Pharmacological Research, Vol. 22, No . 4, 1990
381
HUMAN HEPATOCYTES IN GENOTOXICITY ASSAYS GIOVANNI BRAMBILLA* and ANTONIETTA MARTELLI Institute of Pharmacology, University of Genoa, 1-16132 Genoa, Italy Received in final form 12 December 1989
SUMMARY Human hepatocyte primary cultures, because of their comprehensive hiotransformation capability, represent an experimental model particularly useful to gain direct information on the genotoxic risk of chemicals to humans . For this purpose they have been used in the last few years either as target cells to evaluate the induction of DNA damage and/or DNA repair synthesis, or as metabolic activation system in mutagenicity assays. The number of compounds so far tested is rather limited, and for the large majority of them the assays have been performed only on hepatocytes from a few donors . A comparison with the data obtained in rat hepatocytes indicates that quantitative differences in the genotoxic effects induced in cultures derived from different donors of the same species are usually greater than interspecies differences . However, the results- provided by some chemicals suggest the possibility that in certain cases rat hepatocytes might be inappropriate predictors of the genotoxic hazard for humans . Kt woKos : human hepatocytes, genotoxicity tests, DNA damage, DNA repair, mutagenic activity . INTRODUCTION A main problem in the assessment of the genotoxic risk of chemicals is the extrapolation to man of results obtained in laboratory animals, or in in vitro systems employing prokaryotes, lower eukaryotes or mammalian cells . Because many xenobiotics are activated and/or detoxified by cytochrome P-450 dependent mixed function oxidases or by the subsequent conjugation with glycuronic acid, sulphate and reduced glutathione, the extent of genotoxicity essentially depends on the balance between these activation and detoxification processes . A growing evidence indicates the existence of interspecies differences in the metabolic activation/detoxification processes [1-3] . These differences are presumably one of the main causes of the non-satisfying predictiveness for rats of data provided by carcinogenesis assays carried out in mice, and vice versa [4, 5] . Since it is probable that biotransformation processes are more similar in these two species than in such *To whom all correspondence should he addressed at : Istituto di Farmacologia . dell'Universita . Viaic Benedetto XV, 2,1-16132 Genova, Italia . 1043-6618/90/040381-12/$03 .00'0
© 1990 The Italian Pharmacological Society
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Pharmacological Research, Vol. 22, No. 4, 1990
phylogenetically distant species as rat and man, the extrapolation to humans of results obtained in rodents may be subjected to substantial errors . Experimental systems employing human cells offer the advantage of directly assessing the genotoxic effects of chemicals in our species [6]. Human hepatocytes, because of their comprehensive metabolic capability, represent an ideal model system . Compared with rodent hepatocytes, they have been shown to maintain higher levels of cytochrome P-450 while in culture, and to metabolize some xenobiotics to a greater extent [7-9] . Methods used to obtain suspensions of viable human hepatocytes are essentially similar to those employed for the preparation of rat liver cells ; usually they are prepared by collagenase perfusion either of liver fragments discarded during the course of prescribed surgery, or of entire liver lobes obtained from organ donors . Intact hepatocytes, as well as liver slices and subcellular fractions, have been employed to evaluate cytotoxicity, genotoxicity and metabolic activation/detoxification of several xenobiotics . The objective of this short review is limited to the comparison of results provided by the evaluation of the genotoxic effects of chemicals in primary cultures of human and rat hepatocytes . Three end-points are considered : induction of DNA repair synthesis as evaluated by quantitative autoradiography ; DNA fragmentation measured by the alkaline elution assay ; induction of mutations in both bacterial and mammalian cells using intact hepatocytes as activation system . Due to the large variability of results from different laboratories, only data obtained in identical experimental conditions were compared .
INDUCTION OF DNA REPAIR SYNTHESIS Table I compares the potencies in inducing unscheduled DNA synthesis (UDS) in human and rat hepatocytes of 51 chemicals . UDS-inducing potency was calculated by the formula (NG, - NGJ/concentration (mm) where (NG,-NG C ) is the increase over controls in the number of net nuclear grains at the concentration level producing the maximal response . UDS-inducing potency is given as mean ± SD when at least three data were available ; it was considered zero when (NG, - NGC ) < 5, this value being the minimal indicative of DNA repair . The data listed in Table I indicate that 19 of the 51 compounds tested do not induce DNA repair synthesis in hepatocytes of both species . The UDS-inducing potencies of the 32 active chemicals range from 0 . 54 to 29 716 in human, and from 0 . 97 to 60 781 in rat hepatocytes, metronidazole being the least potent in both species, and 1,6-dinitropyrene and aflatoxin B, the most potent in human and rat, respectively . Twenty-six of these 32 chemicals were tested in only one laboratory . 2,6-Diaminotoluene and 5-methylchrysene were active only in human hepatocytes ; 2,7-dinitrofluorene, 3-nitrofluoranthene, ß-naphthylamine, unleaded gasoline, cimetidine and tripelennamine only in rat hepatocytes . The other 18 chemicals were active in both species and can be divided into three classes : eight compounds display a similar UDS-inducing potency in human and rat hepatocytes
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Pharmacological Research, Vol. 22, No. 4, 1990
(man/rat ratio from 0 . 5 to 1 . 5 ) ; five demonstrate a greater effect in human hepatocytes (man/rat ratio > 1 . 5) ; five are definitely more active in rat liver cells (man/rat ratio
