Chemotherapy-induced mucositis development in a murine model of colitis-associated colorectal cancer

Abstract

Objectives: Ulcerative colitis is an incurable inflammatory bowel disease that increases the risk of colorectal cancer (CRC). 5-Fluorouracil (5-FU) is the predominant chemotherapy for CRC patients; however, undesirable side-effects, including mucositis, are common. This study utilised 5-FU-treatment in a model of colitis-associated CRC to develop a pre-clinical setting of intestinal mucositis coincident with manifestation of CRC.

Materials/methods: On day 0, female C57BL/6 mice (n?=?10/group); (1) saline control, (2) AOM/DSS control, or (3) AOM/DSS + 5-FU were injected with saline or AOM (i.p; 7.4?mg/kg). Groups 2 and 3 underwent cycles of seven days 2%w/v DSS followed by 14?days plain water. After three cycles, 5-FU was administered weekly (i.p; 75?mg/kg) to group 3 for five weeks. Clinical indicators were measured daily and colonoscopy performed at four time-points. Mice were euthanized at 13?weeks (day 91). Intestinal sections were collected for histological and biochemical analyses. p?<?.05 was considered significant.

Results: AOM/DSS resulted in bodyweight loss, increased disease activity index, colitis-severity and tumour number compared to saline controls (p?<?.05). 5-FU-treatment in AOM/DSS mice decreased bodyweight and disease activity index at selected time-points compared to AOM/DSS controls (p?<?.05). 5-FU did not impact colitis-severity or overall tumour burden; although, resulted in fewer small tumours compared to AOM/DSS controls (<2mm; p?<?.05). AOM/DSS increased histological severity scores in intestinal sections (p?<?.05), however, 5-FU-treatment did not further increase histologically-assessed disease severity (p?>?.05).

Conclusion: Weekly 5-FU administration at a dose of 75?mg/kg was insufficient to reduce overall tumour burden or induce intestinal mucositis in the AOM/DSS mouse model.     

Effect of cisplatin in advanced colorectal cancer resistant to 5-fluorouracil plus (S)-leucovorin

Modulation of 5-fluorouracil (5-FU) is currently being investigated in advanced colorectal cancer. In an attempt to improve the results obtainable for the association of 5-FU and leucovorin, we decided to add cisplatin to 5-FU and (6S)-leucovorin (S-LV) after disease progression. The hypothesis was that a pharmacological enhancement of the efficacy of 5-FU would result in responses in 5-FU-unresponsive patients or in a second response in previously responding patients. A group of 28 5-FU+S-LV-pretreated patients, with advanced measurable colorectal cancer, were treated with 80 mg/m² cisplatin on day 1, 80 mg/m² S-LV every 4 weeks. We obtained 3 partial responses (response rate: 11±11%), while 11 patients had stable disease (39±18%). Among the 3 responders, 1 patient had earlier achieved a partial response, a second stable disease and 1 had disease progression after the previous 5-FU+S-LV treatment. The median survival time for all 28 patients was 11 months. Toxicity was minimal and consisted of mild and reversible gastrointestinal symptoms and myelosuppression. We believe that further studies must be carried out to establish the real impact of the synergism between cisplatin, 5-FU and S-LV in untreated patients.Abbreviations S-LV (6S) leucovorin - 5-FU 5 fluorouracil - PD progressive disease - PR partial response - SD stable disease     

Changes in the mucus barrier of the rat during 5-fluorouracil-induced gastrointestinal mucositis

