Evaluation of the potential in vivo genotoxicity of quercetin.

Quercetin, a naturally occurring flavonol commonly detected in apples, cranberries, blueberries, and onions, has been reported to possess antioxidant, anti-carcinogenic, anti-inflammatory, and cardioprotective properties. While positive results have been consistently reported in numerous in vitro mutagenicity and genotoxicity assays of quercetin, tested in vivo, quercetin has generally produced negative results in such studies. Furthermore, no evidence of carcinogenicity related to the oral administration of quercetin was observed in chronic rodent assays. In order to further define the in vivo genotoxic potential of quercetin, a bone marrow micronucleus assay and an unscheduled DNA synthesis (UDS) assay were conducted in Wistar rats. Administered orally to male rats at dose levels of up to 2000 mg/kg body weight, quercetin did not increase the number of micronucleated polychromatic erythrocytes (MN-PCE) 24 or 48 h following dosing in the micronucleus assay. Likewise, orally administered quercetin (up to 2000 mg/kg body weight) did not induce UDS in hepatocytes of male or female rats. While measurable levels of metabolized quercetin were observed in rat plasma samples for up to 48 h after dosing, peaking at 1h following treatment administration, the unmetabolized aglycone was not identified in either plasma or bone marrow. With the exception of only a few rats, the aglycone was also not detected in liver tissue. These results demonstrate that quercetin is not genotoxic under the conditions of these assays and further support the negative results of previously conducted in vivo assays.

Metabolic activity of fresh and cryopreserved cynomolgus monkey (Macaca fascicularis) hepatocytes.

1. The effect of cryopreservation on the metabolic capacity of monkey hepatocytes over 4 h in suspension and 24 h in culture was determined. Hepatocytes were diluted in a buffer containing 10% DMSO and frozen in a computer-controlled chamber. 2. Initial ethoxyresorufin and ethoxycoumarin O-deethylase (ECOD) activities were the same in fresh and cryopreserved (CP) hepatocytes. ECOD activity in suspensions declined over 4 h but was the same in fresh and CP hepatocytes. 3. The formation of testosterone hydroxy (OHT) metabolites (namely 6beta-OHT, 2beta-OHT, 16beta-OHT, 16alpha-OHT, 15beta-OHT, 2alpha-OHT and 6beta-OHT) was unaffected by cryopreservation. The loss of OHT activities over 4 h in CP and fresh whole cell suspensions was attributed to a loss of cofactor. CP hepatocyte cultures had equivalent OHT activities to freshly isolated hepatocytes. 4. Initial UDP-glucuronyltransferase (UGT) activities, using the substrates 4-methylumbelliferone, ethoxycoumarin and hydroxycoumarin, were equivalent in fresh and CP whole hepatocytes. At later times, UGT activity was lower in CP than fresh hepatocytes but this was due to a loss of UDPGA. Initial sulphotransferase (SULT) activities, using the substrates 2-naphthol, ethoxycoumarin and hydroxycoumarin, were equivalent in fresh and CP hepatocytes. SULT activities were less stable than UGT activities but were the same in fresh and CP hepatocytes throughout the 4-h incubation. 5. Initial glutathione S-transferase activities (using 1-chloro-2,4-dinitrobenzene) were the same in fresh and CP hepatocytes and both did not decrease over 4 h. 6. CP monkey hepatocytes are a useful model for metabolic and cytotoxicity studies. These cells can be can be used either in suspension or in culture.

In vitro micronucleus assay with Chinese hamster V79 cells - results of a collaborative study with in situ exposure to 26 chemical substances.

A collaborative study with 10 participating laboratories was conducted to evaluate a test protocol for the performance of the in vitro micronucleus (MN) test using the V79 cell line with one treatment and one sampling time only. A total of 26 coded substances were tested in this study for MN-inducing properties. Three substances were tested by all 10 laboratories and 23 substances were tested by three or four laboratories in parallel. Six aneugenic, 7 clastogenic and 6 non-genotoxic chemicals were uniformly recognised as such by all laboratories. Three chemicals were tested uniformly negative by three laboratories although also clastogenic properties have been reported for these substances. Another set of three clastogenic substances showed inconsistent results and one non-clastogenic substance was found to be positive by one out of three laboratories. Within the study, the applicability of the determination of a proliferation index (PI) as an internal cytotoxicity parameter in comparison with the determination of the mitotic index (MI) was also evaluated. Both parameters were found to be useful for the interpretation of the MN test result with regard to the control of cell cycle kinetics and the mode of action for MN induction. The MN test in vitro was found to be easy to perform and its results were mainly in accordance with results from chromosomal aberration tests in vitro.

