Relationship between p53 status and 5-fluorouracil sensitivity in 3 cell lines.

Mouse lymphoma L5178Ytk+/- (MOLY) cells and human lymphoblastoid TK6 and WTK-1 cells are widely used to detect mutagens in vitro. MOLY and WTK-1 cells have a p53 mutation, while TK6 cells, which were derived from the same parental line as WTK-1 cells, do not. In this study, we tested the clastogen 5-fluorouracil (5-FU) in the Tk assay and the in vitro micronucleus (MN) assay in MOLY, TK6, and WTK-1 cells to clarify whether differential responses were related to p53 gene status. We also determined the effect of 5-FU on the frequency of apoptotic cells and on cell cycle distribution in each cell line. Furthermore, we measured the activity of the 5-FU metabolizing enzymes (thymidylate synthetase (TS), dihydrouracil dehydrogenase (DPD), orotate phosphoribosyl transferase (OPRT), and thymidine phosphorylase (TP)) in each cell line. We treated MOLY cells with 1.0-8.0 microg/mL 5-FU for 3 h and TK6 and WTK-1 cells with 1.56-25 and 3.13-50 microg/mL, respectively, for 4 h. In MOLY cells, the mutation frequency (MF) and MN frequency increased. In WTK-1 cells, the MN frequency but not the MF increased. In TK6 cells, neither the MF nor the MN frequency increased. Furthermore, the IC50 of 5-FU was lower in MOLY cells than in the human cells. The response to 5-FU treatment differed in other ways as well. At the same level of cytotoxicity, the frequency of apoptotic cell was highest in TK6 cells. The cell cycle was delayed just after treatment in MOLY cells while the delay appeared 24 h later in TK6 and WTK-1 cells. Nothing in our analysis, however, revealed marked differences between the cell lines that could account for the severe cytotoxic and mutagenic responses that 5-FU elicited only in MOLY cells. 5-FU is phosphorylated by OPRT and TP and detoxified by DPD. MOLY cells have higher OPRT activity and markedly lower DPD and TP activity than TK6 and WTK-1 cells. The content of TS, however, the target enzyme of 5-FU, was similar in all cell lines, suggesting that 5-FU was more readily phosphorylated and less readily detoxified in MOLY cells than in TK6 and WTK-1 cells. MOLY cells were more sensitive to 5-FU than WTK-1 cells even though both have a mutated p53 gene, suggesting that the different responses to 5-FU were due to differences in 5-FU metabolism rather than the p53 status.

[DLST as a method for detecting TS-1-induced allergy].

Drug-induced allergic adverse events including rash, interstitial pneumonia and hepatic injury are often observed in a few patients treated with anticancer drugs that are 5-FU derivatives, including TS-1. In patients suspected to be liable to develop allergic reactions, the drug-induced lymphocyte stimulation test (DLST), based on the (3)H-thymidine incorporation ratio into the DNA of lymphocytes derived from the patients, is generally employed to identify drugs that could induce allergy. In this case report, we conducted the DLST on 20 healthy volunteers without TS-1 treatment in order to obtain reference data on the evaluation of TS-1-induced allergy. Even though all 20 volunteers were healthy, there were 6 positive responses to the DLST. In view of the positive response to TS-1 in healthy volunteers undergoing the DLST, it is suggested that the DLST could show a false positive response through an intracellular function that accelerates incorporation of thymidine in the lymphocytes by the salvage pathway after inhibition of DNA de novo synthesis caused by 5-FU derivative anticancer, including TS-1. Therefore, such a positive response to the drugs is considered, in fact, to be false-positive in the DLST. In view of the occurrence of false-positive results, the possibility of drug-induced allergy in patients receiving TS-1 should be carefully evaluated using a combination of other clinical examinations.

Diamine oxidase, a plasma biomarker in rats to GI tract toxicity of oral fluorouracil anti-cancer drugs.

Diamine oxidase (DAO; EC 1.4.3.6), which catabolizes a variety of substrates including histamine and diamines, is the degradative enzyme of the catabolic pathway of polyamines found in high activity in the mature upper villus cells of the rat intestinal mucosa [Luk, G.D., Bayless, T.M., Baylin, S.B., 1983. Plasma post-heparin diamine oxidase. Sensitive provocative test for quantitating length of acute intestinal mucosal injury in the rat. J. Clin. Invest. 71, 1308-1315; Wolvekamp, M.C.J., de Bruin, R.W.F., 1994. Diamine oxidase: an overview of historical, biochemical and functional aspects. Dig. Dis. 12, 2-14]. Rats were given 1-week repeated oral administration of anti-cancer drugs S-1, containing FT+CDHP+Oxo, and FCD, containing FT+CDHP, and the ameliorating effect of Oxo on the rat gastrointestinal (GI) tract toxicity from 5-FU was evaluated by measuring plasma DAO activity which is related to the enzyme located in the rat intestinal mucosa. Plasma DAO activity in the FCD-treated group was significantly less than that in the S-1-treated group while the jejunal mucosal area in the FCD group was significantly smaller than that in the S-1 group. In addition the histopathological findings in the FCD group showed villus atrophy in the jejunal mucosa which was not observed in the S-1 group. The degree of these findings correlated with the plasma DAO levels. Therefore, the protective effect of Oxo on 5-FU-induced GI tract toxicity was clarified by measuring plasma DAO activity in rats. In summary, DAO is a very sensitive plasma biomarker and will be useful for the quantitative evaluation of the small intestinal mucosal lesions induced by the anti-cancer drug, 5-FU, in rats.

Potassium oxonate modulation of 5-fluorouracil-induced myelotoxicity in murine and human colony forming assays of hematopoietic precursor cells.

Potassium oxonate (Oxo) is one of three components of S-1, an anticancer drug developed to improve the selective toxicity of 5-fluorouracil (5-FU). Oxo has been shown to reduce gastrointestinal toxicity. In this study, using murine and human granulocyte/macrophage colony forming (mCFU-GM, hCFU-GM) assays, we investigated whether Oxo can reduce 5-FU-induced myelotoxicity. The respective concentrations of 5-FU for 50% reduction (IC(50)) in mCFU-GM and hCFU-GM assays were 1.1 and 0.76 microM. The concentration-response curve was substantially steeper in the mCFU-GM assay than in the hCFU-GM assay. In the mCFU-GM assay, Oxo prevented growth suppression by 1 and 2 microM 5-FU, and at greater than 1 microM, it prevented suppression by 1 microM 5-FU almost completely. In the hCFU-GM assay, in contrast, Oxo prevented 5-FU suppression only slightly, and without a dose-response relationship. The difference between the mCFU-GM and hCFU-GM results may have stemmed from the spontaneous decomposition of Oxo during the longer culture period and Oxo's toxicity for human cells. Considering the human pharmacokinetic data, we concluded that Oxo has the potential to reduce 5-FU-induced myelotoxicity in human.