The enterohepatic circulation of methotrexate in vivo: Inhibition by bile salt

Summary We examined the enterohepatic circulation of methotrexate (MTX) in the rat in vivo and determined the effect of the unconjugated bile salt, cholate, on the process. MTX (70 mg/kg body weight) was administered i.v. and the bile salt (1 mM) was delivered through intestinal perfusion. In the control group 38.43%±4% of the administered dose of MTX appeared in bile 2 h after administration of the drug. In the bile salt-treated group 21.4%±3.7% of the administered does of MTX appeared in bile, which was significantly lower (P<0.01) than the proportion in the control group. The liver content of MTX was depressed by 23% in the bile salt-treated group compared with the control group. This study demonstrates, in vivo, the important role that the enterohepatic circulation plays in exposing the small intestine to toxic levels of MTX and shows that the unconjugated bile salt, cholate, inhibits the process.This study was supported by NIH Grant NIADDKD 5P30AM 26657. Donald Griffin is a recipient of an NIH student training award, grant no. 5R25CA 19429-10     

Effect of repeated intraperitoneal chemotherapy with epirubicin on the hepatic transport of bile acids and their enterohepatic circulation

The effect of repeated intraperitoneal perfusion with epirubicin on the clearance of 14C-taurocholate by the liver and the enterohepatic circulation of the synthetic bile acid 75-SeHCAT were investigated using a rat model. After six treatments there was no significant difference in the transport rate constants (plasma to liver, liver to bile and liver to plasma) between and within the saline and epirubicin groups. Similarly, there were no differences detected between the groups for the parameters derived from these transport rate constants. Thus the initial plasma clearance after six perfusions was 39 +/- 9, and 36 +/- 11 ml/min/kg in the epirubicin and saline groups respectively. The excretory efficiency of the liver at this stage was identical: 67 +/- 10% in the epirubicin treated animals and 67 +/- 6% in the saline controls. A deterioration in the SeHCAT retention was observed after repeated intraperitoneal perfusion in both groups. This was significant only in the cytotoxic group, between the first and sixth epirubicin perfusion: 59 +/- 9% vs 48 +/- 9% at 24 h (P = 0.03), 31 +/- 8% vs 22 +/- 5% at 48 h (P = 0.019) and was not cumulative beyond this stage. These findings indicate that repeated intraperitoneal perfusion chemotherapy with epirubicin does not impair bile acid clearance by the hepatocyte. The decrease in the retention of SeHCAT is unlikely to be the result of epirubicin-induced ileal mucosal damage since the reduction was not cumulative beyond 48 h of administration of the compound.     

Inhibition of intestinal P-glycoprotein and effects on etoposide absorption

 P-glycoprotein (Pgp) actively pumps a number of antineoplastic drugs, such as etoposide, out of cancer cells and causes multidrug resistance. Pgp is also expressed at the brush–border membrane of the small intestine under normal physiological conditions. We hypothesized that inhibition of intestinal Pgp might decrease the efflux of etoposide from the blood into the intestinal lumen, thereby, increasing the bioavailability of etoposide. The absorption of etoposide was studied using everted gut sacs prepared from rat jejunum and ileum. The addition of C219, a monoclonal antibody of Pgp, at 100 ng/ml or of 0.2 M 5′-adenylylimidodiphosphate, a nonhydrolyzable adenosine triphosphate (ATP) analog, increased the absorption of etoposide. Quinidine, an antiarrythmic agent, has been demonstrated to circumvent multidrug resistance in cell lines, possibly by interfering with Pgp function. Adding quinidine at 1 mg/ml to the everted gut sac increased the absorption of etoposide. In vivo absorption of etoposide was also studied by intraluminal perfusion of the drug in the small intestine of anesthetized rats. Intravenous infusion of quinidine at either 1 or 2 mg/h increased the serum level of etoposide in a dose-dependent manner. Intravenous infusion of etoposide at 0.2 mg/h resulted in luminal exsorption of the drug in the small intestine. The intestinal clearance of etoposide was 41.7±7.2 ml kg^-1, which decreased to 18.4±3.9 ml kg^-1 with the infusion of quinidine at 1 mg/h. The present data confirm that intestinal Pgp mediates the efflux of etoposide and that the use of Pgp-inhibiting agents such as quinidine may increase the bioavailability of etoposide. Received: 19 May 1994/Accepted 16 August 1994     

The effect of methotrexate on folate metabolism in the rat.

