EDIT: A New International Multicentre Programme to Develop and Evaluate Batteries of In Vitro Tests for Acute and Chronic Systemic Toxicity.

The Multicenter Evaluation of In Vitro Cytotoxicity (MEIC) programme provided a battery of three basal cytotoxicity tests with a good (R2 = 0.77) prediction of human acute lethal blood concentrations. The predictive power of this battery would be considerably improved by the addition of new supplementary in vitro tests. The development of these new tests will be facilitated by a close coupling of test development to evaluation. The Cytotoxicology Laboratory, Uppsala (CTLU), is therefore inviting all interested in vitro toxicologists to take part in the Evaluation-guided Development of In Vitro Toxicity and Toxicokinetic Tests (EDIT). All EDIT activities (subprojects) will be designed on a case-by-case basis, but will follow a common pattern. The CTLU will use the accumulated MEIC/EDIT data, and its experience from the previous MEIC evaluation, to suggest priority areas, i.e. the need for certain in vitro toxicity data/tests as supplements to existing in vitro models/batteries on human systemic toxicity. Detailed research programmes corresponding to these areas will be published on the Internet. The CTLU will also try to raise funds for some projects and will coordinate multilaboratory studies. Interested laboratories developing or already using priority tests are encouraged to join the subprojects and to test specific sets of substances (usually sets of MEIC reference chemicals) in their new assays. The CTLU will provide adequate human reference data and will also evaluate results as single components of complex models, together with the laboratory conducting the test. At present, ten priority areas have been identified: a) repeat dose toxicity in vitro; b) urgent mechanistic information from in vitro studies of protein denaturation, morphology of cell injury, differential toxicity between various rapidly measured endpoints (10-60 minutes) and 24-hour cytotoxicity, toxicity to aerobic cells, and discrimination between rapid and slow cytotoxic mechanisms; c) in vitro tests on vitally important, specific receptor toxicity in humans; d) excitatory cytotoxicity; e) reversibility of cell toxicity; f) in vitro tests on passage across the blood-brain barrier; g) in vitro tests on absorption in the gut; h) protein binding in vitro; i) in vitro tests on distribution volumes (Vd); and j) in vitro tests on biotransformation to more-toxic metabolites (hepatocytes plus target cells). This paper gives a short presentation of the rationale for each subproject and reports on ongoing activities.

Screening for agents inhibiting the mutagenicity of extracts and constituents of tobacco products.

The aim of this study was to screen for potential agents affecting the mutagenicity of tobacco products. The influence of a number of compounds which have been suggested to be antimutagenic some of which are present in tobacco products, was investigated on the mutagenicity of a cigarette smoke condensate (CSC) and, in some cases, an extract of oral Swedish moist snuff (SNUS), using a screening procedure of the Ames Salmonella/microsome assay (STY). For some of the compounds the V79/hprt mutagenicity assay with benzo[a]pyrene metabolites as mutagens was used to obtain complementary and confirmatory information on mammalian cells. The antimutagens used included two selenium compounds, sodium selenite and ebselen; the flavonoids and polyphenols, ellagic acid, (+)-catechin hydrate, scopoletin, chlorogenic acid and rutin trihydrate; the porphyrin derivatives, bovine hemin, biliverdine dihydrochloride, chlorophyllin and a plant extract containing chlorophyll; the terpenoids, beta-carotene, retinol and a mixture of the two epimers (4R) and (4S) of (1S,2E,6R,7E,11E)-cembra-2,7,11-triene-4,6-diols (CBD); and cyclohexanol and ubiquinone. Screening of antimutagenic activities using the STY involves problems with toxicity. In several cases in this study mutagenicity was decreased below the control level without signs of toxicity in the background growth of bacteria. Since the survival of mutants and slight bacteriostatic effects on the background growth cannot be determined accurately in the STY, a reduction in mutagenicity may simply be due to toxicity. Only in cases where a dose-response curve declines to a level at or above the background and then levels off, can toxicity be excluded. An antimutagenic effect determined using this test system is therefore often not sufficient for classifying a compound as antimutagenic until these findings are confirmed in other test systems and, preferably, the mechanism behind this effect is clarified. The results obtained with the selenium compounds were considered to be inconclusive since the reduction in the mutation rate declined below the background level and might only reflect the toxic effects of these compounds. For ellagic acid an almost complete inhibition of the mutagenicity of CSC and SNUS in STY was indicated. This indication of antimutagenicity was confirmed in V79 cells using two metabolites of the CSC constituent benzo[a]pyrene, i.e., trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene and (+)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-oxy-7,8,9,10- tetrahydrobenzo[a]pyrene (BPDE). Chlorogenic acid and (+)--catechin reduced the mutagenicity of CSC and chlorogenic acid also strongly inhibited SNUS mutagenicity. Scopoletin and rutin trihydrate inhibited the mutagenicity of CSC, but showed confounding effects with SNUS.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparison of co-cultivation of V79 cells with rat hepatocytes and rat H4IIE hepatoma cells for studying nitrosamine-induced hprt gene mutations.

