Thymidylate Synthetase Activity Research Articles

Distribution of the most common polymorphisms in TYMS gene in Slavic population of central Europe.

Thymidylate synthetase (TS) plays a critical role in the de novo synthesis of dTMP inside the cell. Therefore, TS is a suitable target for cytotoxic drugs such as fluoropyrimidines. Drug efficacy and toxicity depend on the intracellular level of TS, which is significantly influenced by the polymorphisms in the 5'UTR (TSER - rs45445694, TSER*3G>C - rs2853542) and 3'UTR (1494del TTAAAG - rs151264360) of TYMS gene. Polymorphic variants of TYMS gene affect TS activity via gene expression and transcript stability. Patients who undergo fluoropyrimidine therapy may benefit from genetic testing prior to the administration of chemotherapy. At the 5' terminus of TYMS, there is a polymorphic region represented by a variable number of 28bp long tandem repeats (2-9 tandems) with the G or C nucleotide variant (SNP G>C). The 3'end of TYMS gene may decrease the stability of mRNA in the case of 6 base deletion (1494del6, D). In our study, we have focused on testing of TYMS gene polymorphisms, determination of TYMS variant frequencies in Western Slavic population and comparison of Slovak population with other populations.We performed identification of 5'UTR (rs45445694 - TSER*2 or TSER*3; rs2853542 - TSER*3G>C; TSER*3+ins6) and 3'UTR (rs151264360/1494del6/D) polymorphic regions of TYMS gene among 96 volunteers by PCR-RFLP and fragment analysis. Slovak frequencies of selected polymorphisms were established as follows: the frequency of TSER*2, TSER*3, TSER*3G>C, 1494del6/D and I to be 41%, 59%, 34%, 37.5% and 62.5% respectively. The high resolution of the capillary electrophoresis technique allowed among TSER*3 group identification of a subgroup of four individuals with rare 6bp insertion in 3R allele, id est 2.1% TSER*3+ins6 allele frequency. In our study, we have revealed individuals with rare G>C substitution in the first 28bp tandem repeat of TSER*2 promoter enhancer region (rs183205964) as well, the overall frequency of this polymorphic allele in Slovak population was 2.1%. Our results proved that Slovak population is in Hardy-Weinberg equilibrium and proportion of TYMS polymorphisms is in accordance with other published data.

A significance of some prognostic immunomorphologic factors in adenocarcinoma of the lung

The aim of this study was to search tumor expression of thymidylate synthetase and some other immunomorphological markers and to determine their prognostic role for pulmonary adenocarcinoma. Methods. Histological and immunohistochemical study of pulmonary adenocarcinoma has been conducted using several markers such as thymidylate synthetase, Ki67, p53, Bcl2, Bax. Results. Thymidylate synthetase and cellular prolif eration activity marker Ki67 were revealed in 100 % of cases; p53, Bcl2 and Bax were revealed in 48 %, 7.9 % and 6.3 %, respectively. The results demonstrated a tendency to decrease 3year eventfree survival by 14 % (p = 0.1007) in patients with high thymidylate synthetase expression in comparison to patients with low expression. Conclusion . Thymidylate synthetase could be used as a prognostic factor of pulmonary adenocarcinoma course.

Clinical implications of thymidylate synthetase, dihydropyrimidine dehydrogenase and orotate phosphoribosyl transferase activity levels in colorectal carcinoma following radical resection and administration of adjuvant 5-FU chemotherapy