Evaluation of the in vitro micronucleus test as an alternative to the in vitro chromosomal aberration assay: position of the GUM Working Group on the in vitro micronucleus test. Gesellschaft für Umwelt-Mutations-forschung.

In order to license a pharmaceutical or chemical, a compound has to be tested for several genotoxicity endpoints, including the induction of chromosomal aberrations in vitro. A working group within the GUM has evaluated published data on the in vitro micronucleus test with the aim of judging its suitability as a replacement for the in vitro chromosomal aberration test. After strict rejection criteria were applied, a database including 96 publications and 34 compounds was obtained. For 30 of these compounds, data on both tests were available. For 24 of the 30, concordant results in both test systems were obtained (80% correlation). The discordant results in 6 compounds can be explained by a known or suspected aneugenic potential of these compounds. Considering that cell types and test protocols were extremely heterogeneous, this correlation is rather encouraging. Comparison of the different protocols, and experience established within the working group yielded several recommendations for the routine use of the in vitro micronucleus test. Although many cell lines are suitable, those most often used in genotoxicity testing (e.g. CHL, CHO, V79, human lymphocytes, L5178Y mouse lymphoma cells) are recommended. Cytochalasin B may be used in the case of human lymphocytes; however, the possibility of its interaction with aneugenic test compounds should be considered. For continuously dividing cell lines, cytochalasin B is not recommended by the working group. Although, there seems to be flexibility in the choice of treatment and sampling times, the average generation time of the chosen cell line of choice should be taken into account when determining sampling time, and treatment of cells for at least one cell cycle duration is recommended. The use of appropriate cytotoxicity tests is strongly recommended. Although studies on some parameters of the test protocol may be useful, the introduction of the in vitro micronucleus test into genotoxicity testing and guidelines should not be delayed. Even in its present state, the in vitro micronucleus is a reliable genotoxicity test. Compared with the chromosomal aberration test, it detects aneugens more reliably, it is faster and easier to perform, and it has more statistical power and the possibility of automation.

The gap junctional intercellular communication is no prerequisite for the stabilization of xenobiotic metabolizing enzyme activities in primary rat liver parenchymal cells in vitro.

In primary monocultures of adult rat liver parenchymal cells (PC), the activities of the xenobiotic metabolizing enzymes microsomal epoxide hydrolase (mEHb), soluble epoxide hydrolase (sEH), glutathione S-transferases (GST), and phenolsulfotransferase (ST) were reduced after 7 d to values below 33% of the initial activities. Furthermore, the gap junctional intercellular communication (GJIC), measured after microinjection by dye transfer, decreased from 90% on Day 1 to undetectable values after 5 d in monoculture. Co-culture of PC with nonparenchymal rat liver epithelial cells (NEC) increased (98% on Day 1) and stabilized (82% on Day 7) the homotypic GJIC of PC. Additionally, most of the measured xenobiotic metabolizing enzyme activities were well stabilized over 1 wk in co-culture. Because GJIC is one of several mechanisms playing an important role in cell differentiation, the importance of GJIC for the stabilization of xenobiotic metabolizing enzymes in PC was investigated. PC in monoculture were, therefore, treated with 2% dimethyl sulfoxide (DMSO), a differentiation promoting factor, and 1,1,1-trichloro-2,2,-bis (p-chlorophenyl) ethane (DDT) (10 micrograms/ml), a liver tumor promotor and inhibitor of GJIC, was given to co-cultures of PC with NEC. DMSO significantly stabilized (68% on Day 7), while DDT significantly inhibited (8% on Day 7) homotypic GJIC of PC in the respective culture systems. In contrast, the activities of mEHb, sEH, GST, and ST were not affected in the presence of DMSO or DDT. These results lead to the assumption that the differentiation parameters measured in this study (i.e., homotypic GJIC and the activities of xenobiotic metabolizing enzymes) are independently regulated in adult rat liver PC.

Gap junctional intercellular communication of cultured rat liver parenchymal cells is stabilized by epithelial cells and their isolated plasma membranes.

The gap junctional intercellular communication (GJIC) determined by measuring dye coupling with Lucifer yellow, decreased within 3 d from 66% to 28% in monocultures of rat liver parenchymal cells. Coculturing of the parenchymal cells with a nonparenchymal epithelial cell line from rat liver resulted in increased and stabilized intercellular communication (83% after 3 d). The presence of isolated plasma membrane vesicles of the nonparenchymal epithelial cells also stabilized the intercellular communication between the liver parenchymal cells (70% after 3 d). When liver parenchymal cells were cocultured with a rat liver fibroblast cell line the gap junctional communication between the parenchymal cells was not stabilized (43% after 3 d), and isolated plasma membrane vesicles of the fibroblast were also unable to support the GJIC in parenchymal cells (35% after 3 d). It is concluded that plasma membrane constituents of the nonparenchymal epithelial cells were responsible for the stabilization of the GJIC between parenchymal cells. A heterotypic gap junctional communication between parenchymal and nonparenchymal cells was not observed.

