaluation of its real efficacy, (b) development of a few new antidotes, and (c) innovative approaches in antidote administration. A meeting of the World Federation of Associations of Poisons Centres and Clinical Toxicology and a number of poisons centres, hosted by the IPCS (Geneva, October 1985), recognized the need to define antidotes and evaluate their efficacy. As a ~sult, during the last decade antidotes have become a subject of international evaluation (the results of which are to be presented in another communication). In recent decades, a number of antidotes have been developed and proven to be effective for specific poisoning: Naloxone, N-acetylcisteine, Fab fragments, new chelators, flumazenil, Fab fragment antivenom and 4-methyl pyrazole. Although the clinical benefits in the use of these compounds have been shown, some controversial issues still exist (e.g. doses, routes of administration, opportunity for use). The approach to the use of a number of antidotes has changed in recent years. Some examples are presented, namely in the antidotal management of poisoning by organophosphorous pesticides, cyanide, carbon monoxide, and also of the local effects produced by exposure to bites, stings and to some corrosive agents. Unfortunately no major developments have taken place in the last years concerning (a) the development of antidotes for some extremely hazardous substances, (b) expanding the availability of antidotes, especially to areas of need, or (c) dissemination of up-to-date information concerning the real role of antidotes and their efficacy in the management of poisoned patients.
Keywords: antidote; treatment; poisoning