Lennart Romert

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 1Multicenter 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.TABLE 1The First 50 Reference Chemicals of the MEIC Project1. Acetaminophen26. Arsenic trioxide2. Aspirin27. Cupric sulfate3. Ferrous sulfate28. Mercuric chloride4. Diazepam29. Thioridazine HCl5. Amitriptyline30. Thallium sulfate6. Digoxin31. Warfarin7. Ethylene glycol32. Lindane8. Methyl alcohol33. Chloroform9. Ethyl alcohol34. Carbon tetrachloride10. Isopropyl alcohol35. Isoniazid11. 1,1,1-Trichloroethane36. Dichloromethane12. Phenol37. Barium nitrate13. Sodium chloride38. Hexachlorophene14. Sodium fluoride39. Pentachlorophenol15. Malathion40. Verapamil HCl16. 2,4-Dichlorophenoxyacetic acid41. Chloroquine pholphate17. Xylene42. Orphenadrine HCl18. Nicotine43. Quinidine sulfate19. Potassium cyanide44. Diphenylhydantoin20. Lithium sulfate45. Chloramphenicol21. Theophylline46. Sodium oxalate22. Dextropropoxyphene HCl47. Amphetamine sulfate23. Propranolol HCl48. Caffeine24. Phenobarbital49. Atropine sulfate25. Paraquat50. Potassium chloride

Palmitoyl coenzyme A: A possible physiological regulator of nucleotide binding to brown adipose tissue mitochondria

The requirement for extramitochondrial purine nucleotides in order to obtain energy conservation in freshly isolated brown adipose tissue mitochondria is now well documented [1 -3 ] . The specificity of the action correlates well with the effect of these nucleotides on inhibition of passive anion permeability in these mitochondria [4,5]. This inhibition has been demonstrated for chloride, bromide and nitrate but not for phosphate [4]. The site of action of the nucleotides has been shown to be outside the mitochondrial matrix [3,6] and thus, if the effect is of physiological importance, must be elicited by cytoplasmic nucleotides. Their action could be envisaged as inhibition of hydroxyl ion permeability. The concentrations of these nucleotides in vivo are well in excess of those required to accomplish energy conservation or inhibition of anion permeability in vitro [3,4,6]. It is therefore necessary to demonstrate the existence of a physiological antagonist to the nucleotide binding, which would allow a high effective proton conductance (alternatively hydroxide ion permeability) giving loose coupling and consequently thermogenesis, in spite of high cytoplasmic nucleotide concentrations. As stated above, the nucleotide action on energy conservation correlates well with the inhibition of passive anion permeability. This ion permeability can be visualised as mitochondrial swelling in suitable media. It was therefore decided in this study to con-

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.

Morphology and biochemical properties of perirenal adipose tissue from lamb (Ovis aries). A comparison with brown adipose tissue

Abstract 1. 1. Perirenal adipose tissue from lamb resembles brown adipose tissue from other species in many respects: morphological appearance; double sympathetic innervation; mitochondrial energy conservation; pattern of fatty acid oxidation. 2. 2. The tissue differs significantly from brown adipose tissue in having a high mitochondrial ATP synthetic capacity; elementary particles on the mitochondrial inner membrane; possessing the specific mitochondrial ATPase inhibitor protein. 3. 3. The simple delineation of a definition for brown adipose tissue is not possible. 4. 4. The tissue appears useful in studying the postulated interconvertibility of brown and white adipose tissue.

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)

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.

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.

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.

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. When the ability of PAM to metabolize B[a]P and the 7,8-diol to mutagenic products was compared with that of primary embryonic fibroblasts from Syrian hamsters, PAM were found to be one-tenth as efficient. Experiments were performed to find out how the phagocytic process could affect the metabolic activation of polycyclic aromatic hydrocarbons. The results showed that PAM-mediated mutagenesis of the 7,8-diol was enhanced 5-10-fold if PAM were fed with opsonized particles. The mechanism by which the phagocytosis of PAM enhanced the mutagenicity of 7,8-diol, as detected in co-cultivated V79 cells, can so far only be a matter for speculation.

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).