Effects of sodium butyrate on DNA content, glutathione S-transferase activities, cell morphology and growth characteristics of rat liver nonparenchymal epithelial cells in vitro.

The effects of sodium butyrate, which has been shown to act as a differentiation promoting agent in several different tumor cell lines, were studied in a rat liver nonparenchymal epithelial cell line. Exposure of these cells to 3.75 mM butyrate resulted in an inhibition of cell proliferation and, at the same time, an increase in cell diameter (2- to 6-fold) and size of the nuclei (approximately 2-fold) after 3 days in culture. Binucleated cells arose, comprising approximately 12% of the cells investigated, and the number of cells with an abnormal set of chromosomes was increased. Intercellular communication, measured by dye transfer of Lucifer Yellow, was unchanged. From the various xenobiotic metabolizing enzyme activities measured, only those of glutathione S-transferases were significantly altered (increases of 4- to 9-fold) by butyrate treatment. These increases were mainly due to the predominant rise in the pi class isoenzyme which is a well-known tumour marker in rat hepatocarcinogenesis. Thus, our results cannot be interpreted as being either due to promotion of differentiation or due to transformation. The state and type of cell under study has to be considered and investigations of further differentiation parameters are needed to obtain a deeper insight into the biological activity and the underlying mechanisms of cell state modifying agents like butyrate.

Molecular and cellular basis for adequate metabolic design of genotoxicity studies.

Genotoxic species and metabolites are usually under the control of a complex set of activating, inactivating and precursor sequestering enzymes. These enzymes differ greatly between test systems, animal species and man. An adequate metabolic design of genotoxicity studies requires careful attention to factors such as: Dilution of cofactors in in vitro tests which are present in much higher concentrations in the intact cell; Induction in high dose carcinogenicity bioassays of enzymes, which are constitutively not expressed and not induced at such doses of the compound, which occur in the situations of the practical use of the compound; Modifications of control enzymes, which are effected by hormones or other endogenous factors, which are differently influenced by high dose (bioassay) versus moderate dose (real exposure) or by in vivo (endocrine regulation) versus in vitro (no endocrine regulation) conditions.

Xenobiotic-metabolizing enzyme activities in hybrid cell lines established by fusion of primary rat liver parenchymal cells with hepatoma cells.

1. The activities of xenobiotic-metabolizing enzymes were determined in hybrid cell lines (hepatocytoma, HPCT) which have been established by fusion of liver parenchymal cells from adult rat (PC) with cells from a Reuber hepatoma cell line (FAO). 2. Cytochrome P450 was not measurable spectrophotometrically in FAO and HPCT. P450-dependent conversion of testosterone was below the detection limit in FAO and only marginally present in HPCT. 3. Microsomal and cytosolic epoxide hydrolase, glutathione S-transferase and phenol sulphotranserase were low or even below detection limit in FAO. These enzyme activities were significantly higher in HPCT and correspond to about 1-10% the activities measured in PC. 4. 1-Naphthol UPD-glucuronosyl transferase activity was about 20% in FAO and about 100% in HPCT compared to PC. 5. Metabolic conversion of benzo[a]pyrene was low in FAO, high in PC, and intermediate in HPCT. The presented data, however, do not allow the conclusion whether this intermediate rate is catalyzed by similar P450 isoenzymes as in PC. 6. Due to the easily measurable phase II-metabolizing enzyme activities HPCT may, however, be useful for in vitro enzyme induction or repression studies.

Mutagenicity experiments on L-cysteine and D-penicillamine using V79 cells as indicators and for metabolic activation.

Previous studies have shown that cysteine and penicillamine induce gene mutations in Salmonella typhimurium, the effect being strongly potentiated in the presence of mammalian tissue preparations. It has now been demonstrated that homogenate of V79 Chinese hamster cells is an efficient activator of thiol amino acids as well. Nevertheless, L-cysteine and D-penicillamine did not induce gene mutations (acquisition of resistance towards 6-thioguanine) in V79 cells. This was true even in the presence of the most efficient activating system, kidney postmitochondrial fraction. The result suggests the existence of an effective protective system in mammalian cells against the natural amino acid L-cysteine and its therapeutically used derivative, D-penicillamine.

