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The mutagenic potential of arbutine, a naturally occurring hydroquinone glycoside
Abstracts/Mutation Research 360 (1996) 201-300
increased after pretreatment of the rats with PCB. Furthermore, reactive B[j]A intermediates caused more mutations in Salmonella typhimurium upon co-incubation with PCB-lung-microsomes compared to control-lung microsomes (total lung). Thus, the results indicate that inducers of P450 may increase the activation of these compounds in rat lung cells. 8-7
Comparative effects of 2-AAF and DEN on unscheduled DNA synthesis in primary cultures of rat and monkey hepatocytes E. Lorge, et al.; Biologic Servier, BP255, 45403 Fleury les Aubrais, France 2-AFF (2-acetylaminofluorene) and DEN (N-nitrosodiethylamine) have been extensively studied in a number of mutagenicity systems. The monkey is one of the species known to be resistant to the carcinogenicity of 2-AAF. That species-specificity has been shown to be due to the monkey's capacity to repair specifically induced 2-AAF lesions. It has also been argued that the low level of N-hydroxylase activity in the monkey contributes to the lack of the promutagen 2-AAF carcinogenicity in this species. Consequently, it was considered worthwhile to compare the abilities of the arylamide 2-AAF and of the nitrosamine DEN to induce primary DNA lesions in monkey and in rat hepatocytes. This comparison was made by measuring thymidine incorporation due to unscheduled DNA synthesis. Cells were obtained by collagenase perfusion and seeded in chamber slides. They were incubated with the compound from the 2nd to the 22nd hour of culture in the presence of tritiated thymidine. Repair activity was evaluated by autoradiography. In our experiment, the lack of 2-AAF mutagenicity in the monkey was confirmed at concentrations which have been proved to be highly mutagenic in the rat. DEN, on the other hand, was shown to be mutagenic in the monkey, inducing high levels of thymidine repair incorporation in monkey hepatocytes.
291
8-8
The mutagenic potential of arbutine, a naturally occurring hydroquinone glycoside L. Miiller, P. Kasper; Federal Institute for Drugs and Medical Devices, Berlin, Germany Arbutine is a naturally occurring hydroquinone glycoside. It is present in some plants (e.g., Arctostaphylos uva ursi leaves) which are used in traditional medicine for the treatment of urinary tract infections. In principle the ability of these traditional phytotherapeutics to act against urinary infections is believed to be the result of the action of free hydroquinone cleaved from the arbutine molecule in the urinary tract. It is not known in which tissue this cleavage occurs in vivo and what amount of hydroquinone is present in the organisms after treatment with arbutine. Investigations with people having drunken tea from uva ursi leaves showed that significant amounts of hydroquinone are present in the urine. Given the known mutagenic properties of hydroquinone across all in vitro and in vivo systems, it was of interest for us, whether a mutagenic potential of arbutine could also be detected in vitro and in vivo. The cleavage of the glycosidic moiety from arbutin is mediated via [3-glycosidase, an enzyme which is usually not present in mammalian cells in vitro or in vivo but which is readily present in some bacteria. In a gene mutation assay with V79-cells arbutine itself did not induce mutations in concentrations up to 10 -2 M but when the arbutine solution was preincubated with [3-glycosidase an effect was obtained. An increase in mutation frequency was observed with concentrations of 10 3 M arbutine and higher. Hydroquinone which was used as positive control exhibited clear effects with a LOEC of about 10 -5 M. We attempted to trail this in vitro effect in a micronucleus assay in vivo. Hydroquinone as a positive control (50 and 100mg/kg i.p.) induced clearly elevated micronucleus incidences in the bone marrow of treated mice. There was no induction of
292
Abstracts/Mutation Research360 (1996)201-300
micronuclei by arbutine when mice were treated orally with doses ranging from 0.5 up to 2 g / k g bodyweight. Leaves of uva ursi contain up to 6.0% hydroquinone glycosides. 4 times daily up to 3 g of the drug drunk as tea correspond to a dose of about 720 mg arbutine (approx. 12 m g / k g bodyweight). Arbutine itself is not mutagenic, but when the glycoside moiety is cleaved from the molecule, the generated hydroquinone can exert its mutagenic potential. [3Glycosidase which is necessary to cleave arbutine is present in microorganisms which occur in the gastrointestinal tract or possibly in the urinary tract, when infected. The negative micronucleus test at doses up to 2 g / k g might give a reassurance that there is not much hydroquinone present in the organism after oral treatment with arbutine. However, the potential of hydroquinone induced genotoxicity is there and should therefore not be neglected especially for tissues in the urinary tract. 8-9
Is the algal cell/microbe assay suitable for metabolic activation of promutatagens S. Podstavkovfi, E. Miadokovfi, D. V1Eek; Dept. of Genetics, Faculty of Science Comenius Univ., Bratislava, Slovak Republic Plewa et al. developed a genetic assay that employed living plant cell cultures to investigate the metabolic activation of non-mutagenic environmental pollutants (promutagens) to mutagenic product. We replaced the plant cell/microbe coincubation system by the algal cell/microbe coincubation system. This assay is based on coincubating unicellular green algal cells Chlamydomonas reinhardtii (as a metabolic activating system), tester strains of Salmonella typhimurium (as the genetic indicator organism) together with the test promutagen. The objective of our experiments was to find out if some promutagens (i.e., diphenylamines) can be activated in this assay. Results obtained showed that algal cell/microbe coincubation system is equally effective as tabasco cell/microbe one.
