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The present review describes available evidence about the fate of micronuclei and micronucleated cells. Micronuclei are small, extranuclear chromatin bodies surrounded by a nuclear envelope. The mechanisms underlying the formation of micronuclei are well understood but not much is known about the potential fate of micronuclei and micronucleated cells. Many studies with different experimental approaches addressed the various aspects of the post-mitotic fate of micronuclei and micronucleated cells. These studies are reviewed here considering four basic possibilities for potential fates of micronuclei: degradation of the micronucleus or the micronucleated cell, reincorporation into the main nucleus, extrusion from the cell, and persistence in the cytoplasm. Two additional fates need to be considered: premature chromosome condensation/chromothripsis and the elimination of micronucleated cells by apoptosis, yielding six potential fates for micronuclei and/or micronucleated cells. The available data is still limited, but it can be concluded that degradation and extrusion of micronuclei might occur in rare cases under specific conditions, reincorporation during the next mitosis occurs more frequently, and the majority of the micronuclei persist without alteration at least until the next mitosis, possibly much longer. Overall, the consequences of micronucleus formation on the cellular level are still far from clear, but they should be investigated further because micronucleus formation may contribute to the initial and later steps of malignant cell transformation, by causing gain or loss of genetic material in the daughter cells and by the possibility of massive chromosome rearrangement in chromosomes entrapped within a micronucleus by the mechanisms of chromothripsis and chromoanagenesis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Based on new scientific developments and experience of the regulation of chemical compounds, a working group of the Gesellschaft fuer Umweltmutationsforschung (GUM), a German-speaking section of the European Environmental Mutagen Society, proposes a simple and straightforward approach to genotoxicity testing. This strategy is divided into basic testing (stage I) and follow-up testing (stage II). Stage I consists of a bacterial gene mutation test plus an in vitro micronucleus test, therewith covering all mutagenicity endpoints. Stage II testing is in general required only if relevant positive results occur in stage I testing and will usually be in vivo. However, an isolated positive bacterial gene mutation test in stage I can be followed up with a gene mutation assay in mammalian cells. If this assay turns out negative and there are no compound-specific reasons for concern, in vivo follow-up testing may not be required. In those cases where in vivo testing is indicated, a single study combining the analysis of micronuclei in bone marrow with the comet assay in appropriately selected tissues is suggested. Negative results for both end points in relevant tissues will generally provide sufficient evidence to conclude that the test compound is nongenotoxic in vivo. Compounds which were recognized as in vivo somatic cell mutagens/genotoxicants in this hazard identification step will need further testing. In the absence of additional data, such compounds will have to be assumed to be potential genotoxic carcinogens and potential germ cell mutagens.

Metabolism in fresh and cryopreserved (CP) rat, dog and monkey hepatocyte suspensions and cultures was measured using midazolam (CYP3A), tolbutamide (CYP2C), dextromethorphan (CYP2D) and p-nitrophenol (glucuronosyl S-transferases (UGT), sulphotransferases (ST)). CYP3A, CYP2C9, CYP2D6, UGT and ST enzyme functions in fresh and CP rat, dog and monkey hepatocyte suspensions were retained - CP rat hepatocytes lost some CYP2C activity but this was restored by adding NADPH or by placing the cells in culture, suggesting that the enzyme was still functional. Phase 2 activities were equivalent in fresh and CP hepatocyte suspensions. In some cases, incubation conditions increased the rate of metabolism, possibly reflecting de novo cofactor synthesis. However, this effect was substrate and species dependent and was not always the same in fresh and CP cells. CYP3A, CYP2C, CYP2D, UGT and ST activities at 24 hours of culture of rat and monkey hepatocytes were not compromised by cryopreservation. CYP3A, CYP2D but not CYP2C were lower in 24-hour cultures of CP dog hepatocytes than in fresh cells. Despite being lower than fresh cells, UGT activity in dog CP hepatocytes did not decrease from 0 to 24 hours of culture. Species-specific metabolism of p-nitrophenol could be demonstrated in both CP cell suspensions and cultures. In conclusion, these data suggest that the enzyme characteristics of fresh and CP hepatocytes from each species and under specific incubation conditions should be considered when carrying out metabolism studies of new compounds.