Objective. A frequent complication of antineoplastic chemotherapy (CT) is gastrointestinal (GI) mucositis. Although clinically this mucositis can be treated, data on the effect of CT on the mucosal defense mechanisms are scant, so the effects of 5-fluorouracil (5-FU) on mucin, one of the principal defense factors of the GI mucosa, were investigated. Material and methods. 5-FU was administered orally to rats at a dose of 50 mg/kg once daily for 5 days. Using anti-mucin monoclonal antibodies, the immunoreactivity in different areas of the rats’ GI tracts was compared, as well as the mucin content. Changes in the GI mucin during the process of recovery from the injury were also investigated. Immunohistochemical analysis of proliferating cell nuclear antigen (PCNA) was used to determine whether or not the effects of 5-FU on cell proliferation contributed to the changes in mucin. Results. 5-FU caused significant alterations of the immunoreactivity and content of mucin in the rat GI mucosa, especially in the jejunum. The jejunal mucin content was most markedly reduced on day 1 after drug withdrawal, and increased thereafter. By day 7, the content had transiently but significantly increased approximately 1.5-fold, and returned to the basal level by day 13. The number of PCNA-positive cells strikingly decreased at day 1, but by day 7 had increased approximately 2-fold, compared with the control. Conclusion. The activation of mucus cells in the jejunum, if appropriately manipulated, could lead to more effective prevention of CT-induced GI mucositis.     

High-dose chronomodulated infusion of 5-fluorouracil (5-FU) and folinic acid (FA) (FF<Subscript>5–16</Subscript>) in advanced colorectal cancer patients

Purpose . The best way to deliver infusional 5-fluorouracil (5-FU) and folinic acid (FA) has yet to be determined. The aim of this prospective phase II trial was to verify the tolerability, activity and efficacy of chronomodulated 5-FU-FA (FF_5–16) every 3 weeks in 48 untreated patients (group A), and 28 pretreated and four non-measurable, advanced colorectal cancer (ACC) patients (group B).Methods The sinusoidal delivery of both drugs started at 10.00 p.m. and ended at 10.00 a.m., with peak flow at 4.00 a.m. for 5 consecutive days. The initial 5-FU dose was 900 mg/m²/day with intra-patient dose increase at 1,000 and 1,100 mg/m²/day, at the second and third course, respectively; FA was injected at a fixed dose of 150 mg/m²/day (Garufi et al.1997).Results Neither death from toxicity nor hematological toxicities were encountered. Maximal toxicity consisted of Grade 3 oral mucositis in 41% of patients, in only 8% of 535 courses. It was possible to achieve objective responses in 31% of untreated patients, with a progression free survival (PFS) of 7 months, median survival of 14 months and a 2-year survival rate of 28%. Similar results for PFS and survival were obtained in pretreated patients as well. Univariate analysis and multivariate analysis showed that response was related to the occurrence of mucositis and diarrhea (p=0.03 and p=0.0007) and to performance status (PS) (p=0.01). Quality of life, measured with the EORTC QLQ-C30+3 questionnaire, was unaffected by treatment and was better in patients with good PS and responsiveness.Conclusions In this chronomodulated FF_5–16 phase II study, the probability of obtaining a relevant tumor reduction was significantly correlated with a patient variable such as PS, and toxicity variables such as mucositis and diarrhea. This observation and the validation of predictive factors for QoL deserve further investigation in ACC patients.     

Phase I study of continuous mitomycin-C infusion in concomitant radiochemotherapy of primary inoperable advanced head and neck cancer

Purpose: A phase I trial to evaluate the maximum tolerated dose (MTD) of continuous mitomycin-C infusion in radiochemotherapy of inoperable HNSCC. Methods: Twenty-one patients were treated with 70 Gy (2 Gy/day) and simultaneous chemotherapy (5-FU 600 mg/m2/day and MMC, both as continuous infusion on days 1–5 and 36–40. The MMC dose was dependent on dose escalation levels I–IV: 2/2.6/3.2/4 mg/m2/day. Results: Dose limiting toxicity (DLT) (grade 3 mucositis and/or dysphagia) occurred at dose level IV (MMC 4 mg/m2/day). Accordingly, dose escalation level III (MMC 3.2 mg/m2/day) was set as the MTD. One and 2-year survival rate: 66.7 and 29.5%, disease free survival: 47.6 and 22.8%, respectively. Conclusions: Our study demonstrates that continuous infusion of 5-FU/MMC can be safely administered at a MMC dose of 3.21 mg/m2/day on days 1–5 and 36–40 in concomitant radiochemotherapy. A phase II study should be initiated to establish the role of this regimen in the treatment of head and neck cancer.     