Regio- and stereoselective regulation of monooxygenase activities by isoenzyme-selective phosphorylation of cytochrome P450.

The phosphorylation of the two major phenobarbital-inducible cytochrome P450 isoenzymes IIB1 and IIB2 was increased in hepatocytes by the action of the membrane permeating cAMP derivatives N6-dibutyryl-cAMP and 8-thiomethyl-cAMP. Under these conditions the dealkylation of 7-pentoxyresorufin, a selective substrate of cytochrome P450IIB1 and P450IIB2 was markedly reduced. 16 beta-Hydroxylation of testosterone which is catalyzed specifically only by cytochrome P450IIB1 and IIB2 was strongly reduced; for 16 alpha-hydroxylation which is also catalyzed by cytochrome P450IIB1 and IIB2 but additionally by 3 further cytochrome P450 isoenzymes, this reduction was less pronounced; for the oxidation of the 17 beta-hydroxyl group which besides cytochromes P450IIB1 and IIB2 is additionally catalyzed not only by other cytochromes P450 but also by 17 beta-hydroxysteroid dehydrogenase there was a clear tendency of reduction which, however, no longer reached statistical significance. Hydroxylation at other positions of testosterone which are catalyzed by other cytochrome P450 isoenzymes were not significantly changed. Hence isoenzyme-selective phosphorylation of cytochrome P450 leads to a corresponding isoenzyme-selective modulation of monooxygenase activity which holds promise to be especially important as a fast regulation of the control of genotoxic metabolites.

Phosphorylation of carcinogen metabolizing enzymes: regulation of the phosphorylation status of the major phenobarbital inducible cytochromes P-450 in hepatocytes.

We present data showing that the major phenobarbital inducible cytochromes P-450 (cytochrome P-450IIB1 and cytochrome P-450IIB2) were phosphorylated in intact hepatocytes. This phosphorylation was greatly increased by the cAMP derivatives N6-dibutyryl-cAMP and 8-thiomethyl-cAMP mediated by a cAMP-dependent protein kinase. Most importantly the phosphorylation status of cytochromes P-450 was shown to change in the hepatocytes after treatment with glucagon, which is known to increase the level of cAMP in hepatocytes. The observed impact of the hormone glucagon on the phosphorylation of distinct cytochrome P-450 forms in intact hepatocytes reveals the possibility that the enzyme activity of cytochromes P-450 could be rapidly and differentially regulated by their phosphorylation and therefore dependent on the hormonal status of the organism.

Metabolic perspectives on in vitro toxicity tests.

1. The toxic effects of chemicals arise either from the chemical itself or from metabolites. In either event, the concentration of the molecular species responsible for toxicity is under the control of metabolism. This metabolism is highly variable between animals and humans, and in human populations may exhibit substantial inter-individual differences. The range of human variability can be covered by resorting to a number of animal models. 2. For many purposes, it is desirable to have in vitro procedures to allow detailed examination of mechanisms, but for a long time it was commonly the case that results from in vitro systems were misleading. It was realised that this problem arose at least in part from the absence of appropriate metabolism in many in vitro systems, and to overcome this, it has become common to add metabolizing enzymes in the form of the so-called S9 fraction--the 9000 g supernatant containing both microsomal and cytosolic enzymes--of rat liver. These enzyme preparations contain both the cytochrome P-450 mono-oxygenases and the obligatory NADPH-generating system. This system is thereby optimized for the mono-oxygenase reactions which are frequently responsible for the metabolic activation of toxic chemicals. 3. It is extremely important to appreciate that such systems are highly artificial, being far removed from the situation operating in the intact cell and the whole body. The levels of reactive metabolites in the whole cell are determined by the balance between activating and inactivating enzyme systems. When the S9 fraction is used for metabolism, the detoxication enzymes are essentially inactive, so that a misleading emphasis is thrown upon metabolic activation. Although the inactivating enzymes, such as the glutathione S-transferases, the sulphotransferases and the glucuronosyltransferases, are present in the S9 fraction, their cofactors are diluted so far below the relevant Km values as to render them completely inoperative. This absence of metabolic inactivation in in vitro test systems very commonly underlies observed discrepancies between the results of bacterial mutagenicity tests and carcinogenicity studies in whole animals. 4. Present and future demands upon the toxicologist require the use of rapid in vitro tests for the screening of chemicals, the results of which have predictive value for in vivo adverse effects, e.g. general toxicity, carcinogenicity or mutagenicity. The key role of metabolic information in such procedures is illustrated in this report with reference to the series of methylated derivatives of the polycyclic aromatic hydrocarbon benz(a)anthracene.(ABSTRACT TRUNCATED AT 400 WORDS)

Rat hepatocyte-mediated bacterial mutagenicity in relation to the carcinogenic potency of benz(a)anthracene, benzo(a)pyrene, and twenty-five methylated derivatives.