The induction of mutations by nitrosamines in the hprt locus of V79 Chinese hamster cells was examined after metabolic activation in a co-cultivation system using either freshly isolated rat hepatocytes or H4IIE rat hepatoma cells and the results obtained were compared with systems which employ the rat liver microsomal fraction (S9-mix). This study was also designed as a first approach to investigating the induction of point mutations by tobacco-specific nitrosamines in mammalian cells in order to obtain information about the significance of these compounds in connection with the carcinogenicity of tobacco smoke. The mutagenicity of two tobacco-specific nitrosamines, 4-(methylnitroso)-1-(3-pyridol)-1-butanone (NNK) and N'-nitrosonornicotine (NNN), were investigated and compared to two extensively investigated nitrosamines, i.e. dimethylnitrosamine (DMN) and diethylnitrosamine (DEN). DMN was activated to mutagenic species by primary hepatocytes at mumolar concentrations, i.e. 1/100 of the concentrations required for mutagenesis by DEN and NNK. NNN was not activated to mutagenic species by liver S9 or primary hepatocytes. The findings shown here on the mutagenicities of NNK and NNN with liver preparations are in agreement with their relative carcinogenic potencies. When the established liver cell line H4IIE was used for metabolic activation, DMN and was found to be mutagenic, whereas the results for NNN were borderline and for DEN and NNK were without effect. The fate of these compounds via different metabolic pathways is discussed in terms of systems for detection of mutagenic metabolites and type of mutation induced.

The cytotoxicity of 50 chemicals from the MEIC study determined by growth inhibition of ascites sarcoma BP8 cells: a comparison with acute toxicity data in man and rodents.

In this study, 50 chemicals selected on the basis of existence of particularly reliable human toxicity data were screened in a cytotoxicity test involving inhibition of the growth of Ascites Sarcoma BP8 cells. These test results are part of an international validation program, the Multicenter Evaluation of In Vitro Cytotoxicity (MEIC), the aim of which is to recommend batteries of in vitro tests to be used for prediction of human toxicity. The cytotoxicities (expressed as the concentrations causing 50% inhibition of cell growth) were compared to acute toxicity data in humans (LDL0) and rodents (LD50), showing the best correlation to rodent data. The results are discussed in relationship to what is usually referred to as basal cytotoxic mechanisms as a cause of in vivo toxicity. It could be concluded that the predicted results on the basis of mechanistic reasoning were not always obtained.

Biotransformation of carbon tetrachloride in cultured neurons and astrocytes.

The ability of brain neuronal cells to metabolize carbon tetrachloride (CCl(4)) has been studied in an attempt to explain earlier observed toxic effects of CCl(4) on these cells. The expression of cytochrome P-450, the glutathione (GSH) content and the activity of glutathione-S-transferase (GST) were measured in cultured neurons and astrocytes from chick embryo cerebral hemispheres. The metabolism of CCl(4) in the neuron and astrocyte cultures was also assessed by determining the formation of: CCl(2) in membrane preparations of these cells. In the membrane fractions of neurons and astrocytes, no measurable levels of cytochrome P-450 were observed. Nevertheless, neurons as well as astrocytes had a capacity for the metabolism of CCl(4). The metabolic capacity of the neurons was significantly greater than that of the astrocytes. The neuron cultures had a higher initial content of GSH and a higher control activity of GST than had the astrocytes. Neither the GSH level nor GST activity were significantly affected in the neuron cultures after exposure to CCl(4). In astrocyte cultures 2 mm CCl(4) slightly depleted the GSH level and significantly induced GST activity. At 3 mm CCl(4), GSH was depleted by 30% and by more than 50% at 4 mm CCl(4). It can be concluded that the metabolic activation of CCl(4) was higher in neurons than in astrocytes. This can explain the earlier observation of CCl(4)-induced lipid peroxidation in cultured neurons. Moreover, neuron GSH was not able to protect these cells against CCl(4)-induced peroxidative damage. In the astrocytes, on the other hand, GSH and GST appeared to have a role in detoxification of CCl(4).