Chemotherapy-induced nuclear alterations of morphologic and genomic characteristics in a human colon cancer grafted onto nude mice

PURPOSE: A human Dukes B colonic adenocarcinoma was grafted onto 40 nude mice. The mice were divided into four groups, one control and three representing experimental conditions. Animals in the three experimental groups received either adriamycin (ADR), 5-fluorouracil (5-FU), or camptothecin (CPT) over a 25-day period beginning 34 days after grafting. Control animals received saline on an identical schedule. Animals were killed 105 days after grafting. METHODS: The effect of therapy was assessed by three techniques: 1) tumor size was periodically measured during the life of the animals, 2) modifications of APC, Ki-ras, and p53 genes were studied by polymerase chain reaction, dotblot analysis, restriction analysis, and DNA sequencing, and 3) image cytometry of Feulgen-stained material was used to characterize 15 parameters describing morphometric, densitometric, and textural features of tumor nuclei. RESULTS: When compared with controls, tumor growth (size) was maximumly suppressed by treatment with CPT (P <-0.001). Growth was inhibited significantly by treatment with 5-FU ( P <-0 0.01); no statistical difference in tumor size was observed between controls and animals treated with ADR. Modifications of APC, Ki-ras, and p53 genes were not observed; however, treatment did inhibit amplification of APC and p53 genes. CONCLUSIONS: The 15 morphonuclear parameters were assessed to define populations of cell nuclei altered by chemotherapy. Although CPT maximally suppressed growth, it did not alter nuclear morphology when compared with controls. Treatment with either 5-FU or ADR resulted in nuclear morphologic alterations defined as distinct populations using multivariate analysis. Nonsupervised linear discriminant analysis was used to quantify the relative proportions of these populations. Four morphonuclear parameters were identified, which discriminated nuclei exposed to either ADR or 5-FU from controls.     

Reduced incidence of acute graft versus host disease (GVHD) of the gut in Chinese carriers of<Emphasis Type="Italic"> Helicobacter Pylori</Emphasis> during allogeneic bone marrow transplantation

Helicobacter pylori (H. Pylori) infection is associated with gastritis and peptic ulcer, but its relationship with gut graft versus host disease (GVHD) is unknown. We investigated the association between H. Pylori carriage and incidence and severity of mucosal toxicity and GVHD in 128 consecutive matched sibling stem cell transplantation (SCT) recipients. Using a verified enzyme linked immunosorbant assay (ELISA), 43.5% of patients had H. Pylori exposure before SCT. There was absolute concordance between serological and breath test data in 40 prospective cases. There was no increased risk in WHO grade 3 or 4 mucositis in H. Pylori carriers. Significant (grade II or above) overall GVHD was only predicted by preceding mucositis (p<0.001), while gut GVHD was associated with increased age (p=0.001) and mucositis (p=0.022). Despite increased incidence with age, H. Pylori carriage was associated with significantly reduced risk of gut GVHD (p=0.04) but not overall GVHD. The reduced risk of immune-mediated gut inflammation in H. Pylori carriers after SCT may be related to the known reduced incidence of inflammatory bowel disease in chronic H. Pylori carriers.     

Tauromustine is more effective than conventional chemotherapy in the treatment of colonic tumors

Despite recent advances in chemotherapy, the prognosis of advanced colorectal cancer remains poor. Although the taurine-based nitrosourea tauromustine (TCNU) has demonstrated schedule-dependent synergism with 5-fluorouracil (5-FU) and leucovorin (LV) against a variety of tumors in vitro,its efficacy relative to and in combination with these drugs in vivo remains unknown. To study this, BALB/C mice had C26 tumor implanted subcutaneously five days prior to the following treatment (doses and route of administration being the same in all groups): Group 1—no treatment; Group 2-TCNU (30 mg/kg by gavage); Group 3-LV (100 mg/kg intraperitoneally [IP]) and, one hour later, LV plus 5-FU (100 mg/kg IP); Group 4—LV and, one hour later, LV plus 5-FU and TCNU; and Group 5-TCNU and, on the following day, LV and, one hour later, LV plus 5-FU. All treatments were repeated seven days later. Body weight and tumor weight were measured twice weekly, and survival was noted. Postmortems were performed in all animals, and evidence of primary or secondary tumor was recorded. All surviving animals were sacrificed at 60 days. We found that 1) 87 percent of animals receiving TCNU and 60 percent of animals receiving LV, 5-FU, and TCNU survived to day 60; none of these animals had evidence of tumor when sacrificed; 2) animals in all other groups died by day 34; 3) evidence of metastases was found in five animals in Group 1 and one each in Groups 2 and 5; and 4) administering TCNU 24 hours prior to 5-FU plus LV resulted in death from toxicity in all animals. Thus, while synergism between TCNU and 5-FU plus LV was not seen, the antitumor properties of TCNU are significantly greater than those of conventional chemotherapy.     