The metabolism of 2-[14C]-folic acid, 2-[14C]-5-methyltetrahydrofolate 5-[14C]-methyltetrahydrofolate, and a mixture of 2-[14C]-folic acid and 3'',5'',7,9-[3H]-folic acid has been studied in rats that were dosed with methotrexate (MTX) 24 h before receiving the radioactive folate. Methotrexate increases urinary excretion of radioactivity in rats given 2-[14C]-folic acid, but there was no significant increase in urinary radioactivity in animals given 5-methyltetrahydrofolate. Animals dosed with MTX had less of the dose in the liver, and excreted more of the dose via the faeces. These results are consistent with the known biochemical effects of methotrexate. Experiments with a mixture of 2-[14C]-folic acid and 3'',5'',7,9-[3H]-folic acid indicate that there is an increase in scission of the folate molecule following a dose of MTX.     

The effect of pelvic irradiation on the absorption of bile acids

The pathophysiology of radiation-induced diarrhea was evaluated in 17 patients undergoing pelvic irradiation for gynecological malignancies. The glycine conjugates of cholic acid (GC) and chenodeoxycholic acid (GCDC) were measured in serum by radioimmunoassay. Fasting and 2 hr post prandial (pp) determinations were performed prior to and in the fifth week of radiotherapy. The pre-treatment fasting and 2 hr pp GC levels were 0.20 ± 0.29 (mean ± SD) and 0.48 ± 0.47 μM. In the fifth week the fasting and 2 hr pp GC levels were 0.16 ± 0.23 and 0.25 ± 0.27 μM. The first week fasting and 2 hr pp GCDC levels were 0.32 ± 0.47 and 0.80 ± 0.83 μM: in the fifth week they were 0.10 ± 0.06 and 0.33 ± 0.27 μM. The differences between the first and the fifth week post prandial increases in serum GC and GCDC levels were significant (p < 0.02). The reduced post prandial increases in serum GC and GCDC in the fifth week of radiotherapy occurred at a time when the patients' daily stool frequencies were significantly increased (P < 0.01).The data suggest that a cholerrheic enteropathy is the major determinant in the pathophysiology of radiation-induced diarrhea.     

Inhibition of MRP1-mediated efflux in human erythrocytes by mono-anionic bile salts

BACKGROUND: Multidrug resistance-associated protein 1 (MRP1 or ABCC1) -mediated transport is an important mechanism in multidrug resistance during cancer treatment. One strategy for the reversal of MRP1-mediated multidrug resistance is inhibition of this efflux pump. Therefore, efficient inhibitors are searched for and the structure-activity relationships of inhibitors are studied. In the present work, the ability of a series of mono-anionic bile salts to inhibit MRP1-like substrate transport was examined. MATERIALS AND METHODS: The effect of bile salts on the efflux of the MRP1 substrate 2,7'-bis-(carboxypropyl)-5(6)-carboxyfluorescein (BCPCF) from human erythrocytes was fluorimetrically monitored. RESULTS: All bile salts inhibited the BCPCF efflux. The most efficient inhibitor, lithocholic acid, decreased the BCPCF efflux by 50% (IC50) at 4 microM during 60 min of incubation at 37 degrees C. The most efficient bile salt inhibitors showed high haemolysis start/IC50 concentration ratios and did not induce membrane bending or phosphatidylserine (PS) exposure at IC50. CONCLUSION: The overall hydrophobicity, as well as the orientation of the hydroxyl groups and conjugation with glycine or taurine per se, affect the inhibitory potency of bile salts. The most efficient inhibitors apparently interact with MRP1 in a specific way.     

Inhibitory effect of androgens on DMBA-induced mammary carcinoma in the rat

Summary Constant release of the androgen 5-dihydrotestosterone (DHT) in ovariectomized rats bearing DMBA-induced mammary carcinoma caused a marked inhibition of tumor growth induced by 17-estradiol (E₂). That DHT acts through interaction with the androgen receptor is supported by the finding that simultaneous treatment with the antiandrogen Flutamide completely prevents DHT action. Particularly illustrative of the potent inhibitory effect of DHT on tumor growth are the decrease by DHT of the number of progressing tumors from 69.2% to 29.2% in E₂-treated animals and the increase by DHT of the number of complete responses (disappearance of palpable tumors) from 11.5% to 33.3% in the same groups of animals. The number of new tumors appearing during the 28-day observation period in E₂-treated animals decreased from 1.5 ± 0.3 to 0.7 ± 0.2 per rat during treatment with DHT, an effect which was also reversed by the antiandrogen Flutamide. The present data demonstrate, for the first time, that androgens are potent inhibitors of DMBA-induced mammary carcinoma growth by an action independent of inhibition of gonadotropin secretion, and suggest an action exerted directly at the tumor level.     