Glutathione content, glutathione transferase activity and lipid peroxidation in acrylamide-treated neuroblastoma N1E 115 cells.

Acrylamide is a well known neurotoxic compound that produces central and peripheral distal axonopathy. Degenerative changes of this type can be induced in the neuroblastoma cell line C 1300, clone N1E 115 and have been extensively studied in our laboratory particularly with regard to the interference by acrylamide with cellular metabolism. In the present study the mechanism of acrylamide-induced neurite degeneration in N1E 115 cells is elucidated further. Acrylamide concentrations were selected that were not cytotoxic but caused an increasingly severe neurite degeneration. The rate of protein synthesis was decreased in a concentration-dependent manner in response to acrylamide exposure (0-2.5 mm). Detoxification of acrylamide in vivo occurs mainly through conjugation with glutathione, (GSH) both non-enzymatically and enzymatically by glutathione S-transferases (GST). Cells grown in the presence of acrylamide showed a concentration-dependent decrease in GSH content. At the highest acrylamide concentration tested this was accompanied by an increased GST activity. Despite the reduced level of GSH and possible impaired protection of the plasma membrane against oxidative stress no elevated level of lipid peroxidation could be observed in the acrylamide-treated cells.

Metabolism of polycyclic aromatic hydrocarbons to mutagenic species by rat and porcine ovarian granulosa cells: detection by cocultivation with V79 Chinese hamster cells.

The capacity of ovarian granulosa cells from rat and pig to release reactive metabolites produced from polycyclic aromatic hydrocarbons with the ability to cause mutations in neighbouring cells has been studied. For this purpose we have used cocultivation with V79 Chinese hamster cells as a detection system. The cells were treated with two different polycyclic aromatic hydrocarbons (PAHs), 7,12-dimethylbenz(a)anthracene (DMBA) or (-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene (BP-7,8-diol). The resulting mutation frequency in the V79 cells after cocultivation, as a function of granulosa cell number, DMBA, or BP-7,8-diol concentration and time, was determined. The progesterone concentration in the medium after cocultivation was also analyzed as a measure of the differentiation of the granulosa cells. These studies demonstrate an increasing frequency of mutations in the V79 cells with an increasing number of granulosa cells. The maximal number of mutations were achieved with a DMBA or BP-7,8-diol concentration of 5 or 2 microM, respectively. The optimal cocultivation time was 24 h. These results clearly show that the granulosa cells can bioactivate PAHs to reactive metabolites with the capacity to migrate into surrounding cells and cause mutations in these cells. Compounds metabolized to mutagenic products by granulosa cells might thus cause mutations in the neighbouring germ cells, with possible consequences for the offspring.

Studies on glutathione transferases belonging to class pi in cell lines with different capacities for conjugating (+)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene.

The glutathione transferases (GST) belonging to class pi are primarily responsible for the intracellular detoxification of the highly mutagenic and carcinogenic compound (+)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). The aim of the present investigation was to study the nature and function of the GST pi gene in relation to the mutagenicity of BPDE in different cell lines. The studies were performed on three cell lines commonly used in toxicological studies, i.e. rat hepatoma cells (H4IIE), human mammary carcinoma cells (MCF-7) and Chinese hamster lung fibroblasts (V79). Western blotting with antisera against GST pi revealed a high level of reaction with cytosol from V79 and H4IIE cells. Furthermore, cytosol from the V79 cells demonstrated low levels of GSTs belonging to the alpha and mu classes, suggesting that a considerable portion of the total capacity of these cells to conjugate chlorodinitrobenzene (CDNB) was provided by GST pi. The level of mRNA for GST pi, as measured by Northern blots, was high in V79 and H4IIE and undetectable in the MCF-7 cell line. Analysis of the DNA fragment patterns using a series of restriction enzymes, revealed that all three cell lines have the pi class gene, although with different band patterns. The findings with H4IIE and MCF-7 cells with respect to their expression of the GST pi gene and their ability to conjugate BPDE were in agreement with the mutagenic effects of BPDE, produced by metabolic activation of (-)-7 beta, 8 alpha-dihydroxybenzo[a]-pyrene in the cells. In contrast, V79 cells although expressing high levels of GST pi, showed no ability to conjugate BPDE or to inhibit the mutagenicity of this compound. Based on these results, we suggest that V79 Chinese hamster lung cells contain a GST pi with a different substrate specificity from those of the human and rat GST pi enzymes.