Different effects of sequential combinations ofN-methylformamide with 5-fluorouracil on human colon carcinoma cells growing in nude mice

Summary The effects of the combination ofN-methylformamide (NMF) with 5-fluorouracil (5-FU) on tumor growth and morphological features of human colon carcinoma cells (HT29) implanted in nude mice were assessed. Both agents were administered i.p. at tolerable doses: 5-FU at 19 mg/kg for 5 days and NMF at 200 mg/ kg for 12 days. Four main schedules were tested: 5-FU alone, NMF alone, NMF followed by 5-FU and 5-FU followed by NMF. The last sequence was the most effective, as compared with the other treatment regimens. In particular, the 5-FU NMF combination induced a tumor inhibition of about 75% at the end of the treatments (17th day) versus an inhibition of 23%–43% in the other schedules. Morphological observations, carried out by light and electron microscopy, indicated a possible relationship between the presence of structural changes and tumor growth inhibition. The results of this study renew interest in the use of NMF in sequential combination confirming sequence as a critical factor for the optimal combination of NMF and 5-FU.Abbreviations 5-FU 5-fluorouracil - NMF N-methylformamide - DMF N,N-dimethylformamide - cisplatin cis-diamminedichloroplatinum - m _T/m _c tumor weight inhibition - t _T-t _c tumor growth delay This work was supported by grants from the Italian National Research Council, contract 89.03979.CT04; Ministero della Sanità; Istituto Oncologico Romagnolo n 90152.1     

Pharmacokinetics of excretory passage of 5-fluorouracil (i.v.) into the pancreatic juice of patients with pancreatic or biliary carcinoma: Evaluation of possible adjuvant chemotherapy

The drug, 5-fluorouracil (5-FU), is thought to be efficacious in treating human pancreatic or biliary carcinomas; therefore, to determine the optimal dosage for chemotherapeutic use in these conditions, we performed this pharmacokinetic study in which we investigate the passage of various doses of intravenously administered 5-FU into the pancreatic juice of 11 patients with pancreatic or biliary carcinoma. Whenever possible, all 11 patients, who had undergone a pancreaticoduodenectomy and had an external drainage tube, received the following three regimens: (1) a bolus injection of 5-FU, 185 mg/m² per day; (2) a continuous infusion of 5-FU, 185 mg/m² per day over 48h (CIV-I), and (3) a continuous infusion of 5-FU, 370 mg/m² per day over 48 h (CIV-II), with a sufficient wash-out period of 2 weeks between each regimen. The major findings were: (i) the percentage of the administered 5-FU dose excreted (pancreatic passage fraction; Fp) was strongly correlated with the total amount of pancreatic juice excreted over the 24-h period (Vp) of drug testing; (ii) the Fp per 100 ml Vp (Fp') was greater after the bolus treatment than after either CIV treatment; (iii) 90% of the 5-FU excreted into the pancreatic juice was present within 30min of the bolus injection; and (iv), the entire body clearance (CL_total) of 5-FU was significantly lower after the bolus injection than after either CIV treatment. It was concluded that the Fp' value was dependent on the method of 5-FU administration, that a 5-FU bolus injection probably inundates the hepatic metabolic capacity, and that the Fp' of 5-FU largely depends on the patient's ability to metabolize the drug. Therefore, the efficacy of 5-FU as an anticancer agent appears to be time-rather than dose-dependent.     

