Co-cultivation of cells as a promising tool in cancer research in vitro

Epidemiological data show that in western societies I out of 3 persons gets cancer and I out of 4 persons dies of cancer. This makes cancer, next to heart and vascular diseases, a major cause of death. There are three major factors contributing to the cancer death rate in humans:1. ionizing radiation2. certain viruses3. chemicalsIntensive research efforts for many decades have led to the development of a generalized concept which implies that the process of tumor formation proceeds stepwise. This stepwise development includes both genetic and epigenetic factors.In this thesis studies are described which were aimed at the development of an in vitro system enabling the study of the effects of compounds on various important aspects of the cancer process. In the first chapter a description is given of the cancer process as it emerges from epidemiological data and data from studies with experimental animals. The process can be divided in three more or less distinct phases: the initiation phase, the promotion phase and the progression phase. Within the concept presented, the role of intercellular communication was emphasized and inhibition of intercellular communication has been described as an essential step in tumor promotion. In addition the role of drug metabolism in the formation of reactive intermediates is described briefly and the advantages of the principle of using intact cells, compared with subcellular fractions is discussed. Finally the objectives of the investigations are presented.In chapter II the principles of the test methods used in the investigations are described, including methods determining interactions of chemicals with DNA of cells as well as methods designed to study effects of compounds on intercellular communication.Chapter III deals with the relation between the sensitivity of the co- cultivation system, the genetic endpoint and the type of xenobiotic. It was shown that the use of primary chick-embryo hepatocytes as an activation system showed the best performance compared with subcellular fractions from chick-embryo liver and rat liver pretreated with Aroclor 1254. Induction of SCEs in V79 Chinese hamster cells is no good endpoint to determine the potential of cigarette-smoke condensate (CSC) to interact with DNA. The small but consistently observed increase in the number of SCEs after exposure could not be ascribed to a reaction of CSC with DNA of V79 cells. In contrast, the complex mixture was clearly positive in the test on forward mutation at the HGPRT locus.When a co-cultivation system is used to study the interactions of reactive intermediates with DNA of cells, the transport of reactive intermediates is of vital importance for an adequate measurement of these interactions. Any factor disturbing the transport from drug metabolizing cells to target cells influences the outcomtive intermediates. From the data obtained with time-course studies and those obtained with different mutagens; it was concluded that this type of transport is not restricted to specific types of ultimate mutagens but is a phenomenon of a more general nature. If this transport occurs in vivo this might have consequences as to which cells in a given tissue are at risk to become neoplastic upon exposure to mutagens. For example, drug metabolizing cells like the primary chick-embryo hepatocytes are protected more efficiently against toxic intermediates than non metabolizing cells like the V79 cells.Definitive evidence that functional heterologous gap junctions are formed between the two cell types used in the co-cultivation system is given in chapter V.The purpose of the studies presented in this chapter was twofold. The first aim was to investigate whether functional gap junctions are really formed between the two cell types and the second aim was to study the possibility to develop a test system measuring inhibition of metabolic cooperation using the co-cultivation system. The presence of gap junctions was visualized with transmission electron microscopy. The formation of heterologous gap junctions was difficult to determine. In contrast to homologous gap junctions, the heterologous gap junctions were very small and could be recognized merely by the closeness of the opposing membranes. Studies on the transfer of labeled nucleotides showed unequivocally that the heterologous gap junctions were functional. Addition of the phorbolester TPA inhibited this transfer, which was accompanied by the disappearance of all gap junctions. Upon longer treatment times transfer was partly restored and also morphologically the gap junctions reappeared. It was shown that this adaptation is associated with the mitotic index of the cells.A model system was developed, enabling the study of the role of drug metabolism in the formation of compounds with the potential to inhibit intercellular communication. This model is based on the use of a mutant (HGPRT-) V79 cell line which lacks the capacity to phosphorylate hypoxanthine. These mutant V79 cells are co-cultured with primary chick- embryo hepatocytes in the presence of [3H] hypoxanthine. The HGPRT- V79 cells are not capable of phosphorylating this hypoxanthine and cannot incorporate the label in the genetic material. Only when the hypoxanthine is phosphorylated by the primary chick-embryo hepatocytes and is transported through gap junctions to neighbouring HGPRT-V79 cells, incorporation will occur. Incorporation of label is visualized with auto radiographic methods and the accurate number of grains can be determined by counting them under the microscope. When TPA and CSC were tested with this method, both were able to inhibit this transfer. When the effect of CSC on disappearance of gap junctions was investigated at cytotoxic concentrations, no decrease in the number of gap junctions was found. In the same system the complex mixture inhibits