The effect of different salts of saccharin on the rat urinary bladder

Weanling male F344 rats were fed the sodium, potassium, or calcium salf of saccharin or acid saccharin as 5% of the diet by weight for 10 weeks. Sodium saccharin induced significant urinary bladder epithelial proliferation as determined by the labelling index following [³H] thymidine incorporation and by light and scanning electron microscopic evaluation. Potassium saccharin also significantly increased the labelling index but less than the sodium salt. Calcium saccharin and acid saccharin did not significantly increase the labelling index. Similar differences were evident by light and scanning electron microscopy. These differences were not due to differences in the level of saccharin excretion in the urine following feeding of the various salts. Differences in urinary pH and urinary concentration of sodium and calcium were observed between rats fed the various forms of saccharin.     

The effect of timing of a single injection on the toxicity of methotrexate in the rat

Summary The possibility of a circadian rhythm in the toxicity of methotrexate was investigated in rats after a single intravenous bolus. Indices of haematological, renal and hepatic toxicity were studied, as were the pharmacokinetics of the drug. All the parameters showed a circadian variation, with maximum toxicity occurring after dosage at 06.00 h and minimum toxicity after dosage at midnight. Administration at the other two time points, 12.00 h and 18.00 h, gave intermediate results.     

The suppressive effect of piroxicam on autochthonous intestinal tumors in the rat

Male Lobund strain Sprague-Dawley (SD) rats respond to single doses of methylazoxymethanol acetate (MAM) with high incidence of intestinal tumors within 5 months. Some non-steroidal anti-inflammatory drugs (NSAIDs) can block the synthesis of prostaglandins (PGs) and also interfere with tumor growth. A new class of NSAID, piroxicam, was added to the feed of MAM-treated rats. When examined 150 days later, there was a significant reduction of tumor-bearing rats and of tumors/rat compared to controls on drug-free feed; and there was no evidence of toxicity.     

The effect of abolition of the endogenous corticosteroid rhythm on the circadian variation in methotrexate toxicity in the rat

Summary Monitoring of indices of haematological, renal and hepatic toxicity in rats after a single i.v. bolus of methotrexate has shown that they vary with the time of day at which the drug is administered. Maximum toxicity occurs after administration at 0600 h. Further experimentation has shown that the amount of corticosteroid present in the blood has a profound effect on the toxicity of methotrexate in the rat. If the endogenous production of corticosterone is suppressed by treatment with dexamethasone the toxicity of methotrexate is markedly increased at whatever clock time it is administered. However, if constantly high plasma levels are achieved by giving supplementary corticosterone methotrexate toxicity is diminished regardless of what time it is given.Since the timing of maximum methotrexate toxicity corresponds to the circadian nadir of endogenous plasma corticosterone concentration in the rat the possibility that it might be related to corticosterone production must be considered. Whether this phenomenon occurs in man and has any clinical relevance has yet to be investigated.     

Inhibition of intestinal carcinogenesis in rats: effect of difluoromethylornithine with piroxicam or fish oil

An inhibitor of ornithine decarboxylase, difluoromethylornithine (DFMO), and two inhibitors of prostaglandin biosynthesis, piroxicam and menhaden fish oil, were examined for their effect on intestinal tumorigenesis in male Sprague-Dawley rats fed a 5% fat semisynthetic diet. Each agent was given individually in one of two doses as follows: DFMO, 0.05% and 0.1% in the drinking water; piroxicam, 65 mg/kg diet and 130 mg/kg diet; and menhaden fish oil, 1.25% and 2.50% of the diet. Additional animal groups were given combinations of the lower dose of DFMO and the lower dose of either piroxicam or fish oil. Intestinal tumors were induced by sc injections of azoxymethane (AOM; CAS: 25843-45-2) at 8 mg/kg (body wt) weekly for 8 weeks. Test diets were started 1 week prior to the first dose of AOM, and the rats were sacrificed 26 weeks later. Rats that received either dose of DFMO or the high dose of piroxicam developed significantly fewer intestinal tumors compared to controls. The low dose of piroxicam and the fish oil given at either dose level had no effect. The combination of the low dose of DFMO and the low dose of piroxicam reduced tumor formation more than either dose of DFMO alone, whereas the low dose of DFMO and fish oil together was no more effective than either dose of DFMO alone. These results show that a combination of a small amount of DFMO and piroxicam, each acting through a different mechanism, exerts an additive inhibitory effect on intestinal tumor formation in rats.     