381
HUMAN HEPATOCYTES IN GENOTOXICITY ASSAYS GIOVANNI BRAMBILLA* and ANTONIETTA MARTELLI Institute of Pharmacology, University of Genoa, 1-16132 Genoa, Italy Received in final form 12 December 1989
SUMMARY Human hepatocyte primary cultures, because of their comprehensive hiotransformation capability, represent an experimental model particularly useful to gain direct information on the genotoxic risk of chemicals to humans . For this purpose they have been used in the last few years either as target cells to evaluate the induction of DNA damage and/or DNA repair synthesis, or as metabolic activation system in mutagenicity assays. The number of compounds so far tested is rather limited, and for the large majority of them the assays have been performed only on hepatocytes from a few donors . A comparison with the data obtained in rat hepatocytes indicates that quantitative differences in the genotoxic effects induced in cultures derived from different donors of the same species are usually greater than interspecies differences . However, the results- provided by some chemicals suggest the possibility that in certain cases rat hepatocytes might be inappropriate predictors of the genotoxic hazard for humans . Kt woKos : human hepatocytes, genotoxicity tests, DNA damage, DNA repair, mutagenic activity . INTRODUCTION A main problem in the assessment of the genotoxic risk of chemicals is the extrapolation to man of results obtained in laboratory animals, or in in vitro systems employing prokaryotes, lower eukaryotes or mammalian cells . Because many xenobiotics are activated and/or detoxified by cytochrome P-450 dependent mixed function oxidases or by the subsequent conjugation with glycuronic acid, sulphate and reduced glutathione, the extent of genotoxicity essentially depends on the balance between these activation and detoxification processes . A growing evidence indicates the existence of interspecies differences in the metabolic activation/detoxification processes [1-3] . These differences are presumably one of the main causes of the non-satisfying predictiveness for rats of data provided by carcinogenesis assays carried out in mice, and vice versa [4, 5] . Since it is probable that biotransformation processes are more similar in these two species than in such *To whom all correspondence should he addressed at : Istituto di Farmacologia . dell'Universita . Viaic Benedetto XV, 2,1-16132 Genova, Italia . 1043-6618/90/040381-12/$03 .00'0
© 1990 The Italian Pharmacological Society
382
Pharmacological Research, Vol. 22, No. 4, 1990
phylogenetically distant species as rat and man, the extrapolation to humans of results obtained in rodents may be subjected to substantial errors . Experimental systems employing human cells offer the advantage of directly assessing the genotoxic effects of chemicals in our species [6]. Human hepatocytes, because of their comprehensive metabolic capability, represent an ideal model system . Compared with rodent hepatocytes, they have been shown to maintain higher levels of cytochrome P-450 while in culture, and to metabolize some xenobiotics to a greater extent [7-9] . Methods used to obtain suspensions of viable human hepatocytes are essentially similar to those employed for the preparation of rat liver cells ; usually they are prepared by collagenase perfusion either of liver fragments discarded during the course of prescribed surgery, or of entire liver lobes obtained from organ donors . Intact hepatocytes, as well as liver slices and subcellular fractions, have been employed to evaluate cytotoxicity, genotoxicity and metabolic activation/detoxification of several xenobiotics . The objective of this short review is limited to the comparison of results provided by the evaluation of the genotoxic effects of chemicals in primary cultures of human and rat hepatocytes . Three end-points are considered : induction of DNA repair synthesis as evaluated by quantitative autoradiography ; DNA fragmentation measured by the alkaline elution assay ; induction of mutations in both bacterial and mammalian cells using intact hepatocytes as activation system . Due to the large variability of results from different laboratories, only data obtained in identical experimental conditions were compared .
INDUCTION OF DNA REPAIR SYNTHESIS Table I compares the potencies in inducing unscheduled DNA synthesis (UDS) in human and rat hepatocytes of 51 chemicals . UDS-inducing potency was calculated by the formula (NG, - NGJ/concentration (mm) where (NG,-NG C ) is the increase over controls in the number of net nuclear grains at the concentration level producing the maximal response . UDS-inducing potency is given as mean ± SD when at least three data were available ; it was considered zero when (NG, - NGC ) < 5, this value being the minimal indicative of DNA repair . The data listed in Table I indicate that 19 of the 51 compounds tested do not induce DNA repair synthesis in hepatocytes of both species . The UDS-inducing potencies of the 32 active chemicals range from 0 . 54 to 29 716 in human, and from 0 . 97 to 60 781 in rat hepatocytes, metronidazole being the least potent in both species, and 1,6-dinitropyrene and aflatoxin B, the most potent in human and rat, respectively . Twenty-six of these 32 chemicals were tested in only one laboratory . 2,6-Diaminotoluene and 5-methylchrysene were active only in human hepatocytes ; 2,7-dinitrofluorene, 3-nitrofluoranthene, ß-naphthylamine, unleaded gasoline, cimetidine and tripelennamine only in rat hepatocytes . The other 18 chemicals were active in both species and can be divided into three classes : eight compounds display a similar UDS-inducing potency in human and rat hepatocytes
383
Pharmacological Research, Vol. 22, No. 4, 1990
(man/rat ratio from 0 . 5 to 1 . 5 ) ; five demonstrate a greater effect in human hepatocytes (man/rat ratio > 1 . 5) ; five are definitely more active in rat liver cells (man/rat ratio