Lactose Hydrogen Breath Test in <Emphasis Type="Italic">Giardia lamblia</Emphasis>-Positive Patients

The study was conducted in 54 adult patients with Giardia lamblia infection and 54 adult controls to detect lactose maldigestion employing the noninvasive lactose hydrogen breath test. Forty of 54 (74%) patients with Giardia lamblia and 24 of 54 (44.4%) controls showed lactose maldigestion (P < 0.01). In conclusion, this study shows that the frequency of lactose maldigestion is significantly higher in adult Indians suffering from Giardia lamblia infection compared to healthy individuals.     

Histological evaluation of colonic anastomotic healing in the rat following preoperative 5-fluorouracil, fractionated irradiation, and combined treatment

There is a growing interest in neoadjuvant chemo- and radiotherapy as a treatment modality for colorectal cancer which could affect mechanical and biochemical parameters of anastomotic healing. This study investigated the effect of such protocols on colonic anastomotic healing by evaluating the histopathological parameters. One hundred and sixty male Wistar rats were divided into six groups: a control group (I, n=20), a saline group (II, n=30) which received 1 ml NaC1 intraperitoneally, a sham-irradiated group (III, n=20), a 5-fluorouracil (5-FU) group (IV, n=30), which received 5-FU (20 mg/kg) intraperitoneally for 5 consecutive days, an irradiated group (V, n=40) which received fractionated irradiation to the whole pelvis to a totaldose of 22 Gy, 5.5 Gy per fraction on 4 consecutive days, and a concomitant 5-FU + irradiation group (VI, n=20) which received 5-FU as in group IV and irradiated as in group V. All groups underwent left colonic resection with primary anastomosis, and the last fraction of irradiation and the last injection were given 4 and 3 days before the operation, respectively. Within each group one half of the animals were killed on the third postoperative day and the other half on the seventh postoperative. day. After the resection of the anastomotic segments, histopathological examination was evaluated. Apposition of the wound edges of the mucosa and the muscularis were not affected by the therapy. The level of granulocytes was high, inflammatory exudate and necrosis persisted, granulation tissue formation was delayed, and the levels of macrophages and fibroblasts were low. We conclude that colonic anastomotic healing can be affected by the administration of preoperative chemotherapy, irradiation, and chemoirradiation. Accepted: 17 July 1998     

Experimental model of colon cancer: Recurrences after surgery alone or associated with intraperitoneal 5-fluorouracil chemotherapy

The liver is the most frequent site of metastases in colon cancer. No good animal model has been available to help improve the treatment of liver metastases or their prevention after resection of a primary colon cancer. The aim of this study was to develop a model of colon cancer induced by azoxymethane in the rat and to study the outcome after surgical resection alone or in association with intraperitoneal chemotherapy (5-fluorouracil (5-FU)). Three hundred male Wistar rats received subcutaneous azoxymethane (10 mg/kg body weight/week) for 12 weeks. Eighty-three rats with isolated colon cancer underwent total colectomy; 40 of these rats with no metastases were randomized into two groups: surgery alone or surgery plus 5-FU (5 mg/kg body weight/day) for 5 days after surgery. Thirty rats were able to be evaluated. At autopsy, peritoneal carcinomatosis and liver metastases were more frequent in the control group than after adjuvant treatment with 5-FU (27.7 percent vs. 0,P <0.05; and 22.2 percent vs. 0,P <0.05, respectively). The rates of peritoneal and hepatic recurrence after resection of the primary cancer indicate that the model mimics the natural history of human colon cancer. In this model, 5-FU reduced the rate of peritoneal carcinomatosis and liver metastases but did not influence survival.Supported by Grant Nr 6444 R11 from University Paris VI and a grant from the Institut National de la Santé et de la Recherche Médicale (INSERM).     