谷氨酰胺对甲氨蝶呤所致大鼠肠黏膜炎症的保护作用

目的:通过研究甲氨蝶呤(methotrexate,MTX)模型鼠小肠炎症,观察谷氨酰胺(glutamine,GLN)对MTX所致大鼠肠黏膜的保护作用及肠黏膜炎时紧密连接蛋白的变化,探讨GLN保护作用的可能机制。方法:SD大鼠随机分为三组,每组8只。CON组(control group):腹腔注射生理盐水(normal saline,NS)1ml连续2天,每日口服灌胃NS 1ml。MTX组:腹腔注射MTX 1ml(20mg/kg)连续2天,每日口服灌胃NS 1ml。GLN组:腹腔注射MTX 1ml(20mg/kg)连续2天,每日口服灌胃GLN 1ml[2g/(kg·d)]。各组大鼠于注射MTX后第8天处死,观察MTX模型鼠小肠炎症时一般状态、免疫组化、荧光实时定量PCR检测紧密连接蛋白的表达。结果:GLN组、MTX组与对照组比较Occludin和Claudin-1的基因表达水平均有减低,较对照组有显著差异(P<0.05)。MTX组的基因表达均较GLN组有所下调,差异显著(P<0.05),GLN组表达相对增加,提示肠黏膜炎时对紧密连接蛋白表达有保护作用。结论:MTX可诱导鼠发生小肠炎症,GLN对肠黏膜炎症有保护作用,其作用机制可能与肠上皮细胞间紧密连接蛋白调节有关。     

The effect of cotrimoxazole on the absorption of orally administered 6-mercaptopurine in the rat

Summary The effect of cotrimoxazole (CTX) on plasma levels of 6-mercaptopurine (6-MP) was studied in the rat. Animals receiving CTX in conjunction with 6-MP were found to have a marked but non-significant decrease in the area under the plasma time curve as compared with animals receiving 6-MP alone. It is suggested that the bioavailability and thereby, the antileukaemic effect) during maintenance therapy of ALL of 6-MP may be decreased by the co-administration of CTX.     

Inhibitory effect of some intestinal bacteria on liver tumorigenesis in gnotobiotic C3HHe male mice

Liver tumorigenesis in gnotobiotic $\text{C3H}\text{He}$ male mice was markedly promoted by association with a bacterial combination of Escherichia coli, Streptococcus faecalis, and Clostridium paraputrificum. This study demonstrated that this promoting effect was suppressed by addition of certain intestinal bacteria, such as Bifidobacterium longum, Lactobacillus acidophilus, and Eubacterium rectale.     

The effect of antineoplastic agents on the healing of small intestinal anastomoses in the rat

The influence of an intravenous 5-day combined chemotherapy with bleomycin (2 mg/kg/d), 5-fluorouracil (10 mg/kg/d) and cis-diamminedichloroplatinum (0.35 mg/kg/d) on the healing of ileal anastomoses was investigated in rats. Ninety-six male Wistar rats were used, divided into four groups. The rats in the control group had surgery without administration of cytostatic agents. The other rats were operated either 2 days after, 2 days before, or during the 5-day chemotherapy course. In each group, rats were killed after 3, 7, and 21 days. Anastomotic healing was assessed by measurement of bursting pressures and hydroxyproline levels. Intestinal healing appeared to be impaired most if the operation was performed in the middle of the antineoplastic chemotherapy course. The effects were most pronounced on the seventh postoperative day. Surgery on the second day after the chemotherapy course led to a slight and early delay in wound healing as measured by the hydroxyproline content. Seven days postoperatively, concentrations had returned to preoperative values. Surgery 2 days before chemotherapy induced only minor differences with respect to the control group. In all groups, bursting pressure and hydroxyproline content at 21 days were similar. Thus, antineoplastic agents retard but do not prevent healing of intestinal anastomoses. The effects are most pronounced when surgery is performed during chemotherapy. If possible, surgery should be performed prior to chemotherapy. Increasing the time interval between surgery and chemotherapy may decrease the delay in intestinal woundhealing.     

