Development of an in vitro test battery for the estimation of acute human systemic toxicity: An outline of the EDIT project. Evaluation-guided Development of New In Vitro Test Batteries.

The aim of the Evaluation-guided Development of new In Vitro Test Batteries (EDIT) multicentre programme is to establish and validate in vitro tests relevant to toxicokinetics and for organ-specific toxicity, to be incorporated into optimal test batteries for the estimation of human acute systemic toxicity. The scientific basis of EDIT is the good prediction of human acute toxicity obtained with three human cell line tests (R(2) = 0.77), in the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) programme.

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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 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).

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.

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.

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.

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.

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.

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.

Rabbit alveolar macrophage-mediated mutagenesis of polycyclic aromatic hydrocarbons in V79 Chinese hamster cells.

Rabbit pulmonary alveolar macrophages (PAM) were used as a metabolizing device in combination with V79 Chinese hamster cells as a mutational indicator system. The capacity for metabolic activation of benzo[a]pyrene (B[a]P), its 7,8-diol and 2-aminoanthracene by PAM was investigated. Because of the high variation between different PAM preparations, a statistically significant effect of the 3 compounds could only be demonstrated in a series of 4 or 5 experiments.

Effects of air pollutants on the oxidative metabolism and phagocytic capacity of pulmonary alveolar macrophages.

Isolated rabbit pulmonary alveolar macrophages were found to be a convenient biological model system, relevant for studies of the toxicity of air pollutants. The phagocytic capacity and the oxygen consumption were used as test parameters and studied simultaneously on the same cells. The toxicity of extracts of airborne particles (phi less than 15 microns) collected in urban and rural areas was investigated and compared to a cigarette-smoke condensate.