BckgroundA number of studies have investigated whether the activity levels of enzymes involved in 5-fluorouracil (5-FU) metabolism are prognostic factors for survival in patients with colorectal carcinoma. Most reports have examined thymidylate synthetase (TS) and dihydropyrimidine dehydrogenase (DPD) in unresectable or metastatic cases, therefore it is unclear whether the activity of these enzymes is of prognostic value in colorectal cancer patients treated with radical resection and adjuvant chemotherapy with 5-FU.MethodsThis study examined fresh frozen specimens of colorectal carcinoma from 40 patients who had undergone curative operation and were orally administered adjuvant tegafur/uracil (UFT) chemotherapy. TS, DPD and orotate phosphoribosyl transferase (OPRT) activities were assayed in cancer tissue and adjacent normal tissue and their association with clinicopathological variables was investigated. In addition, the relationships between TS, DPD and OPRT activities and patient survival were examined to determine whether any of these enzymes could be useful prognostic factors.ResultsWhile there was no clear relationship between pathological findings and TS or DPD activity, OPRT activity was significantly lower in tumors with lymph node metastasis than in tumors lacking lymph node metastasis. Postoperative survival was significantly better in the groups with low TS activity and/or high OPRT activity.ConclusionTS and OPRT activity levels in tumor tissue may be important prognostic factors for survival in Dukes' B and C colorectal carcinoma with radical resection and adjuvant chemotherapy with UFT.

Phase I Trial of the Cyclin-Dependent Kinase Inhibitor and Protein Kinase C Inhibitor 7-Hydroxystaurosporine in Combination With Fluorouracil in Patients With Advanced Solid Tumors

Preclinical studies indicate that the cyclin-dependent kinase and protein kinase C inhibitor 7-hydroxystaurosporine (UCN-01) potentiates the cytotoxic effects of fluorouracil (FU). We designed a phase I clinical trial of FU in combination with UCN-01. FU was administered as a weekly 24-hour infusion. Doses were escalated in successive cohorts according to a modified Fibonacci design. UCN-01 was administered once every 4 weeks, immediately after disconnection from FU, at a dose of 135 mg/m(2) over 72 hours in cycle 1 and 67.5 mg/m(2) over 36 hours in subsequent cycles. FU and UCN-01 pharmacokinetics were obtained on all patients, and thymidylate synthetase (TS) activity was measured in peripheral-blood mononuclear cells by reverse-transcriptase polymerase chain reaction. We escalated the weekly FU dose to 2,600 mg/m(2) in combination with once a month infusions of UCN-01. Dose-limiting toxicity included arrhythmia and syncope. Other toxicities included hyperglycemia, headache, and nausea and vomiting. The mean maximal plasma concentration of UCN-01 was 33.5 micromol/L. There was significant interpatient variability, which correlated with plasma concentrations of alpha-1 acid glycoprotein. FU was rapidly cleared and the dose had no effect on the area under the curve of UCN-01. Changes in TS expression were detectable in peripheral-blood mononuclear cells after administration of UCN-01 but did not correlate with toxicity or activity. We observed no objective response, although seven patients had stable disease, six of whom had received prior fluoropyrimidines. The combination of weekly infusions of FU and monthly UCN-01 can be administered safely and warrants further study in phase II trials. The recommended phase II dose of FU in combination with monthly UCN-01 is 2,600 mg/m(2).

An optimal dosing schedule for a novel combination antimetabolite, TAS-102, based on its intracellular metabolism and its incorporation into DNA

TAS-102 is a combination drug consisting of alpha,alpha,alpha-trifluorothymidine (FTD) and thymidine phosphorylase inhibitor (TPI). FTD is converted to F3TMP by thymidine kinase and inhibits the thymidylate synthetase (TS) activity by binding to TS. In addition, FTD triphosphate form, F3TTP is incorporated into DNA, which leads to cytocidal effects. Therefore, the incorporation of FTD into DNA is expected to be an important factor, discriminating it from 5-FU showing TS inhibitory activity as their main mechanism of action. To assess a clinically more effective regimen protocol, the intracellular metabolism and the incorporation of FTD into DNA were investigated using human cancer cell lines in vitro and in vivo. FTD was incorporated into DNA in a time-dependent manner, but not in a concentration-dependent manner. FTD was the most efficiently incorporated into DNA after treatment with a several-micro molar level of FTD for around 8 h. The intracellular F3TTP was rapidly eliminated from tumor cells, as soon as FTD was washed out from the culture medium, whereas the FTD incorporated into DNA was retained by 80% or more even at 24 h after a washing-out procedure. When TAS-102 was administered into tumor-bearing mice once daily or three times daily at 3-h intervals at a dose of 150 mg/kg/day for one or 3 consecutive days, incorporation of FTD into tumor DNA by divided dosing significantly higher than that of single dosing. Based on our findings, the antitumor effects of TAS-102 against 3 different human cancer cell xenografts into mice were examined. The divided daily dosing resulted in enhancement of the antitumor effects of TAS-102 without any additional side effects. It is concluded that multiple daily dosing may result in better clinical benefits of TAS-102, when compared with single daily dosing and TAS-102 is a promising candidate for not only FU-sensitive but also FU-resistant cancer patients.