Chlorophyllin is both a positive and negative modifier of mutagenicity.

The mechanism responsible for the modification of mutagenicity by chlorophyllin has been investigated using mutagenic compounds with different mechanisms of action, including the monofunctional alkylating agents, N-methyl-N'-nitrosourea (MNU) and ethylmethanesulphonate (EMS); nitrosamines related to tobacco products, i.e. dimethyl-nitrosamine (DMN), N-nitrosonornicotine (NNN) and 4-(N-methyl-N-nitrosoamino)-1-(3-pyridinyl)-2-butanone (NNK); the polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (B[a]P) and two of its metabolites, i.e. (-)-7 beta,8 alpha-dihydroxy-7,8-dihydrobenzo[a]pyrene (7,8-diol) and (+)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-oxy-7,8,9,10- tetrahydrobenzo[a]pyrene (BPDE); and a complex mutagenic mixture, an extract and subfractions of Swedish moist oral snuff (SMOS). Mutagenicity was monitored with the Ames Salmonella/microsome assays (STY) and hprt V79 point mutation assay (V79). The effects of chlorophyllin on the mutagenicity of the nitrosamines in the STY assays were found to be complex. In the presence of either NNN or NNK, low concentrations of chlorophyllin actually potentiated the mutagenicity > 2-fold. However, at higher, but still non-toxic concentrations, chlorophyllin decreased the mutagenicity of both compounds. The same type of dose-response relationship for chlorophyllin was indicated in the V79 assay system with DMN, although the effect was much weaker. The results with STY were further confirmed by replacing chlorophyllin with another porphyrin compound, hemin. In contrast, biliverdin, a porphyrin structure without the central metal ion, was unable to potentiate the mutagenicity of NNK in STY.(ABSTRACT TRUNCATED AT 250 WORDS)

Genotoxicity testing of extracts of a Swedish moist oral snuff.

The present study was designed to investigate the potential genotoxicity of aqueous and methylene chloride extracts of Swedish moist oral snuff. The test systems were selected to provide optimal data for the prediction of carcinogenicity in rodents and included assays for the induction of mutation in bacteria, sister-chromatid exchanges (SCE) in human lymphocytes, of chromosome aberrations and gene mutations in V79 Chinese hamster cells and of micronuclei in mouse bone marrow cells. In addition, the methylene chloride extract was tested for the induction of sex-linked recessive lethal mutations in Drosophila melanogaster. The aqueous extract of 'Snus' induced SCE in human lymphocytes and chromosome aberrations in V79 cells, the latter effect being observed both with and without metabolic activation. No induction of point mutations was detected with the Ames test or in V79 cells and the micronucleus test in mice was negative. It was demonstrated that the induction of chromosome aberrations without metabolic activation may be due to a high salt concentration, indicating that the clastogenic agent(s) in this extract required metabolic activation. The methylene chloride extract showed genotoxicity in the Ames test, the SCE test and the chromosome aberration test, whereas no induction of gene mutations in V79 cells was observed. Once again, the results suggested that metabolism is required for genotoxicity. The methylene chloride extract did not cause induction of micronuclei in mice or of sex-linked recessive lethal mutations in Drosophila melanogaster. These combined data on genotoxicity were analyzed using various models for the prediction of carcinogenicity. In a sequential testing model, the probabilities that the aqueous and methylene chloride extracts of 'Snus' are carcinogenic due to a genotoxic mechanism were both predicted to be low. Using carcinogenicity prediction by battery selection (CPBS), the probabilities of the methylene chloride and aqueous extracts being correctly identified as non-carcinogens are 71 and 77%, respectively. Up to date, the CPBS approach has been validated primarily for individual compounds, so some caution should at present be exercised in interpreting the results using this method. Based on these results, the carcinogenic potential of Swedish 'Snus' should be considered to be low, a conclusion in agreement with the low incidence of oral cancer in Sweden compared to other countries.