D-Xylose hydrogen breath tests compared to absorption kinetics in human patients with and without malabsorption

Thed-xylose breath H₂ test may be useful in characterizing intestinal absorptive function. Our aim was to determine whether breath H₂ followingd-xylose administration reflects the extent to which thed-xylose is absorbed by comparing it to a kinetic model ofd-xylose absorption. Twenty-five subjects were studied. They ingested 15 gd-xylose on the first day and 25 gd-xylose on the third day. On the second day they received 10 g intravenousd-xylose along with 15 g oral lactulose. Multiple serum and urine samples were obtained ford-xylose content to calculate its rate constants and extent of absorption by multicompartmental analysis. Breath H₂ determinations were obtained every 15 min for 3 hr following the 15 gd-xylose and lactulose ingestion. Peak breath H₂ concentration correlated with extent of absorption (r=–0.787,P<0.001),K ₀, the rate constant for nonabsorptive loss (r=0.744,P<0.001), and 5-hr urine content (r=–0.705,P<0.001). Area under the breath H₂ curve also correlated with these parameters: extent of absorption (r=–0.770,P<0.001),K ₀ (r=0.662,P<0.001), 5-hr urine content (r=–0.629,P<0.012). Peakd-xylose breath H₂ to peak lactulose breath H₂ showed no correlation with extent of absorption. The extent of absorption was higher with the 15-g dose than the 25-g dose in all patients tested (P<0.01). This was the result of decreased nonabsorptive loss (lowerK ₀), as the rate constant for absorption,K _a , was not statistically different (P>0.05). Peakd-xylose breath H₂ can be used as an inverse estimate ofd-xylose absorption. Lactulose breath H₂ cannot be used as a standard for comparison ford-xylose. The three compartment kinetic model ford-xylose absorption with passive absorption of this carbohydrate is supported by similar rate constants for absorption for the twod-xylose doses used.d-xylose at 15 g may be a more appropriate dose than 25 g for H₂ breath testing as it does not lead to increased nonabsorptive losses.Supported in part by grant RR0048, National Institutes of Health, National Center for Research Resources.     

Pre-treatment with insulin-like growth factor-I partially ameliorates 5-fluorouracil-induced intestinal mucositis in rats.

Insulin-like growth factor-I (IGF-I) has been demonstrated to enhance mucosal repair following intestinal damage induced by chemotherapeutic agents (intestinal mucositis). However, the potential for prophylactic IGF-I to protect the intestine remains undefined. We investigated the effects of IGF-I pre-treatment on chemotherapy-induced mucositis in rats. Male Sprague Dawley rats were treated for 7 days with 0 or 4.3mg/kg/day IGF-I delivered systemically via osmotic mini-pump. Rats received an intraperitoneal injection of 0 or 150 mg/kg 5-fluorouracil (5-FU) on day 7 and were killed 48 h later for assessment of intestinal damage and repair. Compared to normal controls, 5-FU decreased epithelial proliferation by 86%, concurrently increasing the incidence of apoptosis 87-fold, whilst decreasing small intestinal (SI) length by 14%, SI weight by 30% and total gut weight by 24%. 5-FU decreased villus height in the duodenum (23%), jejunum (20%) and ileum (30%) with crypt depths decreased by 31%, 27% and 33% in these gut regions. These effects were less profound in IGF-I pre-treated rats in which apoptosis was increased 48-fold, with SI length decreased by 7%, SI weight by 18% and total gut weight by 15% accompanied by decreases in villus height of 8% (duodenum), 14% (jejunum) and 21% (ileum), and crypt depth decreases of 23%, 16% and 17% for the same gut regions, compared to normal controls. We conclude that IGF-I pre-treatment only partially attenuates features of intestinal mucositis when assessed 48 h after 5-FU chemotherapy.     

In vivo and in vitro responses of the bovine corpus luteum after exposure to exogenous gonadotropin-releasing hormone and prostaglandin F(2α)