Effect of a combined administration of 5-fluorouracil and medroxyprogesterone acetate on pyrimidine nucleoside phosphorylases and thymidylate synthetase in 7,12-dimethylbenz.

The effect of a combined therapy of medroxyprogesterone acetate (MPA) and 5-fluorouracil (5-FU) on tumor size, pyrimidine nucleoside phosphorylase (PyNPase) activity, and thymidylate synthetase (TS) activity was examined in Sprague-Dawley (SD) rats with 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors. MPA augmented the antitumor activity of 5-FU and protected against body weight-loss due to 5-FU administration. PyNPase activity of both the MPA group and the MPA+5-FU group tended to increase compared with that of the 5-FU alone group. TS inhibition levels in the MPA+5-FU group tended to increase compared with those in the 5-FU alone group. These results indicate that MPA tended to augment antitumor activity of 5-FU and to reduce the side effects caused by 5-FU.

UCN-01 (7-hydroxystaurosporine) enhances 5-fluorouracil cytotoxicity through down-regulation of thymidylate synthetase messenger RNA.

UCN‐01 (7‐hydroxystaurosporine) is a newly developed cell cycle inhibitor known to have several modes of action, including inhibition of cyclin‐dependent kinase, induction of p21 and suppression of pRb phosphorylation. In order to test a combination therapy of UCN‐01 and 5‐fluorouracil (5‐FU), growth inhibition of CRL 1420 (MIA PaCa‐2; undifferentiated pancreatic carcinoma) by four different treatments was measured using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT) assay. The treatments used were UCN‐01 alone, 5‐FU alone, 5‐FU followed by UCN‐01 (5‐FU/UCN‐01) and UCN‐01 followed by 5‐FU (UCN‐01/5‐FU). We also assessed changes in thymidylate synthetase (TS) mRNA levels, TS activity, and 5‐FU incorporation by RNA (FRNA) for each treatment. Although treatment with UCN‐01 alone, 5‐FU alone, and 5‐FU/UCN‐01 inhibited CRL 1420 growth in a concentration‐dependent manner, treatment with UCN‐01/5‐FU inhibited the growth of CRL 1420 synergistically at less than 1 μg/ml drug concentration. The down‐regulation of TS mRNA by UCN‐01 resulted in stable total TS and decreased free TS, and UCN‐01/5‐FU resulted in enhanced thymidylate synthetase inhibition rate (TSIR) compared to UCN‐01 alone and 5‐FU/UCN‐01. This increased TSIR due to UCN‐01 pretreatment was accompanied by elevated F‐RNA concentrations in the UCN‐01/5‐FU treatment. The suppression of TS mRNA and TS activity by UCN‐01 may lead to higher sensitivity of tumor cells to 5‐FU and may explain the synergistic antitumor effect of UCN‐01/5‐FU. In conclusion, low concentrations of UCN‐01 (from 0.01 to 1 μg/ml) may be clinically useful, affording low cytotoxicity of UCN‐01, while enhancing the antitumor effect of 5‐FU.

Dihydropyrimidine dehydrogenase and messenger RNA levels in gastric cancer: possible predictor for sensitivity to 5-fluorouracil.