Thiol-enhanced decomposition of MNNG, ENNG, and nitrosocimetidine: relationship to mutagenicity in V79 Chinese hamster cells.

The nitrosated form of cimetidine (Tagamet), nitrosocimetidine (NC), belongs to a group of nitrosoamidines in which the mutagenic and carcinogenic properties of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) have been studied in detail. The common mechanism of action of these compounds is that nucleophilic atoms can attack their iminocarbon, thereby leading to the formation of alkyldiazohydroxide and, subsequently of an alkylating and mutagenic diazonium ion. A competitive, non-mutagenic pathway involves denitrosation, which is strongly dependent on pH and can be enhanced by glutathione transferase. The influence of different thiols (e.g. glutathione and the L- and D-forms of N-acetylcysteine (L-NAC and D-NAC respectively] at different extra- and intracellular concentrations on the mutagenicity of these nitrosoamidines in V79 cells has been studied in the present investigation. The results demonstrate that the mutagenicity of MNNG and ENNG is highly dependent on where their reaction with thiols takes place. Thus, an increase in the intracellular glutathione level in combination with treatment with MNNG (or ENNG) in thiol-free medium elevated the mutagenicity, whereas treatment with thiols in the medium reduced mutagenicity. The mutagenicity of NC was, on the other hand, only slightly affected by increasing extra- or intracellular thiol levels. The dependence of NC-induced mutagenicity on thiols was indicated, however, by the finding that depletion of intracellular glutathione reduced this mutagenicity almost completely. The effects of treatments with thiols alone or in combination with glutathione transferases suggest that, under our assay conditions (e.g. physiological pH and thiol levels, in combination with low levels of the nitrosoamidines), no denitrosation occurs. On the contrary, our results indicate that intracellular thiols, and possibly glutathione transferases, potentiate the production of mutagenic species from these nitrosamidines.

The importance of glutathione and glutathione transferase for somatic mutations in Drosophila melanogaster induced in vivo by 1,2-dichloroethane.

Two principal pathways of metabolism of the carcinogenic compound 1,2-dichloroethane (DCE) have been proposed. One is a mixed function oxidase dependent pathway requiring oxygen and NADPH. The other pathway depends on the presence of glutathione (GSH) and glutathione transferase (GST). The aim of this study was to investigate the role of the latter pathway for the in vivo mutagenicity of DCE in the somatic wing spot test in Drosophila melanogaster. DCE caused a dose-dependent increase of wing spots. In order to investigate the role of cellular GSH for the mutagenicity, the level of GSH was decreased by 24 h pretreatment with buthionine sulfoximine (BSO), an efficient inhibitor of GSH synthesis. This pretreatment decreased the GSH level to approximately 6% as compared to the control. The pretreatment also resulted in a significant decrease of the mutagenicity of DCE. Treatment of the larvae with phenobarbiturate (PB) resulted in approximately 200% induction of cytosolic GST, and a corresponding increase in the DCE mutagenicity. These results indicate that the important pathway in vivo for the mutagenicity of DCE is dependent on GSH and GST. A similar experimental protocol was used to study interactions between aflatoxin B1 (AFB) and GSH and GST. No effect of the treatment with BSO on the mutagenicity of AFB was observed, while pretreatment with PB caused a decrease of the mutagenicity of AFB.

Preliminary results from the Scandinavian multicentre evaluation of in vitro cytotoxicity (MEIC).

The multicentre evaluation study of in vitro cytotoxicity tests (MEIC) is organized by the Scandinavian Society of Cell Toxicology. All interested laboratories are invited to test a published list of 50 reference chemicals in their various in vitro assays with a bearing on general toxicity. Submitted results will be centrally evaluated for their relevance to human toxicity, including a comparison with the efficiency of conventional animal tests. This brief communication presents the very first preliminary results of the study, that is, prediction of human acute lethal toxicity for the first 10 MEIC chemicals by all the results submitted to date, that is, five in vitro cytotoxicity assays. As a baseline for judging the efficiency of the cytotoxicity tests, rat and mouse LD(50) values were compared with human acute lethal dosage of the chemicals. Rat LD(50) prediction was relatively poor, but mouse LD(50) values correctly predicted the human lethal dose for six out of the 10 substances. A multivariate method of comparison including all cytotoxicity test results, predicted human lethal blood concentrations as well as the mouse LD(50) prediction of dosage. Since the blood concentrations used in the comparison were derived from human lethal dosage with the help of two simple pharmacokinetic factors (absorbed fraction in the intestine and distribution volume of chemicals), the cytotoxicity assays were found also to be able to predict human dosage, as well as did the mouse LD(50) prediction.