Administration of gonadotropin-releasing hormone (GnRH) early in the estrous cycle has been shown to cause subsequent altered luteal function. To determine whether membrane-related events may be involved in GnRH-attenuated luteal function, corpora lutea (CL) were removed from beef heifers on day seven of the estrous cycle after iv injection of GnRH or saline on day two of the cycle (n=5/group). Luteal slices were incubated with saline (control), luteinizing hormone (LH), or 8-bromo-cAMP for 2h. In vivo administration of GnRH reduced LH and cAMP-stimulated progesterone production by tissue (p<0.01), but basal progesterone production was not affected (p>0.05). Luteal adenylyl cyclase activity did not differ between saline and GnRH-treated animals (p>0.05). Then to examine if early administration of GnRH alters response of the CL to prostaglandin (PG) F_2α, beefheifers were injected with GnRH as described above (n=4/group), and then injected with PGF_2α on day eight and the CL removed 60 min later. Blood samples were collected for oxytocin (OT) analysis at frequent intervals after PGF_2α injection and for progesterone at 0 and 60 min. Induction of the early response gene c-jun or release of OT by PGF_2α was not altered by GnRH injection (p>0.05). Injection of PGF_2α decreased serum progesterone by 60 min postinjection (p<0.05), but concentrations of this steroid were unaffected by GnRH (p>0.05). Collectively, these data suggest that GnRH-induced alteration of bovine luteal function may be owing to events distal to cAMP synthesis that do not interfere with PGF_2α-induced expression of c-jun or OT release, cellular phenomena involved in luteolysis.     

A human leukemia cell line made resistant to two folate analogues,trimetrexate andN 10-propargyl-5,8-dideazafolic acid (CB3717)

Summary We established a novel human acute lympho-blastic leukemia cell line made resistant to two folate analogues, trimetrexate (TMQ) andN ¹⁰-propargyl-5,8-dideazafolic acid (CB3717), by sequential exposure of the 200-fold TMQ-resistant cells (MOLT-3/TMQ₂₀₀) to CB3717. A 30-fold-resistant subline to CB3717 was selected from the TMQ-resistant cells and designated as MOLT-3/TMQ₂₀₀-CB3717₃₀. This double-folate-resistant cell line was 15-fold more resistant to methotrexate (MTX) than MOLT-3/TMQ₂₀₀; however, TMQ resistance was decreased to 10-fold as compared to MOLT-3/TMQ₂₀₀. The doubly resistant cells also showed 2-fold cross-resistance to 5-fluorouracil (5-FU). Equimolar concentrations of leucovorin almost completely reversed the inhibitory effect of MTX on the doubly resistant cells and partially that of CB3717 and TMQ; on the other hand, leucovorin enhanced the inhibitory effect of 5-FU. Thymidylate synthase activities demonstrated little or no difference among these three cell lines, being consistent with no overexpression of mRNA for this enzyme in the doubly resistant cells. MOLT-3/TMQ₂₀₀ cells displayed classical multidrug resistance; sequential development of CB3717 resistance in the TMQ-resistant cells resulted in an enhancement of the multidrug-resistance phenotype and a concomitant increase ofMDR1 mRNA. The development of a complex resistance pattern seen in this double-folate-resistant subline indicates intricacy in the study of drug resistance after multidrug chemotherapy.Abbreviations TMQ trimetrexate 2,4-diamino-5-methyl-6-[(3,4,5-trimethoxyanilino)methyl]quinazoline - MTX methotrexate, 4-amino-N ¹⁰-methylpteroylglutamic acid - CB3717 N ₁₀-propargyl-5,8-dideazafolic acid - MDR1 the gene for multidrug resistance - ID₅₀ 50% inhibiting drug concentration for cell growth on day 3 This work was supported in part by the Japan Research Foundation for Clinical Pharmacology and by USPHS research grant CA-15936 from the National Cancer Institute, Bethesda, Md., USA. Yuzuru Takemura was an Iwama Memorial Sony International Fellow     

Healing of colon anastomoses covered with fibrin glue after immediate postoperative intraperitoneal administration of 5-fluorouracil