We investigated the correlation between tumor sensitivity to 5‐fluorouracil (5‐FU) and enzymatic activities of thymidylate synthetase (TS) and dihydropyrimidine dehydrogenase (DPD) in human gastric cancer specimens. Forty‐one patients with advanced gastric cancer gave informed consent and were enrolled in the study. Biopsy specimens of gastric cancer were obtained preoperatively through gastrofiberscopy and used to determine TS and DPD messenger RNA (mRNA) levels. TS and DPD enzyme activity and mRNA levels were also measured in resected tumor tissue samples obtained after surgical resection. TS and DPD activity were measured using the TS‐binding assay and a radioenzymatic assay, respectively, while mRNA levels were measured by semi‐quantitative reverse transcription‐polymerase chain reaction (RT‐PCR), with co‐amplification of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) as an internal standard. 5‐FU sensitivity of resected tumor specimens was measured by the tetrazolium‐based colorimetric assay (MTT assay). Both TS and DPD mRNA levels correlated well between biopsied and resected tumor specimens. A statistically significant correlation was also observed between mRNA levels in biopsied specimens and enzymatic activities in resected specimens. DPD levels significantly correlated with 5‐FU sensitivity, such that high DPD activity and high DPD mRNA levels resulted in low sensitivity to 5‐FU. In contrast, no correlation was observed between TS activity or TS mRNA levels and 5‐FU sensitivity. We conclude that tumor DPD mRNA level, as assessed from biopsy specimens obtained by gastrofiberscopy, may be a useful indicator in predicting tumor sensitivity to 5‐FU in patients with gastric cancer.

Thymidylate synthetase and dihydropyrimidine dehydrogenase activity in untreated uterine cervical cancer as an indicator of sensitivity to oral fluoropyrimidine chemotherapy

Thymidylate synthetase and dihydropyrimidine dehydrogenase activity in untreated uterine cervical cancer as an indicator of sensitivity to oral fluoropyrimidine chemotherapy

Cytotoxicity of cobalt complexes of furan oximes in murine and human tissue-cultured cell lines

The copper complexes of 2-furaldehyde and furan oximes have previously demonstrated potent cytotoxicity, L1210 DNA synthesis inhibition, DNA topoisomerase II inhibition and DNA fragmentation. Currently a series of cobalt metal complexes of 2-furaldehyde oximes were compared with copper complexes of furan oximes to determine whether the type of metal is important to the cytotoxicity and mode of action of the complexes. The cobalt complexes of furan oximes, like the copper complexes, were shown to be cytotoxic to suspended tumor cell lines, e.g. leukemias, lymphomas, acute monocytic leukemia and HeLa-S3 uterine carcinoma. The cobalt complexes did not demonstrate dramatic cytotoxicity against the growth of tumors derived from solid human tumor lines. The cobalt complexes preferentially inhibited L1210 DNA synthesis, followed by inhibition of RNA and protein synthesis from 25 to 100 µM over 60 min. These agents, like the copper complexes of 2-furaldehyde and furan oximes, were inhibitors of DNA polymerase α activity and de novo purine synthesis with marginal inhibition of ribonucleoside reductase and dihydrofolate reductase activities with DNA fragmentation. Unlike the copper complexes, the cobalt complexes did not inhibit L1210 DNA topoisomerase II activity but did reduce thymidylate synthetase activity. Thus, varying the type of metal within the complexes of 2-furaldehyde and furan oximes produces differences in both cytotoxicity and mode of action. Copyright © 1999 John Wiley & Sons, Ltd.

Protective effects of an antiulcer agent, ecabet sodium on colorectal carcinogenesis in rodents.

A new antiulcer agent, ecabet sodium is one of dehydroabietic acid derivatives prepared from pine resin. The effects of ecabet sodium on colorectal carcinogenesis were investigated in azoxymethane-pretreated mice with chronic ulcerative colitis induced by 3 repeated administration of 3% dextran sulfate sodium and in 1, 2-dimethylhydrazine-treated rats. Although daily treatment with ecabet sodium did not affect the colorectal DNA-synthesizing enzyme activities and bromodeoxyuridine-immunoreactive S-phase cells, high-grade dysplasia in ecabet sodium-treated mice was less frequent than in untreated mice. In rats, ecabet sodium administration reduced the elevated activity of thymidylate synthetase in colorectal tumors.

Modifications of the antioxidant enzymes in relation to chromosome imbalances in human melanoma cell lines.