8-10
Recombinagenic activity of four compounds in the standard and high bioactivation crosses of the wing spot test in
Drosophila melanogaster M. Span6 a.b U. Graf a, F.E. Wiirgler a; ~ Institute of Toxicology, Swiss Federal Institute of Technology and University of Zurich, CH-8603 Schwerzenbach, Switzerland, b Departamento de Bioci~ncias, Universidade Federal de Uberl~ndia, 38400 Uberl~ndia MG, Brazil The wing Somatic Mutation And Recombination Test (SMART) in Drosophila melanogaster is ideally suited for the determination of recombinagenic and mutagenic activity of chemical compounds in an eukaryotic in vivo system. Two different crosses involving the wing cell markers mwh and ,fir 3 are currently used: (1) Standard cross (ST: .flr3/TM3, Bd s females and mwh males; Graf et al., 1989); (2) Improved high bioactivation cross (IHB: ORR; flr3/TM3, Bd s females and mwh males; Graf and van Schaik, 1992). Both crosses produce two types of progeny: marker heterozygous (mwh + / +fir 3) and balancer heterozygous (mwh +/TM3, BdS). The IHB cross is characterized by a high constitutive level of cytochrome P450 which leads to an improved sensitivity to a number of promutagens and procarcinogens. Larvae derived from both crosses were treated with the oxidising agent potassium chromate (POC), with the cytostatic drug cyclophosphamide (CPH), and with the two procarcinogens p-dimethylaminoazobenzene (DAB) and 9,10-dimethylanthracene (DMA). The wings of the resulting two genotypes were analysed for the occurrence of single and twin spots. In the marker heterozygous wings the spots can be due either to mitotic recombination or to mutation. By contrast, in the balancer heterozygous wings only mutational events lead to spot formation; all recombinational events are eliminated. The direct-acting agent POC was equally genotoxic in both crosses. Surprisingly, the promutagen CPH also showed equal genotoxicity in both crosses, whereas DAB was negative in the ST cross,
increased after pretreatment of the rats with PCB. Furthermore, reactive B[j]A intermediates caused more mutations in Salmonella typhimurium upon co-incubation with PCB-lung-microsomes compared to control-lung microsomes (total lung). Thus, the results indicate that inducers of P450 may increase the activation of these compounds in rat lung cells. 8-7
Comparative effects of 2-AAF and DEN on unscheduled DNA synthesis in primary cultures of rat and monkey hepatocytes E. Lorge, et al.; Biologic Servier, BP255, 45403 Fleury les Aubrais, France 2-AFF (2-acetylaminofluorene) and DEN (N-nitrosodiethylamine) have been extensively studied in a number of mutagenicity systems. The monkey is one of the species known to be resistant to the carcinogenicity of 2-AAF. That species-specificity has been shown to be due to the monkey's capacity to repair specifically induced 2-AAF lesions. It has also been argued that the low level of N-hydroxylase activity in the monkey contributes to the lack of the promutagen 2-AAF carcinogenicity in this species. Consequently, it was considered worthwhile to compare the abilities of the arylamide 2-AAF and of the nitrosamine DEN to induce primary DNA lesions in monkey and in rat hepatocytes. This comparison was made by measuring thymidine incorporation due to unscheduled DNA synthesis. Cells were obtained by collagenase perfusion and seeded in chamber slides. They were incubated with the compound from the 2nd to the 22nd hour of culture in the presence of tritiated thymidine. Repair activity was evaluated by autoradiography. In our experiment, the lack of 2-AAF mutagenicity in the monkey was confirmed at concentrations which have been proved to be highly mutagenic in the rat. DEN, on the other hand, was shown to be mutagenic in the monkey, inducing high levels of thymidine repair incorporation in monkey hepatocytes.