metabolic cooperation. Therefore the conclusion was drawn that inhibition of intercellular communication can be accomplished along different metabolic pathways.When dime thy lbenzanthracene, an indirect acting carcinogen was tested at non-cytotoxic concentrations, it inhibited the transfer of labeled nucleotides completely. No effect was found when the compound was tested in the assay using the transfer of a poisonous base analogue between two types of V79 cells. Since V79 cells lack the capacity for drug metabolism it was assumed that not the parent compound but one or more of its metabolites is responsible for the observed effects. This compound is the first example of an indirect acting inhibitor of intercellular communication which emphasizes the importance of the use of adequate drug metabolizing systems.Chapter VI deals with studies on cancer-modulating effects of various compounds. These studies comprize investigations towards enzyme inducing capabilities and the effects of pretreatment on SCE induction by mutagens from different chemical classes. In addition the effects of pretreatment on benzo(a)pyrene **B(a)P¼ metabolism were studied in detail.In the first part the effects of two naturally occurring indoles, indole-3-carbinol (13C) and indole-3-acetonitril (13A) were examined. Both compounds induced the cytochrome P450 associated enzymes EROD and ETCO in cultured primary chick-embryo hepatocytes. Induction was observed also for UDPglucuronyltransferase whereas no Induction of glut athione-S-transferase was found. 13C was a better inducer than 13A; the increases in enzyme activity were larger and obtained at a lower concentration than that of M.The effects of pretreatment with the indoles on SCE induction by mutagens from different chemical classes were studied. Pretreatment with 13C resulted in a decrease in SCE induction for B(a)P and dimethy1nitrosamine (NDMA). No difference was found for 2- aminoanthracene (2-AA) and the direct acting mutagen ethylmethanesulfonate (EMS). On the other hand an increase in SCE induction was found with dibromoethane (DBE).Pretreatment with 13A resulted in a decrease in SCE induction by B(a)P whereas no decrease was observed for NDMA 2-AA and EMS. Apparently there are also qualitative differences between the modulating effects of 13C and DA. Another conclusion is that the choice of the mutagen is crucial. The question whether pretreatment of metabolizing cells leads to decreased or enhanced effects on induction of SCEs, largely depends on the metabolic pathways involved in the biotransformation of the mutagens selected. As was shown, pretreatment with 13C leads to a reduced SCE induction by B(a)P whereas an increase was observed for DBE.The modulating effects of pretreatment with IR on B(a)P-induced SCEs were studied in detail. The observation that both 13C and DA are competitive inhibitors for B(a)P metabolism could imply that the observed inhibitory effects on SCE induction are, at least partly, due to the presence of residual 13C or 13A inside the cells at the time of exposure to B(a)P. Although this cannot be ruled out completely, several lines of evidence argue against this possibility. First, the observed decrease in B(a)P induced SCEs is proportional to the dose applied. If competitive inhibition would play a significant role, increasing doses of B(a)P would decrease the protective effects of pretreatment. Secondly, when microsomes prepared from pretreated chick- embryo hepatocytes were used, the increase in the formation of mutagenic intermediates from B(a)P is not reflected in an increase in SCE induction. This suggests that other enzyme systems play an essential role in the observed effects. Thirdly, the modulating effects of 13C are not restricted to a specific type of mutagen but comprizes mutagens from different chemical classes which are activated via different metabolic pathways. Especially in the case of dibromoethane pretreatment with 13C leads to an increase in SCE induction.Therefore it seems probable that competitive inhibition does not play a significant role in the observed effects. Instead the modulating effects of pretreatment results from a changed balance between the enzyme systems involved in the biotransformation of the mutagens aplied and, in the case of B(a)P, point to an essential role of conjugating enzymes.In the second part of this study the modulating effects of B- naphtoflavone (B-NF), a synthetic flavonol with reported anti' carcinogenic properties, were examined. B-NF treatment of cultured primary chick-embryo hepatocytes led to a large increase in cytochrome P450 content. No effect of pretreatment with B-NF on SCE induction by 2- AA and NDMA were observed. In contrast, a decrease in the number of SCEs, induced by B(a)P was found. Detailed studies with subcellular fractions, prepared from chick-embryo liver pretreated in ovo , showed that when an S9 fraction was used a large decrease in the number of SCEs was found whereas when a microsomal fraction was used, a large increase was found. Combined with the observed large increase in aryl hydrocarbon hydroxylase activity and the increase in formation of the proximate carcinogenic metabolite B(a)P-7,8 dihydrodiol, this has led to the conclusion that enzyme induction by B-NF is not associated with a protective effect. Instead it was postulated that the observed decrease in SCE induction by B(a)P In intact cells, stems from the formation of an intracellular pool of B-NF which acts as a competitive inhibitor for B(a)P metabolism.In conclusion it can be stated that the studies presented in this thesis illustrates that the combined use of different cell types with specific characteristics enables the study of various important aspects of the cancer process in vitro . The results show that the co-cultivation system, consisting of primary chick-embryo hepatocytes and V79 Chinese hamster cells is a promising tool in cancer research.