Five human melanoma cell lines were investigated for their antioxidant activities. These metabolic data were correlated with cytogenetic analysis giving the relative numbers of chromosomes or chromosomal segments carrying the gene encoding for each enzyme. Particular attention was focused on the expression of superoxide dismutase 2 (SOD2), whose gene, located on the long arm of chromosome 6 (6q), has been proposed as a tumour suppressor gene. The activity of glutathione peroxidase (GPX), glutathione reductase (GSR) and catalase appeared to be unrelated to the relative number of 3q, 8p and 11p arms which, respectively, carry their encoding genes. GPX activity paralleled that of total SOD activity, and GSR variations followed those of GPX, suggesting possible metabolic regulation. Both the activity and the amount of SOD1 immunoreactive protein correlated with the number of chromosomes 21, suggesting a gene dosage effect. The three cell lines with deletions of the 6q arm had lower SOD2 activity and less immunoreactive protein than the two cell lines without 6q deletion. In addition, they demonstrated high thymidine kinase and thymidylate synthetase activities, which are directly linked to the cell proliferation rate. These results strengthen the hypothesis that SOD2 has a function as a tumour suppressor gene, but also suggest that the expression of other antioxidant enzymes might be altered in human melanomas.

Additive effects of medroxyprogesterone acetate and 5-fluorouracil derivative on 7,12-dimethylbenz[a]anthracene-induced rat mammary tumors.

Chronic oral administration of 1-(2-tetrahydrofuryl)-5-fluorouracil in combination with uracil suppressed thymidylate synthetase (TS) gene expression followed by reduction of TS activity in rat mammary tumors induced with 7,12-dimethylbenz[a]anthracene. Addition of medroxyprogesterone acetate (MPA) to the anticancer drug caused an additional decrease in TS and thymidine kinase activities in the tumor growth and restoration of bone loss. These results suggest that the simultaneous administration of MPA and anticancer drugs causes increased inhibition of mammary tumor growth and also diminishes the bone loss.

Nucleoside-Mediated Mitigation of 5-Fluorouracil-Induced Toxicity in Synchronized Murine Erythroleukemic Cells

5-Fluorouracil (5-FU) is a chemotherapeutic agent known to retard embryonic growth and induce cleft palate and limb deformities. The predominant mechanism underlying its toxic action is thought to be inhibition of thymidylate synthetase (TS), and hence thymidine triphosphate (dTTP) synthesis, resulting in alteration of the balance of deoxynucleotide (dNTP) pools and disruption of DNA synthesis. Indeed, previously we demonstrated retarded cell-cycle progression concurrent with a 60% decrease in TS activity in rat whole embryos following maternal exposure to 40 mg/kg 5-FU on Gestational Day 14 and in the murine erythroleukemic cell (MELC) suspension culture following exposure to 5–25 μm5-FU for 2 hr. In the study described herein, we used high-performance liquid chromatography (HPLC) to demonstrate in both of these model systems that 5-FU exposure results in similar patterns of dNTP perturbations: a prolonged decrease in dTTP and dGTP levels and an increase in dCTP and dATP. In addition, we used centrifugal elutriation to synchronize MELC in the phases of the cell cycle (G0/G1and early S) most sensitive to 5-FU to investigate the ability of nucleoside supplementation to mitigate 5-FU-induced toxicity. Our data indicate that following a 2-hr exposure to 5–25 μm5-FU, supplementation with 1–10 μmthymidine (TdR) for 24 hr partially reverses 5-FU-induced toxicity as evidenced by increased cellular proliferation and cell-cycle progression and amelioration of 5-FU-induced perturbations of protein synthesis and cellular membrane permeability compared to unsupplemented 5-FU-exposed cells. However, TdR concentrations ≥100 μminhibited growth or were cytotoxic. In comparison, supplementation with 10 μm–10 mmof deoxycytidine (CdR) was not toxic, but effected a dose-dependent recovery from 5-FU-induced toxicity. At 1–100 μm, neither deoxyadenosine nor deoxyguanosine supplementation reduced 5-FU-induced toxicity; at higher concentrations, both purine nucleotides inhibited cell growth. Although these results support the hypothesis that 5-FU disrupts the MELC cell cycle by depleting dTTP (a perturbation that is reversible by TdR supplementation), they also indicate that CdR supplementation offers an additional recovery pathway.

