E.J. Laurensse

In vitro biochemical and in vivo biological studies of the uridine 'rescue' of 5-fluorouracil.

The effect of delayed uridine administration on the in vitro growth inhibitory effects of 5-fluorouracil (5FU) and on the in vivo antitumour activity and toxicity was studied. In vitro growth inhibition of the human intestinal cell lines WiDr and Intestine 407 by 3 microM 5FU could be reversed by 1.0 mM uridine; the effect was more pronounced with WiDr cells. At 0.1 mM uridine an intermediate effect was observed. Inhibition of colony formation in both cell lines could also be reversed by delayed administration of uridine at 0.1 and 1 mM. Incorporation of 5FU into RNA of WiDr cells did not proceed after addition of uridine, in contrast to Intestine 407 cells. In these cells only a partial inhibition was observed. In vivo we studied the effect of uridine on two colon carcinoma tumour lines, the 5FU sensitive Colon 38 and the relatively resistant Colon 26. 5FU was administered i.p. in a weekly schedule. With Colon 26 delayed administration of uridine (3500 mg kg-1) at 2 and 20 h after 5FU enabled us to increase the 5FU dose from 100 to 250 300mg kg-1. The combination of high-dose 5FU and uridine resulted both in a superior antitumour effect and an increase in life span. In the 5FU sensitive Colon 38 we determined whether the sensitivity to 5FU was affected by uridine. Mice were treated at the non-lethal dose of 100 mg kg-1 which inhibited tumour growth almost completely. Delayed administration of uridine did not significantly affect the antitumour effect. In non-tumour bearing mice we studied the time course of the reversal of the haematological toxicity of 5FU. The effective dose of 100 mg kg-1 induced a significant decrease in leukocytes; in combination with delayed uridine the leukopenia was less severe and recovered more rapidly. 5FU also induced a decrease in haematocrit, which could be prevented by delayed administration of uridine. In conclusion, in cell culture the reversal of 5FU cytotoxicity could be achieved at a low concentration of 0.1 mM uridine, the extent of the reversal might be related to the 5FU incorporation into RNA. In vivo the relatively resistant tumour Colon 26 could be treated with a higher dose of 5FU in the presence of uridine. The sensitivity to 5FU of the sensitive Colon 38 was not affected by delayed administration of uridine, while the haematological toxicity of 5FU was less. So, delayed administration of uridine after 5FU resulted in an improved therapeutic effect in both a relatively resistant and sensitive tumour.

Practical Experiences with an Extended Screening Strategy for Genetically Modified Organisms (GMOs) in Real-Life Samples

Nowadays most animal feed products imported into Europe have a GMO (genetically modified organism) label. This means that they contain European Union (EU)-authorized GMOs. For enforcement of these labeling requirements, it is necessary, with the rising number of EU-authorized GMOs, to perform an increasing number of analyses. In addition to this, it is necessary to test products for the potential presence of EU-unauthorized GMOs. Analysis for EU-authorized and -unauthorized GMOs in animal feed has thus become laborious and expensive. Initial screening steps may reduce the number of GMO identification methods that need to be applied, but with the increasing diversity also screening with GMO elements has become more complex. For the present study, the application of an informative detailed 24-element screening and subsequent identification strategy was applied in 50 animal feed samples. Almost all feed samples were labeled as containing GMO-derived materials. The main goal of the study was therefore to investigate if a detailed screening strategy would reduce the number of subsequent identification analyses. An additional goal was to test the samples in this way for the potential presence of EU-unauthorized GMOs. Finally, to test the robustness of the approach, eight of the samples were tested in a concise interlaboratory study. No significant differences were found between the results of the two laboratories.

Toxicity and antitumor effect of 5-fluorouracil and its rescue by uridine.

The pyrimidine analog 5-fluorouracil (5FU) has been used in the treatment against several solid carcinomas, mainly of the colon, for several decades (1). However, overall response rate is only about 20%, while gastrointestinal and myeloid toxicity are dose-limiting (1). Research on 5FU has been directed towards enhancing 5FU anabolism by combination with e.g. methotrexate (2) or thymidine (3) and towards the development of several analogs of 5FU. One recent analog, Doxifluridine (5′-deoxy-5-fluorouridine, 5’dFUR) showed an improved therapeutic index against a broad range of murine and rat tumors (4,5) and therapeutic activity in human advanced rectosigmoid adenocarcinoma (6).

A case study to determine the geographical origin of unknown GM papaya in routine food sample analysis, followed by identification of papaya events 16-0-1 and 18-2-4

During routine monitoring for GMOs in food in the Netherlands, papaya-containing food supplements were found positive for the genetically modified (GM) elements P-35S and T-nos. The goal of this study was to identify the unknown and EU unauthorised GM papaya event(s). A screening strategy was applied using additional GM screening elements including a newly developed PRSV coat protein PCR. The detected PRSV coat protein PCR product was sequenced and the nucleotide sequence showed identity to PRSV YK strains indigenous to China and Taiwan. The GM events 16-0-1 and 18-2-4 could be identified by amplifying and sequencing events-specific sequences. Further analyses showed that both papaya event 16-0-1 and event 18-2-4 were transformed with the same construct. For use in routine analysis, derived TaqMan qPCR methods for events 16-0-1 and 18-2-4 were developed. Event 16-0-1 was detected in all samples tested whereas event 18-2-4 was detected in one sample. This study presents a strategy for combining information from different sources (literature, patent databases) and novel sequence data to identify unknown GM papaya events.