PURPOSE The aim of this experimental study was to investigate whether covering the colonic anastomoses with fibrin glue can protect the colonic healing from the adverse effects of 5-fluorouracil (5-FU), when it is injected intraperitoneally immediately after colon resection. METHODS Sixty-four rats were randomized to one of four groups. After resection of a 1-cm segment of the transverse colon, an end-to-end sutured anastomosis was performed. Rats of the control group and the fibrin glue group were injected with 6 ml of solution 0.9 percent NaCl intraperitoneally. Rats in the 5-FU and the 5-FU + fibrin glue groups received 5-FU intraperitoneally. The colonic anastomoses of the rats in the fibrin glue group and in the 5-FU + fibrin glue group were covered with fibrin glue. All rats were killed on the 8th postoperative day and the anastomoses were examined macroscopically. The bursting pressure measurements were recorded and the anastomoses were graded histologically.RESULTS The leakage rate of the anastomoses was significantly higher in the rats of the 5-FU group than in those of the fibrin glue group and those of the 5-FU + fibrin glue group (37.5 percent vs. 0 percent, P = 0.020). The adhesion formation score was significantly higher in rats of the 5-FU group than in the other groups. Bursting pressures were also significantly lower in the 5-FUgroup than in the other groups (P < 0.001). Rats in the 5-FU + fibrin glue group developed significantly more marked neoagiogenesis than rats in the other groups. Rats in the 5-FU + fibrin glue group also presented significantly more fibroblast activity than those in the 5-FU group. (P = 0.004)CONCLUSIONS The immediate postoperative, intraperitoneal administration of 5-FU inhibited wound healing. However, when the colonic anastomoses were covered with fibrin glue, the injection of 5-FU had no adverse effects on the healing of the anastomoses.     

Yoghurts Containing Probiotics Reduce Disruption of the Small Intestinal Barrier in Methotrexate-Treated Rats

Small intestinal permeability was employed to assess the efficacy of commercially available yoghurts containing probiotics in a rat model of methotrexate (MTX)-induced mucositis. Male Sprague-Dawley rats were allocated to four groups (n = 8): MTX + water, MTX + cow's milk yoghurt (CY; fermented with Lactobacillus johnsonii), MTX + sheep's milk yoghurt (SY; containing Lactobacillus bulgaricus and Streptococcus thermophilus), and saline. Treatment gavage occurred twice daily for 7 days pre-MTX and 5 days post-MTX. Intestinal permeability was assessed on days -7, -1, 2, and 5 of the trial. Intestinal sections were collected at sacrifice for histological and biochemical analyses. Histology revealed that rats receiving CY and SY did not have a significantly damaged duodenum compared to controls. However, an improved small intestinal barrier function was evident, determined by a decreased lactulose/mannitol ratio. Probiotics containing SY and CY may be useful in preventing disruption to intestinal barrier function in MTX-induced mucositis.     

The Effects of N-Acetylcysteine on Bile Duct Ligation–Induced Liver Fibrosis in Rats

Stellate cells are activated by free radicals, and synthesize collagen. N-acetylcysteine (NAC) is a precursor of reduced glutathione and a potent scavenger of hydroxyl radicals and has potential antifibrotic effects. We aimed to test the effects of NAC on bile duct ligation (BDL) induced liver damage in rats. Forty-seven Wistar rats were divided into 5 groups: group 1, BDL + NAC (n = 10); group 2, BDL (n = 10); group 3, sham + NAC (n = 10); group 4, sham (n = 10); and group 5, control group (n = 10). NAC (50 μmol/kg per day) or saline of single doses were administered intraperitoneally for 28 days. Serum biochemical and liver oxidative stress parameters were studied. Liver collagen level was determined by the method of Lopez de Leon and Rojkind. Liver slides were stained by hematoxylin and eosin and Masson trichrome\Gomory reticulum staining. Aspartate aminotransferase (AST) and alkaline phosphatase levels in the BDL + NAC group were lower than the BDL group and were higher than the control groups (all P < .001). Malondialdehyde, luminal, and glutathione levels in group 1 were lower than the BDL group (P = .01, P = .002, and P < .001) and higher than the control groups (all P < .001). NAC had no effect on alanine aminotransferase (ALT), gammaglutamyl transferase, bilirubin, albumin, or lucigenin levels. Liver collagen levels were higher in the BDL groups (P < .001); however, NAC had no effect on the collagen levels. The BDL groups showed stage 3 fibrosis; all the control groups were normal. NAC improved some biochemical parameters (AST, alkaline phosphatase) and oxidative stress parameters (malondialdehyde, luminol, glutathione) in the BDL model. NAC was found to be effective on cholestasis-induced hepatotoxicity. However, NAC was inefficient as an antifibrotic agent within a 1-month period of administration in the BDL model.