Biochemical modulation of 5-fluorouracil with murine interferon-alpha/beta against murine renal cell carcinoma.

Conventional therapy for renal cell carcinoma using interferon (IFN) has shown limited antitumor action. The purpose of our study was to investigate synergistic antitumor effects of IFN and 5-fluorouracil (5-FU), and to elucidate the mechanisms of interaction between the 2 agents in mice. Antitumor effects and biochemical modulation of murine IFN-alpha/beta and 5-FU were determined against the murine renal cell carcinoma cell line, Renca, in vivo. The activity of thymidylate synthetase and thymidine kinase was measured using cytosolic extracts of the tumors. Combination treatment with IFN-alpha/beta and 5-FU produced a significant enhancement of growth inhibition against Renca tumor. Treatment with 5-FU resulted in a 2.7-fold increase in the total amount of thymidylate synthetase and an 11.6-fold increase in the thymidylate synthetase inhibition rate, while the administration of IFN-alpha/beta did not significantly reduce the 5-FU-induced increase in thymidylate synthetase. The administration of IFN-alpha/beta decreased thymidine kinase activity to 65.5% maximally, compared with that in the control mice or the mice treated with 5-FU. The reduction of thymidine kinase caused by treating the mice with IFN-alpha/beta changes the utilization of exogenous thymidine for DNA synthesis, and may represent the mechanism of the additive antitumor effect of the 2 agents, through the suppression of the salvage pathway for deoxythymidine monophosphate induction.

Antineoplastic and Cytotoxic Activities of Nickel(II) Complexes of Thiosemicarbazones

Nickel(II) complexes of thiosemicarbazons were observed to be potent cytotoxic agents in human and rodent tissue cultured tumor cells. Each compound demonstrated a slightly different profile in the various histological types of tumors. The nickel complex of Appip demonstrated the most potent in vivo activity in the Ehrlich ascites carcinoma. This agent selectively inhibited L1210 DNA and purine syntheses, and DNA polymerase α, PRPP-amido transferase, IMP-dehydrogenase, dihydrofolate reductase, TMP-kinase and thymidylate synthetase activities. L1210 DNA strand scission was evident and DNA viscosity was reduced after 24 hr incubation. The nickel complexes were not L1210 DNA topoisomerase II inhibitors.

Effects of conjugated estrogens with or without medroxyprogesterone acetate on mammary carcinogenesis, uterine adenomyosis and femur in mice.

Although combined hormone replacement therapy using estrogens and progestins reduces the risk of endometrial cancer in postmenopausal women, it has been unclear whether the combined therapy is associated with an alteration of the risk of mammary cancer. Virgin mice of the SHN strain, which has a high potential for the development of mammary cancer and uterine adenomyosis, were given diets containing conjugated estrogens with or without medroxyprogesterone acetate for 230 days. The combined administration of conjugated estrogens and medroxyprogesterone acetate completely suppressed the development of uterine adenomyosis with a decrease in uterine thymidylate synthetase activity, significantly enhanced the bone mineral density in the femur and slightly shortened the latent period of mammary carcinogenesis.