MEIC--a new international multicenter project to evaluate the relevance to human toxicity of in vitro cytotoxicity tests.

A new international project to evaluate the relevance for human systemic and local toxicity of in vitro tests of general toxicity of chemicals has been organized by the Scandinavian Society of Cell Toxicology under the title Multicenter Evaluation of In Vitro Cytotoxicity (MEIC). The basic assumptions underlying the project, as well as the practical goals and the design of the program are outlined. The list of the first 50 reference chemicals is presented. The chemicals are an otherwise unbiased selection of compounds with known human acutely lethal dosage and blood concentrations, including LD50-values in the rat or mouse. Most agents also have other data on human toxicity and toxicokinetics, including more extensive animal toxicity data. International laboratories already using or developing in vitro tests of various partial aspects of general toxicity are invited to test the substances, the results of which will be evaluated by us. The predictivity of the in vitro results for both partial and gross human toxicity data will be determined with combined use of univariate regression analysis and soft multivariate modeling. The predictivity of the in vitro results will be compared with the predictivity of conventional animal tests for the same chemicals. Finally, batteries of tests with optimal prediction power for various types of human toxicity will be selected. The need for and possible uses of such batteries are discussed.

Effects of glutathione transferase activity on benzo[a]pyrene 7,8-dihydrodiol metabolism and mutagenesis studied in a mammalian cell co-cultivation assay.

This study deals with the role of glutathione transferase (GST)-mediated conjugation of (+)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-oxy-7,8,9,10- tetrahydrobenzo[a]pyrene (BPDE) in two mammalian cell lines, human mammary carcinoma cells (MCF-7) and rat hepatoma cells (H4IIE), in relation to their capacity to metabolize (-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene [(-)-BP-7,8-diol] to products that induce mutations in co-cultivated V79 cells. Both MCF-7 and H4IIE cells metabolized (-)-BP-7,8-diol to BPDE, but mutations in co-cultivated V79 cells were only detected with MCF-7 cells. However, depletion of glutathione (GSH) in H4IIE cells increased the mutagenicity of (-)-BP-7,8-diol to a similar level to that found with MCF-7 cells. Measurements of GST activity using GSH and post-microsomal supernatants from H4IIE, V79 and MCF-7 cells indicated a substantial difference in conjugation capacity. Although preparations from all three cell-lines showed GST activity with 1-chloro-2,4-dinitrobenzene as the substrate, GST activity towards BPDE could only be detected in supernatants from H4IIE cells. This is consistent with the presence of GST 7-7 an isoenzyme highly efficient in catalysing BPDE-GSH conjugation. The difference in GSH-conjugation activity towards BPDE was confirmed using intact H4IIE and MCF-7 cells in culture. These results indicate that GSH-conjugation plays a pivotal role in mutagenesis induced by polycyclic aromatic hydrocarbons (PAH). Accordingly, a deficiency in GSH-conjugation capacity may be regarded as one important factor in defining a target cell population with an increased risk for tumour initiation following exposure to PAH.

Mechanism of N-acetylcysteine (NAC) and other thiols as both positive and negative modifiers of MNNG-induced mutagenicity in V79 Chinese hamster cells.