In recent years, assessing the photogenotoxic potential of a compound became an issue for certain drugs and cosmetical products. Therefore, existing methods performed according to international guidelines (e.g. OECD guidelines) were adapted to the use of concurrent UV-visible (UV-Vis) light irradiation for the assessment of photomutagenicity/photogenotoxicity. In this review, photobiological bases of the processes occurring in the cell after irradiation with UV- and/or visible (vis)-light as well as a compilation of testing methods is presented. Methods comprise cell free investigations on naked DNA and in vitro methods, such as the photo-Ames test, the photo-HPRT/photo-mouse lymphoma assay (MLA), the photo-micronucleus test (MNT), the photo-chromosomal aberration test (CA) and the photo-Comet assay. A compilation of the currently available international literature of compounds tested on photogenotoxicity is given for each method. The state of the art of photogenotoxicity testing as well as the rational for testing are outlined in relation to the recommendations reached in expert working groups at different international meetings and to regulatory guidance papers. Finally, photogenotoxicity testing as predictor of photocarcinogenicity and in the light of risk assessment is discussed.

Primary hepatocytes represent a well-accepted in vitro cell culture system for studies of drug metabolism, enzyme induction, transplantation, viral hepatitis, and hepatocyte regeneration. Recently, a multicentric research program has been initiated to optimize and standardize new in vitro systems with hepatocytes. In this article, we discuss five of these in vitro systems: hepatocytes in suspension, perifusion culture systems, liver slices, co-culture systems of hepatocytes with intestinal bacteria, and 96-well plate bioreactors. From a technical point of view, freshly isolated or cryopreserved hepatocytes in suspension represent a readily available and easy-to-handle in vitro system that can be used to characterize the metabolism of test substances. Hepatocytes in suspension correctly predict interspecies differences in drug metabolism, which is demonstrated with pantoprazole and propafenone. A limitation of the hepatocyte suspensions is the length of the incubation period, which should not exceed 4hr. This incubation period is sufficiently long to determine the metabolic stability and to allow identification of the main metabolites of a test substance, but may be too short to allow generation of some minor, particularly phase II metabolites, that contribute less than 3% to total metabolism. To achieve longer incubation periods, hepatocyte culture systems or bioreactors are used. In this research program, two bioreactor systems have been optimized: the perifusion culture system and 96-well plate bioreactors. The perifusion culture system consists of collagen-coated slides allowing the continuous superfusion of a hepatocyte monolayer with culture medium as well as establishment of a constant atmosphere of 13% oxygen, 82% nitrogen, and 5% CO2. This system is stable for at least 2 weeks and guarantees a remarkable sensitivity to enzyme induction, even if weak inducers are tested. A particular advantage of this systemis that the same bioreactor can be perfused with different concentrations of a test substance in a sequential manner. The 96-well plate bioreactor runs 96 modules in parallel for pharmacokinetic testing under aerobic culture conditions. This system combines the advantages of a three-dimensional culture system in collagen gel, controlled oxygen supply, and constant culture medium conditions, with the possibility of high throughput and automatization. A newly developed co-culture system of hepatocytes with intestinal bacteria offers the possibility to study the metabolic interaction between liver and intestinal microflora. It consists of two chambers separated by a permeable polycarbonate membrane, where hepatocytes are cultured under aerobic and intestinal bacteria in anaerobic conditions. Test substances are added to the aerobic side to allow their initial metabolism by the hepatocytes, followed by the metabolism by intestinal bacteria at the anaerobic side. Precision-cut slices represent an alternative to isolated hepatocytes and have been used fo the investigation of hepatic metabolism, hepatotoxicity, and enzyme induction. A specific advantage of liver slices is the possibility to study toxic effects on hepatocytes that are mediated or modified by nonparenchymal cells (e.g., by cytokine release from Kupffer cells) because the physiological liver microarchitecture is maintained in cultured slices. For all these in vitro systems, a prevalidation has been performed using standard assays for phase I and II enzymes. Representative results with test substances and recommendations for application of these in vitro systems, as well as standard operation procedures are given.

