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Photochemical genotoxicity and photochemical carcinogenesis — Two sides of a coin?
Symposium 16. Photomutagenicity and Photocarcinogenesis was no evidence of free radicals-mediated apoptosis in astrocytes, free radicals formation in astrocytes howeverresult in the increased depolarization of mitochondrial membrane potentialand in the rapid depletion of glutamine synthetase and ATP, in particular, in striatal astrocytes. The impairment of energy metabolism in striatal astrocytes might thereforelead to an increased sensitivityof dopaminergic terminals to free radicals-mediated apoptotic injuries.
S16. Photomutagenicity and Photocarcinogenesis
IS16/l1 I MECHANISMS OF QUINOLONE PHOTOTOXlClTY K. Shimoda. Drug SafetyResearchLaboratory, Daiichi Pharmaceutical Co., Ltd., Tokyo, Japan Photosensitivity induced by quinolone antibacterial agents has become important problems in humans. Photosensitivity includes both allergic (photoallergy) and non-allergic (phototoxicity) light-induced skin responses, and quinolone-induced photosensitivity has been thought to be mainly in phototoxic nature. We therefore examined mechanisms of quinolone phototoxicity in vivo and in vitro. Simultaneous p.o. administration of a quinolone and ultraviolet-A (UVA) irradiation for 4 hr induced auricular skin inflammation in Balb/c mice, including edema and neutrophil infiltration in the dennis which consisted of the early mild and later exacerbation stages. Antioxidants inhibited the inflammation in the early stage and cyclooxygenase inhibitors did in both the early and later stages, whereas 5-lipoxygenase inhibitors or histamine antagonists had no effect. Further, the phototoxic inflammation was also induced in mast cell-deficient WBB6FI-WlWv mice. Corresponding to the in vivo results, incubation with a quinolone under UVA irradiation for 5 or 10 min stimulated Balblc 3T3 mouse fibroblast cells to release prostaglandin E2 (PGE2) and 6-keto-PGFla, but not leukotrieneB4. In contrast, UVA-preirradiated quinolones did not affect PG release from fibroblasts. The PGE2 release by quinolone phototoxicity was inhibited by cyclooxygenase inhibitors, antioxidants, protein kinase C (PKC) inhibitors and a tyrosine kinase (TK) inhibitor, but not by antibodies against tumor necrosis factor a (TNFa ) and interleukin-I (IL·I ). These results lead a hypothesis that reactive oxygen species generated from quinolones under UVAirradiation trigger PG release from dermal fibroblasts via PKC and TK activation, resulting in skin inflammationand that 5-lipoxygenase products, histamine,TNFa or IL-I is ruled out from the mechanism.
I S16/L21 THE PHOTOMUTAGENIClTY OF FLUOROQUINOLONES AND OTHER DRUGS
E. Gocke *, S. Albertini, A. A. Chetelat, S. Kirchner, W. Muster. PharmaDivision, Preclinical Research, Dep. ofToxicology, F. Hoffmann-La Roche Ltd, CH-4070Basel, Switzerland Induction of DNA damage as a consequence of exposure to UV light has been established as the major and still increasing cause of skin cancer. Absorption of the photon energy may be either directly by the DNA molecules (for wavelenghts <320 nm) or may be by endogenous or exogenous chemicals (sensitizers) with the potential of energy or electron transfer to DNA. Oxygen mediated reactions (often called type II reactions) appear to be the most important mechanism since molecular oxygen is a good and abundantsubstrate for triplet excited sensitizers.Energy transfer to molecular oxygen is possible for wavelenghts in the near UV and in the visible part of the solar spectrum since the energy of the exited oxygen molecule (1 02 *) is comparatively low. A few light-absorbing pharmaceuticals have long been known to cause photo(geno)toxic effects. Notably psoralene and chlorpromazine derivatives have been established as photomutagens and
19
the reaction mechanisms have been identified. The fluoroquinolone antibiotics have more recently been recognized as being photomutagenic. The type of DNA damage and the modulation by antioxidants indicate the involvement of reactiveoxygen species (ROS) but other mechanismsare also reported at least for some derivatives. In routine genotoxicity studies we observed a photomutagenic activity of a compound under developmentas an anxiolyticagent in the Ames tester strain TAlO2 at "normal laboratory illumination" conditions. Further investigations showed strong photogenotoxic activity in tests for gene mutations and chromosomal aberrations in mammalian cells. The compound proved to be a potent 102-producer. The finding led to termination of development but in the course of studies several structural analogues have been tested for which structure activity relationships will be described. The relevanceof photogenotoxicpropertiesof drugs for predicting adverse effects in man will be discussed.