291
8-8
The mutagenic potential of arbutine, a naturally occurring hydroquinone glycoside L. Miiller, P. Kasper; Federal Institute for Drugs and Medical Devices, Berlin, Germany Arbutine is a naturally occurring hydroquinone glycoside. It is present in some plants (e.g., Arctostaphylos uva ursi leaves) which are used in traditional medicine for the treatment of urinary tract infections. In principle the ability of these traditional phytotherapeutics to act against urinary infections is believed to be the result of the action of free hydroquinone cleaved from the arbutine molecule in the urinary tract. It is not known in which tissue this cleavage occurs in vivo and what amount of hydroquinone is present in the organisms after treatment with arbutine. Investigations with people having drunken tea from uva ursi leaves showed that significant amounts of hydroquinone are present in the urine. Given the known mutagenic properties of hydroquinone across all in vitro and in vivo systems, it was of interest for us, whether a mutagenic potential of arbutine could also be detected in vitro and in vivo. The cleavage of the glycosidic moiety from arbutin is mediated via [3-glycosidase, an enzyme which is usually not present in mammalian cells in vitro or in vivo but which is readily present in some bacteria. In a gene mutation assay with V79-cells arbutine itself did not induce mutations in concentrations up to 10 -2 M but when the arbutine solution was preincubated with [3-glycosidase an effect was obtained. An increase in mutation frequency was observed with concentrations of 10 3 M arbutine and higher. Hydroquinone which was used as positive control exhibited clear effects with a LOEC of about 10 -5 M. We attempted to trail this in vitro effect in a micronucleus assay in vivo. Hydroquinone as a positive control (50 and 100mg/kg i.p.) induced clearly elevated micronucleus incidences in the bone marrow of treated mice. There was no induction of
292
Abstracts/Mutation Research360 (1996)201-300
micronuclei by arbutine when mice were treated orally with doses ranging from 0.5 up to 2 g / k g bodyweight. Leaves of uva ursi contain up to 6.0% hydroquinone glycosides. 4 times daily up to 3 g of the drug drunk as tea correspond to a dose of about 720 mg arbutine (approx. 12 m g / k g bodyweight). Arbutine itself is not mutagenic, but when the glycoside moiety is cleaved from the molecule, the generated hydroquinone can exert its mutagenic potential. [3Glycosidase which is necessary to cleave arbutine is present in microorganisms which occur in the gastrointestinal tract or possibly in the urinary tract, when infected. The negative micronucleus test at doses up to 2 g / k g might give a reassurance that there is not much hydroquinone present in the organism after oral treatment with arbutine. However, the potential of hydroquinone induced genotoxicity is there and should therefore not be neglected especially for tissues in the urinary tract. 8-9
Is the algal cell/microbe assay suitable for metabolic activation of promutatagens S. Podstavkovfi, E. Miadokovfi, D. V1Eek; Dept. of Genetics, Faculty of Science Comenius Univ., Bratislava, Slovak Republic Plewa et al. developed a genetic assay that employed living plant cell cultures to investigate the metabolic activation of non-mutagenic environmental pollutants (promutagens) to mutagenic product. We replaced the plant cell/microbe coincubation system by the algal cell/microbe coincubation system. This assay is based on coincubating unicellular green algal cells Chlamydomonas reinhardtii (as a metabolic activating system), tester strains of Salmonella typhimurium (as the genetic indicator organism) together with the test promutagen. The objective of our experiments was to find out if some promutagens (i.e., diphenylamines) can be activated in this assay. Results obtained showed that algal cell/microbe coincubation system is equally effective as tabasco cell/microbe one.
8-10
Recombinagenic activity of four compounds in the standard and high bioactivation crosses of the wing spot test in
Drosophila melanogaster M. Span6 a.b U. Graf a, F.E. Wiirgler a; ~ Institute of Toxicology, Swiss Federal Institute of Technology and University of Zurich, CH-8603 Schwerzenbach, Switzerland, b Departamento de Bioci~ncias, Universidade Federal de Uberl~ndia, 38400 Uberl~ndia MG, Brazil The wing Somatic Mutation And Recombination Test (SMART) in Drosophila melanogaster is ideally suited for the determination of recombinagenic and mutagenic activity of chemical compounds in an eukaryotic in vivo system. Two different crosses involving the wing cell markers mwh and ,fir 3 are currently used: (1) Standard cross (ST: .flr3/TM3, Bd s females and mwh males; Graf et al., 1989); (2) Improved high bioactivation cross (IHB: ORR; flr3/TM3, Bd s females and mwh males; Graf and van Schaik, 1992). Both crosses produce two types of progeny: marker heterozygous (mwh + / +fir 3) and balancer heterozygous (mwh +/TM3, BdS). The IHB cross is characterized by a high constitutive level of cytochrome P450 which leads to an improved sensitivity to a number of promutagens and procarcinogens. Larvae derived from both crosses were treated with the oxidising agent potassium chromate (POC), with the cytostatic drug cyclophosphamide (CPH), and with the two procarcinogens p-dimethylaminoazobenzene (DAB) and 9,10-dimethylanthracene (DMA). The wings of the resulting two genotypes were analysed for the occurrence of single and twin spots. In the marker heterozygous wings the spots can be due either to mitotic recombination or to mutation. By contrast, in the balancer heterozygous wings only mutational events lead to spot formation; all recombinational events are eliminated. The direct-acting agent POC was equally genotoxic in both crosses. Surprisingly, the promutagen CPH also showed equal genotoxicity in both crosses, whereas DAB was negative in the ST cross,