The in vivo metabolism of three pharmaceutical compounds, EMD68843, EMD96785, and EMD128130, was compared in fresh and cryopreserved hepatocyte (CPH) suspensions and microsomes from rat, dog, monkey, and human livers and fresh human and rat hepatocyte collagen gel immobilized cultures (GICs). Half of the major in vivo metabolites was produced by phase 1 (hydroxylation, oxidation, hydrolysis, N-dealkylation) and half by phase 2 metabolism (mostly glucuronidation but also sulfation and glycine conjugation). The identity and percentage of phase 1 and 2 metabolites from each compound produced in hepatocytes compared well with that in each species in vivo. Glucuronidation was more extensive in GICs than in CPHs. In contrast, CPHs but not GICs, produced sulfate metabolites. Microsomes (supplemented with NADPH only) produced most of the phase 1 but no phase 2 metabolites. Metabolism in CPHs was the same as in fresh hepatocyte suspensions. Discrete species differences in metabolism were detected by CPHs and microsomes. Cytochrome P450 and glucuronosyl S-transferase contents of CPHs did not account for species differences in the percentage of phase 1 and 2 metabolites or the rate of disappearance of the parent compounds in these cells. These data show a good correlation between major metabolites formed in vivo and in vitro. CPHs and GICs, unlike microsomes, carried out sequential phase 1 and 2 metabolism. Each in vitro system has its own advantages, however, for short-term metabolism studies CPHs may be more useful since they are readily available, easier and quicker to prepare than GICs, and have more comprehensive enzyme systems than microsomes.

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.

The in vitro micronucleus test (MNT) is a useful assay for the detection of mutagenic events on both the chromosomal and the genomic level. The main disadvantage for introducing the in vitro MNT into official test guidelines seems to be the disparity of existing protocols. To contribute to the aim of standardisation, three different methodological approaches of the in vitro MNT with V79 cells were compared: the standard assay using an asynchronically growing mixed cell population, the cytokinesis block (CB) assay and a modified MNT, the so-called mitotic shake-off (MSO) method. V79 cells were thus treated with two known aneugens (colcemide and griseofulvin) and two clastogens (mitomycin C and cyclophosphamide) over various time periods. The cultures of the CB assay were additionally exposed to cytochalasin B (Cyt-B), an inhibitor of cell, but not of nucleus division. After treatment, the cells were harvested and analysed for the appearance of micronuclei (MN). All three assays yielded positive results for all test substances. These results support the suitability of the MNT with V79 cells with regard to the ability to detect the genotoxic potential of both clastogens and aneugens independent of the test protocol applied. Thus, all three methods are appropriate for MN detection, but due to the fact that the application of Cyt-B has no advantages for a cell line like V79 in which nearly all cells undergo a normal cell cycle, its use is not recommended., (Copyright 1999 Elsevier Science B.V.)

1. Dog hepatocytes were cryopreserved at 6 x 10(6) viable cells/ml in a suspension buffer containing 10% DMSO and were stored in liquid nitrogen. 2. The exclusion of trypan blue dye was 96 +/- 2 and 85 +/- 9% in fresh and cryopreserved (CP) hepatocytes, respectively. Albumin synthesis was unaffected by freezing. 3. Ethoxycoumarin and ethoxyresorufin O-deethylase activities were equivalent in fresh and CP hepatocytes. 4. The profile of testosterone metabolism was unaffected by freezing. Total hydroxylase activities were 815 +/- 33 pmol/min/10(6) cells in freshly isolated whole hepatocytes and 463 +/- 24 pmol/min/10(6) CP whole hepatocytes, but they were equivalent in fresh and CP hepatocyte homogenates supplemented with 250 microM NADPH. 5. Phase 2 enzymes were functional in freshly thawed CP hepatocytes but they required exogenous addition of cofactors (20 microM UDPGA and 1.7 microM PAPS). 6. When placed in suspension for longer times, fresh and CP cell viabilities were 88 +/- 6 and 64 +/- 2% after 4 h. ECOD and EROD activities were equivalent in fresh and CP hepatocyte suspensions, over 4 h. Testosterone hydroxylase activities were well maintained in fresh cell suspensions but they declined to 63 +/- 6% of the initial activity after 4 h in CP hepatocytes. 7. These results indicate that CP dog hepatocytes are a suitable in vitro system for xenobiotic metabolism since enzyme functions in CP hepatocytes were stabilized. Cofactors in freshly thawed CP hepatocytes should be measured and controlled for optimal use.

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.

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.

An optimized computer-controlled freezing protocol for the cryopreservation of rat liver parenchymal cells was developed. The best survival rates were obtained when a slow cooling rate was used and when the supercooling was interrupted with a shock cooling to initiate ice nucleation. Ten percent dimethyl sulfoxide was added and removed gradually for best results. Thawed rat liver parenchymal cells had a viability, as judged by trypan blue exclusion, of 69% (SD = 6) versus 82% (SD = 7) for freshly isolated cells. The content and activities of the xenobiotic metabolizing enzymes, cytochrome P450, UDP-glucuronosyl transferase, and microsomal and cytosolic epoxide hydrolase, were not affected, whereas a slight reduction of glutathione S-transferase and sulfotransferase occurred. If cryopreserved cells were purified by a Percoll centrifugation after thawing the enzyme activities were not significantly different from those of freshly isolated parenchymal cells and also the viability was 86% (SD = 3). Cryopreserved rat liver parenchymal cells only metabolized about 50% of benzo(a)pyrene compared to freshly isolated cells. It is less likely that the reduction in enzyme activities was due to the cryopreservation procedure than that it was due to the loss of NADPH as a cofactor for cytochrome P450 which then resulted in the decreased xenobiotic metabolism. This cryopreservation protocol was also suitable for a variety of liver parenchymal cells from other species when trypan blue exclusion was used as a viability marker.