7,12-Dimethylbenz(a)anthracene, benz(a)anthracene, and benzo(a)pyrene as well as the 24 monomethylbenzo(a)pyrenes (MBPs) and monomethylbenz(a)anthracenes (MBAs), compounds which differ in carcinogenicity from very potent to apparently inactive, were investigated for mutagenicity (reversion to histidine prototrophy) in Salmonella typhimurium TA100 using either intact or NADPH-fortified homogenized rat hepatocytes for metabolic activation. In both systems, all 27 hydrocarbons showed positive responses. Their mutagenic potency in the homogenate-mediated test varied in a narrow range and did not correlate detectably with their reported activity in carcinogenicity experiments. When the cell homogenate was replaced by intact cells, the maximal mutagenic effects were weaker by factors of 1 to 14, depending on the compound, and were seen only at higher substrate concentrations. The differences between cell- and homogenate-mediated mutagenicity were small with the strong carcinogens 7,12-dimethylbenz(a)anthracene 7-MBA, 12-MBA, benzo(a)pyrene, 1-MBP, and 11-MBP. The differences were large with the apparent noncarcinogens and those weak carcinogens that were strongly mutagenic in the homogenate-mediated test. As a result of this differential reduction in activity, the cell-mediated mutagenicity did not correlate with the homogenate-mediated mutagenicity but correlated approximately with the carcinogenic potency. The lowest effects in the cell-mediated experiments were seen with 7-, 8-, 9- and 10-MBP, 2-MBA, and 3-MBA. In these compounds, the methyl group is attached to a carbon of the terminal angular benzo ring, and therefore bay-region diol-epoxides, if formed at all, additionally would carry a methyl group on one of the oxidized positions. On the other hand, among all the compounds tested 7,12-dimethylbenz(a)anthracene, 12-MBA, and 11-MBP, which have the methyl group attached in the bay-region position opposite the terminal benzo ring, showed the highest mutagenic efficacies in the cell-mediated test, as compared to those observed in the homogenate-mediated test. These structure-activity relationships and the previously reported observation that among various promutagenic benzo(a)pyrene metabolites only the 7,8-dihydrodiol was strongly mutagenic in the cell-mediated test would suggest that in the cell-mediated bacterial mutagenicity test, bay-region diol-epoxides are the ultimate mutagens which are preferentially detected.

Metabolizing systems in short-term in vitro tests for carcinogenicity.

Most mutagens require metabolic activation or can be metabolically inactivated. Lipophilic xenobiotics are typically metabolized by introduction and/or modification of a functional group and subsequent conjugation with a hydrophilic endogenous compound to allow excretion. Functional groups are most often introduced by the various cytochromes P-450. The risk that a reactive intermediate is generated is relatively high at this stage. However, many reactive intermediates may be efficiently inactivated by other enzymes, which modulate functional groups or introduce hydrophilic moieties. In other apparently rare cases, activation may also occur during these metabolic steps. Since bacteria and most cultured mammalian cells used as targets in short-term in vitro carcinogenicity tests are largely deficient in the cytochromes P-450, exogenous metabolizing systems (purified enzymes, crude subcellular preparations, intact cells or (in host-mediated tests) whole animals) are routinely used in such tests. Purified enzymes and crude subcellular preparations supplemented with individual or several cofactors are useful in elucidating the enzymatic control of mutagenic metabolites. As crude subcellular preparations are easy to prepare and to store, they are the metabolizing system most frequently used (supplemented with NADPH) in short-term tests. They favour the enzymes preferentially involved in activation. In contrast, intact cells (e.g. freshly isolated hepatocytes) and host animals additionally elicit the various enzyme activities preferentially involved in inactivation, so the results strongly depend on the kind of metabolizing system used. The choice of system plays a pivotal role in tests in bacteria, which are deficient not only in the 'preferentially activating enzymes' but also in the 'preferentially inactivating xenobiotic-metabolizing enzymes'. Mammalian cells in culture retain substantial activity of the latter enzymes. In our experience, results from in vitro mutagenicity tests in which adequate levels of activity of 'inactivating enzymes' are provided, either in the added metabolizing system or in the target cells, correlate better with carcinogenicity than tests in which the 'activating enzymes' are largely favoured.