Carcinogenic xenobiotics can be detoxified by nucleophilic thiols, which interact directly or through enzyme catalyzed reactions with electrophilic metabolites/compounds or metabolically produced oxidants. Formation of such conjugates is assumed to be a dominating competitive pathway reducing the mutagenic and carcinogenic effects of several known carcinogens. In the case of the potent carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) the situation is different since this carcinogen is transformed to reactive intermediates by nucleophilic agents such as thiols. As a consequence of this, the modulating effect of thiols has to be discussed in relation to the reaction kinetics of this nitroso compound. In this report we present data on the mutagenicity of MNNG which was enhanced by intracellular excess of glutathione, achieved by pre-treatment with N-acetylcysteine, cysteine and glutathione. Depletion of GSH by treatment with diethylmaleate or buthioninesulfoximine resulted in a decreased mutagenic effect of MNNG. A decreased effect of MNNG was also obtained by extracellular excess of thiols. The modification of MNNG-mutagenicity was compared to the effect of thiols on the mutagenicity of N-methyl-N'-nitrosourea (MNU) in V79 Chinese hamster cells. No effect of the thiols could be detected on the mutagenicity of MNU, indicating that the intermediates of MNU and MNNG react, in agreement with the reaction kinetics, in favour of water and thiols become less important. The results indicate that the activation of MNNG to mutagenic intermediates is occurring within the cells. This activation is mediated by reaction of MNNG with thiols (and amines) and subsequent release of short-lived alkylating intermediates. The mutagenic effect of MNNG can be reduced or enhanced respectively by decreasing or increasing the intracellular thiol levels. As demonstrated with MNU, intracellular thiol concentrations (in milli/molar range), which is high enough to bring about the activation of MNNG, are not sufficiently high to protect DNA from damage by the alkylating intermediate. Extracellular levels of thiols 'protect' the cells simply by increasing the decomposition of MNNG in the treatment solution.

Mutagen levels in urine from snuff users, cigarette smokers and non tobacco users--a comparison.

The mutagenic activity of concentrates of urine from snuff users, cigarette smokers and non tobacco users has been investigated. A concentration procedure involving use of Sep-Pak C18 columns and elution with methylene chloride was used. The concentrates were assayed for mutagenicity towards strain TA98 of Salmonella typhimurium, both in the presence and absence of a metabolic activation system, the post-mitochondrial liver fraction (S9) from Aroclor 1254 induced rats. The mean mutagenic activity of smokers' urine concentrates was 8.6 X 10(3) revertants per 24 h and significantly higher than the corresponding values for snuff users, abstinent snuff users and non tobacco users, which were (1.3, 1.3 and 0.9) X 10(3), respectively. No significant difference in mutagenic activity was found between urine from snuff users, whether using or abstaining from snuff, and urine from non tobacco users. It could thus be concluded that the level of urinary mutagens, isolated by adsorption on Sep-Pak C18 columns, is not elevated by habitual usage of Swedish wet snuff.

Mutagenicity studies by co-cultivation of bronchoalveolar cells and blood lymphocytes with V79 Chinese hamster cells.

Human bronchoalveolar cells, consisting of approximately 85% pulmonary alveolar macrophages (PAMs), and peripheral blood lymphocytes isolated from healthy volunteers were investigated for their ability to metabolize 7,8-diol of benzo[a]pyrene (B(a)P). The mutagenicity of reactive metabolites was analyzed by employing a co-cultivation system using V79 Chinese hamster cells for the detection of mutations. The metabolic activity of the human cells was compared to PAMs isolated from rabbits. The number of PAMs obtained by bronchoalveolar lavage of smokers was found to be significantly elevated compared with nonsmokers. However, the mean number of induced mutations of the 7,8-diol mediated by PAMs during co-cultivation did not differ significantly between smokers and nonsmokers. The aryl hydrocarbon hydroxylase (AHH) activity of human lymphocytes has been studied by others, but to the best of our knowledge this is the first demonstration that human blood lymphocytes could be successfully used in a co-cultivation assay for the characterization of xenobiotic metabolism in terms of mutations, as illustrated by the linear increase of induced mutations in the V79 cells. Rabbit PAMs were less efficient in mediating mutations as compared to both smokers' and nonsmokers' PAMs or lymphocytes. This can probably be explained by less efficient bioactivation of 7,8-diol in rabbit PAMs, which is supported by the fact that the rabbit PAMs metabolized B(a)P in a different way as compared to human PAMs as revealed by HPLC analysis of ethyl acetate extractable metabolites of 3H-B(a)P. No qualitative or quantitative difference in the patterns of B(a)P metabolism by PAMs isolated from smokers and nonsmokers could be established. In conclusion, human PAMs were found to be more efficient in terms of cell-mediated mutagenicity than human lymphocytes, which are more efficient than rabbit PAMs. The present results differ from previous reports concerning the xenobiotic metabolizing capacity of these cells assessed by other methods. This illustrates the usefulness of the co-cultivation assay, because it measures not only the bioactivating capacity of isolated mammalian cells, but also their detoxifying capacity, the transfer of mutagens to other cells and the ability of their metabolites to cause mutations.