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.

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.

The metabolism of testosterone and benzo(a)pyrene (BaP) which is mediated by diverse enzymes was determined in cryopreserved rat liver parenchymal cells and compared with that found in freshly isolated cells. In addition, the activities of single xenobiotic-metabolizing enzymes were measured by using specific substrates. The cytochrome P450 (P450)-mediated total metabolic conversion of testosterone was reduced to 55% in cryopreserved cells. The metabolite profile, i.e. the formation of single metabolites compared with total metabolic conversion, was however unchanged when compared with freshly isolated cells. A concomitant reduction in the activities of the involved P450 isoenzymes can therefore be postulated. The amount of detected phase I-metabolites of BaP was unaffected by the cryopreservation method. The formation of phase II-metabolites and total metabolic conversion of BaP in cryopreserved cells was however reduced to about 50-60%. The reduced glutathione S-transferase and more obviously phenol sulfotransferase activities measured in cryopreserved cells, may explain the impaired conjugation of BaP. The ratio between phase I- and phase II-metabolites was thus changed by cryopreservation. Density separation on Percoll yielded cryopreserved cells with a viability and metabolic capacity not measurably different from freshly isolated cells. To this extent, cryopreserved, Percoll-purified liver parenchymal cells are a useful in vitro system for drug metabolism studies. However due to the extensive loss in cell number during this procedure (recovery = 22% of freshly isolated cells) the application of this system is limited.

The differentiation status in cultures of primary rat liver parenchymal cells was determined by measuring the activities of various xenobiotic metabolizing enzymes. Most enzyme activities dropped rather rapidly in monocultures of parenchymal cells. The protein content and the activities of cytosolic epoxide hydrolase, glutathione S-transferase, and alpha-naphthol UDP-glucuronosyl transferase were, however, well stabilized in 7-day-old co-cultures of parenchymal cells with two different lines of rat liver nonparenchymal epithelial cells (NEC1 and NEC2). Phenol sulfotransferase and microsomal epoxide hydrolase activity were reduced in this coculture system after 7 days to about 30 and 20% of the initial activity. Generally, higher enzyme activities were measured in co-cultures with one specific epithelial cell line (NEC2) as compared to those with the other line (NEC1). C3H 10T1/2 mouse embryo fibroblasts supported the parenchymal cells even better than the two epithelial lines, because the activity of microsomal epoxide hydrolase was also stabilized. Glutathione transferase activity was increased over time in this co-culture system. Our results show that the differentiation status of liver parenchymal cells was much better stabilized in co-cultures than in monocultures but that, depending on the type of cells used for co-culture, great quantitative differences existed. The entire pattern of xenobiotic metabolizing enzyme activities could not be stabilized at the kind of levels found in freshly isolated parenchymal cells.

Cytochrome P-450 dependent hydroxylation of testosterone was measured in 7-day-old cultures of primary rat liver parenchymal cells. Determinations were carried out in monocultures of parenchymal cells and co-cultures of parenchymal cells with rat liver nonparenchymal epithelial cells, or mouse embryo fibroblasts. In the monoculture system, testosterone metabolism was drastically reduced and hardly measurable after 7 days in culture. In the co-culture systems, individual P-450 isoenzymes were stabilized on different levels. P-450s p and presumably c were well preserved, P-450 a was reduced but clearly measurable, P-450 h was totally lost whereas P-450s b and e were not measurable after 7 days (the activities of these isoenzymes however were already low in freshly isolated parenchymal cells). The results were independent of the cell line used for co-cultivation and of the method of parenchymal cell isolation, that is whether collagenase or EDTA was used as the agent for dissociating the cells from the liver. The results showed that the co-cultivation of liver parenchymal cells with other nonparenchymal cells significantly improved the differentiated status of the former. In this cell culture system however, not every parameter was equally well stabilized.

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.

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)

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.

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.