I S16/LSI PHOTOCHEMICAL GENOTOXICITY AND PHOTOCHEMICAL CARCINOGENESIS - TWOSIDES OFACOIN?
L. MUlier. Federal Institute for Drugs and Medical Devices
(BfArMJ, Seestr: 10, D-13353-Berlin, Germany The direct premutagenic DNA lesions of UVB (290-320 om), i.e. pyrimidine-dimers and 6-4 photo-products, are known to be the most important molecularevents in UVR tumorigenicity. In contrast, the weakly tumorigenic UVA (320-4 00 nm) generates indirectly DNA damage mainly via photodynamic generation of active oxygen species involving endogenous or exogenous photosensitizers. In combination with UVA photoinstable compounds such as some phenotiazides, furocoumarines and f1uoroquinolones are efficient inducers of photo-chemical DNA damage. As a consequence, cellular repair mechanisms can be overloaded and unrepaired primary DNA damage such as thymidineglycolsor 8-oxo-2'-deoxyguanosine residues would lead to mutationscr chromosomal damage. Eventually, skin tumorigenicity of UVA can be substantially enhanced by photochemical genotoxins,Thus, testing for photochemical genotoxicity could identify potential photochemical carcinogens. An easy means for investigation of photo-chemicalgenotoxicity is the in vitro micronucleus test. The test system as well as results for different compounds under various UV radiation conditions are presented by 1. Zhang et al. in their abstract and poster. Hairless mice or XPA (repair) deficient mice have been used as animal models for investigating photochemical carcinogenesis of the skin. This seems to be justified since recent data indicate that p53 mutations in squamous cell carcinomas induced in these mice by UVB have a full equivalent in human skin tumours (Dumaz et al., Carcinogenesis 18, 1997, 897-904). Recent testing of furocoumarines and several fluoroquinolones in hairless mice resulted in a higher incidence and a shorter latent period for skin tumors compared to UVR alone. This appears to be the case even in humans since an elevated skin tumour incidence in patients (squamous cell carcinomas and malignant melanomas) has been reported for longterm treatmentwith the photochemical mutagen8-methoxypsoralene and UVA(Stem et al., New Engl. I. Med. 336, 1997, 1041-1045). Data generated so far show a good correlation between the photogenotoxic and photocarcinogenic potential of photosensitizers. Thus, testing for photochemical genotoxicity in mammalian cells in vitro may be an early warning system for identification of photochemical carcinogens. Eventually, an integrated approach using photostability data, photo-toxicity testing (in vitro and in vivo) and photogenotoxicity test systems may give the same level of safety information for a pharmaceutical or cosmetic product as testing for photocarcinogenicity.
S16. Photomutagenicity and Photocarcinogenesis
IS16/l1 I MECHANISMS OF QUINOLONE PHOTOTOXlClTY K. Shimoda. Drug SafetyResearchLaboratory, Daiichi Pharmaceutical Co., Ltd., Tokyo, Japan Photosensitivity induced by quinolone antibacterial agents has become important problems in humans. Photosensitivity includes both allergic (photoallergy) and non-allergic (phototoxicity) light-induced skin responses, and quinolone-induced photosensitivity has been thought to be mainly in phototoxic nature. We therefore examined mechanisms of quinolone phototoxicity in vivo and in vitro. Simultaneous p.o. administration of a quinolone and ultraviolet-A (UVA) irradiation for 4 hr induced auricular skin inflammation in Balb/c mice, including edema and neutrophil infiltration in the dennis which consisted of the early mild and later exacerbation stages. Antioxidants inhibited the inflammation in the early stage and cyclooxygenase inhibitors did in both the early and later stages, whereas 5-lipoxygenase inhibitors or histamine antagonists had no effect. Further, the phototoxic inflammation was also induced in mast cell-deficient WBB6FI-WlWv mice. Corresponding to the in vivo results, incubation with a quinolone under UVA irradiation for 5 or 10 min stimulated Balblc 3T3 mouse fibroblast cells to release prostaglandin E2 (PGE2) and 6-keto-PGFla, but not leukotrieneB4. In contrast, UVA-preirradiated quinolones did not affect PG release from fibroblasts. The PGE2 release by quinolone phototoxicity was inhibited by cyclooxygenase inhibitors, antioxidants, protein kinase C (PKC) inhibitors and a tyrosine kinase (TK) inhibitor, but not by antibodies against tumor necrosis factor a (TNFa ) and interleukin-I (IL·I ). These results lead a hypothesis that reactive oxygen species generated from quinolones under UVAirradiation trigger PG release from dermal fibroblasts via PKC and TK activation, resulting in skin inflammationand that 5-lipoxygenase products, histamine,TNFa or IL-I is ruled out from the mechanism.