In vitro and in vivo inhibition of thymidylate synthase of human colon cancer by 5-fluorouracil

Thymidylate synthase (TS) is a key enzyme in de novo synthesis of TMP (Fig. 1). TS catalyzes the conversion of dUMP to TMP for which 5, 10-methylenetetrahydrofolate (CH2THF) acts as the methyl-donor; the Km for dUMP is about 1–5 μM (1–5). Inhibition of TS by FdUMP is one of the main mechanisms for the action of 5-fluorouracil (5FU). Without preincubation FdUMP acts as a potent competitive inhibitor of TS with a Ki of about 1 nM. The Km/Ki ratio is about 1000. The inhibition of TS by FdUMP is mediated by the formation of a tight-binding covalent ternary complex of FdUMP with TS and CH THF. Retention of the inhibition of TS is mainly related to the FdUMP/dUMP ratio (6), the FdUMP binding to TS and the stabilization of the ternary complex by CH2 THF or one of its polyglutama-tes (6, 7, 9). In vitro, resistance against 5FU or its analog 5-fluoro-2′-deoxyuridine (FUdR) has been related to altered kinetics of TS with respect to Km values for dUMP and FdUMP binding (2, 5, 8), disturbed folate pools (9), and the level of enzyme before treatment (6, 8). Gene amplification of TS has been demonstrated for FUdR-resistant sub-cell lines (10, 11). Recently evidence for gene amplication has also been obtained in a patient with colon cancer who developed resistance against 5FU (12), while in breast cancer patients binding of FdUMP and the effect of CH2THF decreased during development of resistance (13).

Data on screening and identification of genetically modified papaya in food supplements

This article contains data related to the research article entitled “A case study to determine the geographical origin of unknown GM papaya in routine food sample analysis, followed by identification of papaya events 16-0-1 and 18-2-4” (Prins et al., 2016) [1]. Quantitative real-time PCR (qPCR) with targets that are putatively present in genetically modified (GM) papaya was used as a first screening to narrow down the vast array of candidates. The combination of elements P-nos and nptII was further confirmed by amplification and subsequent sequencing of the P-nos/nptII construct. Next, presence of the candidate GM papayas 16-0-1 and 18-2-4 were investigated by amplification and sequencing of event-spanning regions on the left and right border. This data article reports the Cq values for GM elements, the nucleotide sequence of the P-nos/nptII construct and the presence of GM papaya events 18-2-4 and/or 16-0-1 in five samples that were randomly sampled to be analysed in the framework of the official Dutch GMO monitoring program for food.

Modulation of cytotoxicity and metabolism of 5 fluorouracil in two intestine cell lines

The antitumor activity of 5-fluorouracil (5FU) is dependent on its conversion to active nucleotides. The rate of direct conversion to FUMP, catalyzed by orotate phosphoribosyl transferase (OPRT), depends on the availability of the co-substrate phosphoribosylpyrophosphate (PRPP). The rate of the indirect conversion to FUMP or FdUMP via fluorouridine (FUR) or fluorodeoxyuridine (FUdR), respectively, catalyzed by a pyrimidine nucleoside Phosphorylase (PNP), depends on the availability of the cosubstrates ribose-1-phosphate (Rib-1-P) and deoxyribose-1-phosphate (dRib-1-P). Nucleosides can act as ribose donors. Especially inosine and deoxyinosine are good sources since the major pathway of their metabolism involves phosphorolysis to hypoxanthine (1). Furthermore, these nucleosides are relatively non-toxic (2). In a variety of systems either inosine or deoxyinosine have been shown to increase growth inhibition by 5FU (3,4,5) and to enhance antitumor activity (6). It has also been shown that deoxyinosine or inosine can protect cells against 5FU (4,7).

TOXICITY AND ANTITUMOR EFFECT OF 5-FLUOROURACIL (FU) 157 AND ITS RESCUE BY URIDINE (UR): 157

The application of FU in the treatment of colon cancer is limited because of myeloid and gastrointestinal toxicity. It has been demonstrated that UR is able to rescue mice from the toxic effects of FU, which precludes the use of higher FU doses. Antitumor effect and toxicity were studied in two murine colon carcinomas, both sensitive and resistant, which also show marked differences in FU metabolism (e.g. uridine phosphorylase is 10 times higher in Colon 26 than in Colon 38). Mice received FU weekly. Colon 38, grown in C57B1 mice, was sensitive to FU; at 100 mg/FU kg 70% of the tumors disappeared. UR administration (3500 mg/kg) after 2 and 20 hr, decreased the number of complete responders. Colon 26, grown in Balb/c mice, was not sensitive to 100 mg FU/kg. FU was lethal at 300 mg/kg, but UR increased the life-span, while a significant tumor growth delay was observed. 100 mg FU/kg caused a severe leucopenia which reached a nadir of 10% after the second dose. UR administration prevented this leucopenia and a faster recovery was observed. UR was not lethal, although, a sharp fall in body temperature was observed after 1 hr (± 10°C). However, this hypothermia was not observed with patients in a Phase I trial with UR. In conclusion: hematological toxicity caused by FU can be prevented by delayed administration of UR; the antitumor activity seems to be affected, but this was not significant; the side-effects of UR seem to be acceptable to proceed Phase I trials.