Biochemical modulation applied to experimental cancer chemotherapy

The biochemical modulation (BCM) of the antitumor effect of 5-fluorouracil (5-FU) by leucovorin (LV) was studied with xenografts (MC-1, MC-3, MPC-2) by transplanting human tumors to nude mice and by human tumor clonogenic assay (HTCA). For human tumor transplantation to nude mice, the dose of 5-FU was set at LD50 x 0.6 or LD50 x 0.8 and LV was given in three doses, 0.45, 0.15, 0.06 mg/body. Antitumor effects of combined administration of 5-FU and LV at the same time, as compared with that of administration of LV one hour before 5-FU, were examined. The relationship between the rate of inhibition of thymidylate synthetase (T.S) activity and 5-FU concentration in the neoplastic tissues was also examined. In HTCA the antitumor effects of 5-FU were examined by two methods: 1) limited contact for one four, and 2) continuous contact for two weeks. In the human tumor transplantation to nude mice, the BCM of the antitumor effect of 5-FU by LV was demonstrated in MC-1 and MPC-2. This BCM function of LV was enhanced by administering it one hour before 5-FU. The suitable LV dose was between 0.15 and 0.45 mg/body. Although there was a tendency for the rate of inhibition of T.S to be proportional to the tissue concentration of 5-FU, there was no significant relationship between the T.S inhibition rate and the antitumor effect. In HTCA, the BCM function of LV was suggested by the two-week-continuous contact method with MC-1 and MPC-2. Depending on the method of administering LV, the antitumor activity was higher with two-week continuous contact than with one-hour contact. In conclusion, the BCM effect of LV on the antitumor effect of 5-FU was revealed in MC-1 and MPC-2 strains. Further studies are needed to establish a standard for appropriate dosage and administration of LV.

Biological modeling of 5-fluorouracil developmental toxicity

A biologically-based dose-response (BBDR) model is a mathematical description of the biological events leading to expression of a toxic response. As an alternative to current approaches in non-cancer risk assessment, such models will reduce uncertainty in that they will provide a more comprehensive description of toxicity. We are involved in construction of a BBDR model for the developmental toxicity of 5-fluorouracil (5-FU) in the rat using multiple approaches. First, to identify critical events in the pathogenesis of 5-FU developmental toxicity, thymidylate synthetase (TS) inhibition and alterations in cell cycle kinetics and growth were examined in embryos following maternal administration of 5-FU on day 14 of gestation. A dose-related decline in TS activity was observed within 1 h; however, maximal inhibition and recovery were similar at 10, 20 and 40 mg/kg. Dose-dependent cell cycle alterations were observed within 4 h after exposure and were maximal at 8 h. Hindlimb growth reduction was observed 24 h after exposure to 40 mg/kg, but not at lower doses. At term hindlimb defects were observed at doses above 30 mg/kg. An integrated dose-response model for hindlimb defects was derived from empirical relationships among these events. The resultant dose-response somewhat over-predicted the developmental toxicity of 5-FU, although results of a Monte Carlo simulation indicated that these data were not incompatible with model predictions. Overall, the results suggest that TS inhibition is a key component of the mechanism of 5-FU developmental toxicology, but the model does not capture all of the critical events in the induction of hindlimb defects. A preliminary mechanistic model for the inhibition of embryonic TS, DNA synthesis and cell cycle following maternal exposure to 5-FU, independently derived from literature data to further examine the potential role of this pathway in its developmental toxicity, predicted a dose-response for TS inhibition and DNA synthesis that closely reflected the observed patterns. These results further suggest that TS inhibition, resultant deficits in DNA synthesis and cell cycle perturbations represent a critical mechanistic pathway in the developmental toxicity of 5-FU.

Cytotoxicity of imides-N-alkyl semicarbazones, thiosemicarbazones, acetylhydrazones and related derivatives

The semicarbazones, thiosemicarbazones and acetyl-hydrazones of phthalimide, o-benzosulfimide, naphthalimide and diphenimide demonstrated potent cytotoxicity against murine and human leukemia cell growth and cultured cell growth from human solid tumors. The major site of inhibition in L1210 leukemia cells was DNA synthesis after 60 min incubated with the agents at 25, 50 and 100 microM. De novo synthesis of purines at the regulatory enzyme sites of PRPP amidotransferase and IMP dehydrogenase were the major targets of the agent. Thymidylate synthetase, dihydrofolate reductase and ribonucleoside reductase activities were inhibited by the agents in a manner which would contribute to the overall reduction of DNA synthesis and cell death. d(NTP) pools were significantly reduced and the evidence suggests that the agents interacted with DNA affording DNA strand scission which would interfere with both template utilization by the polymerases and also ultimately reduce nucleic acid synthesis.