I S16/L21 THE PHOTOMUTAGENIClTY OF FLUOROQUINOLONES AND OTHER DRUGS
E. Gocke *, S. Albertini, A. A. Chetelat, S. Kirchner, W. Muster. PharmaDivision, Preclinical Research, Dep. ofToxicology, F. Hoffmann-La Roche Ltd, CH-4070Basel, Switzerland Induction of DNA damage as a consequence of exposure to UV light has been established as the major and still increasing cause of skin cancer. Absorption of the photon energy may be either directly by the DNA molecules (for wavelenghts <320 nm) or may be by endogenous or exogenous chemicals (sensitizers) with the potential of energy or electron transfer to DNA. Oxygen mediated reactions (often called type II reactions) appear to be the most important mechanism since molecular oxygen is a good and abundantsubstrate for triplet excited sensitizers.Energy transfer to molecular oxygen is possible for wavelenghts in the near UV and in the visible part of the solar spectrum since the energy of the exited oxygen molecule (1 02 *) is comparatively low. A few light-absorbing pharmaceuticals have long been known to cause photo(geno)toxic effects. Notably psoralene and chlorpromazine derivatives have been established as photomutagens and
19
the reaction mechanisms have been identified. The fluoroquinolone antibiotics have more recently been recognized as being photomutagenic. The type of DNA damage and the modulation by antioxidants indicate the involvement of reactiveoxygen species (ROS) but other mechanismsare also reported at least for some derivatives. In routine genotoxicity studies we observed a photomutagenic activity of a compound under developmentas an anxiolyticagent in the Ames tester strain TAlO2 at "normal laboratory illumination" conditions. Further investigations showed strong photogenotoxic activity in tests for gene mutations and chromosomal aberrations in mammalian cells. The compound proved to be a potent 102-producer. The finding led to termination of development but in the course of studies several structural analogues have been tested for which structure activity relationships will be described. The relevanceof photogenotoxicpropertiesof drugs for predicting adverse effects in man will be discussed.
I S16/LSI PHOTOCHEMICAL GENOTOXICITY AND PHOTOCHEMICAL CARCINOGENESIS - TWOSIDES OFACOIN?
L. MUlier. Federal Institute for Drugs and Medical Devices
(BfArMJ, Seestr: 10, D-13353-Berlin, Germany The direct premutagenic DNA lesions of UVB (290-320 om), i.e. pyrimidine-dimers and 6-4 photo-products, are known to be the most important molecularevents in UVR tumorigenicity. In contrast, the weakly tumorigenic UVA (320-4 00 nm) generates indirectly DNA damage mainly via photodynamic generation of active oxygen species involving endogenous or exogenous photosensitizers. In combination with UVA photoinstable compounds such as some phenotiazides, furocoumarines and f1uoroquinolones are efficient inducers of photo-chemical DNA damage. As a consequence, cellular repair mechanisms can be overloaded and unrepaired primary DNA damage such as thymidineglycolsor 8-oxo-2'-deoxyguanosine residues would lead to mutationscr chromosomal damage. Eventually, skin tumorigenicity of UVA can be substantially enhanced by photochemical genotoxins,Thus, testing for photochemical genotoxicity could identify potential photochemical carcinogens. An easy means for investigation of photo-chemicalgenotoxicity is the in vitro micronucleus test. The test system as well as results for different compounds under various UV radiation conditions are presented by 1. Zhang et al. in their abstract and poster. Hairless mice or XPA (repair) deficient mice have been used as animal models for investigating photochemical carcinogenesis of the skin. This seems to be justified since recent data indicate that p53 mutations in squamous cell carcinomas induced in these mice by UVB have a full equivalent in human skin tumours (Dumaz et al., Carcinogenesis 18, 1997, 897-904). Recent testing of furocoumarines and several fluoroquinolones in hairless mice resulted in a higher incidence and a shorter latent period for skin tumors compared to UVR alone. This appears to be the case even in humans since an elevated skin tumour incidence in patients (squamous cell carcinomas and malignant melanomas) has been reported for longterm treatmentwith the photochemical mutagen8-methoxypsoralene and UVA(Stem et al., New Engl. I. Med. 336, 1997, 1041-1045). Data generated so far show a good correlation between the photogenotoxic and photocarcinogenic potential of photosensitizers. Thus, testing for photochemical genotoxicity in mammalian cells in vitro may be an early warning system for identification of photochemical carcinogens. Eventually, an integrated approach using photostability data, photo-toxicity testing (in vitro and in vivo) and photogenotoxicity test systems may give the same level of safety information for a pharmaceutical or cosmetic product as testing for photocarcinogenicity.