γ射线对大鼠血管平滑肌细胞增殖和周期影响

目的：探讨γ射线对大鼠血管平滑肌细胞（VSMCs）抑制作用机理。方法：对大鼠VSMCs进行原代培养，经^3H-TdR掺入法观察γ射线对VSMCs增殖情况影响，应用流式细胞仪检测γ射线对VSMCs细胞周期的影响。采用Western Blot技术检测γ射线对VSMCs p53、细胞周期素D（cyclin D)和增殖细胞核抗原（PCNA）表达的影响。结果：γ射线对寻殖的抑制作用呈剂量依赖性。γ射线照射可诱导VSMCs出现G1期阻滞，在照射后一定时间内会导致p53表达增强，cyclinD和PCNA表达减弱，结论：γ射线可抑制VSMCs增殖，其机制可能主要是γ射线诱导VSMCs出现G1期阻滞，抑制VSMCs进行有丝分裂，在这个过程中p53,cyclinD和PCNA等分子起到一定作用。     

Protective effect of aged garlic extract (AGE) on the apoptosis of intestinal epithelial cells caused by methotrexate

Purpose  Methotrexate (MTX) causes intestinal damage, resulting in diarrhea. The side effects often disturb the cancer chemotherapy. We previously reported that AGE protected the small intestine of rats from the MTX-induced damage. In the present paper, the mechanism of the protection of AGE against the MTX-induced damage of small intestine was investigated, using IEC-6 cells originating from rat jejunum crypt. Methods  The viability and apoptosis of IEC-6 cells were examined in the presence of MTX and/or AGE. Results  The viability of IEC-6 cells exposed to MTX was decreased by the increase of MTX concentration. The MTX-induced loss of viable IEC-6 cells was almost completely prevented by the presence of more than 0.1% AGE. In IEC-6 cells exposed to MTX, the cromatin condensation, DNA fragmentation, caspase-3 activation and cytochrome c release were observed. These were preserved to the control levels by the presence of AGE. MTX markedly decreased intracellular GSH in IEC-6 cells, but the presence of AGE in IEC-6 cells with MTX preserved intracellular GSH to the control level. IEC-6 cells in G2/M stage markedly decreased 72 h after the MTX treatment, which was preserved to the control level by the presence of AGE. These results indicated that AGE protected IEC-6 cells from the MTX-induced damage. Conclusions  The MTX-induced apoptosis of IEC-6 cells was shown to be depressed by AGE. AGE may be useful for the cancer chemotherapy with MTX, since AGE reduces the MTX-induced intestinal damage.     

Inhibitory effect of linoleic acid on transformation of IEC6 intestinal cells by in vitro azoxymethane treatment

The effect of linoleic acid (LA) on growth and transformation of IEC6 intestinal cells was examined. IEC6 cells expressed mRNAs of 15-lipooxygenase (LOX15) and peroxisome proliferator-activated receptor (PPAR)gamma but not COX-2. Cell growth was suppressed by LA in a dose-dependent manner in IEC6 cells. Three-week treatment with LA provided IEC6 cells a quiescent state. LA-induced growth inhibition was abrogated by exposure to antisense S-oligodeoxynucleotides (S-ODNs) for LOX15 and/or PPARgamma. In an in vitro carcinogenesis model, IEC6 cells, which had confirmed CYP2E1 expression and activity, were continuously treated with AOM and/or LA for 40 weeks. DNA injury in AOM-treated cells was suppressed to the control level by concurrent LA treatment. Colony formation of AOM-treated cells in soft agar was suppressed by treatment with LA, which was reversed by exposure to antisense S-ODNs for LOX15 and/or PPARgamma. AOM-treated IEC6 cells formed s.c. tumors in 9 of 12 mice, whereas AOM+LA-treated cells formed no tumor. IEC6 cells showed no remarkable alteration of protein production by AOM treatment, whereas cells treated with AOM+LA showed decreased epidermal growth factor receptor (EGFR) and phospho-EGFR and increased BAX. These findings suggest that LA inhibited AOM-induced transformation of COX-2-negative IEC6 cells, which was possibly mediated with PPARgamma ligands generated by LOX15 from LA.