Acta Pbysiol Scand 1995, 153, 249-254

Colchicine induces enhanced intestinal permeability in the rat

A. F R A D K I N , ' J. YAHAV,l A. DIVER-HABER, ' D. Z E M E R ' and A. JONAS ' 'The Paediatric Gastrointestinal Unit and the Heller Institute for Medical Research, Chaim Sheba Medical Center, TeI Hashomer, Israel

FRADKIN, A, , YAHAV, J., DIVER-HABER, A,, ZEMER, D. & JONAS, A. 1995. Colchicine induces enhanced intestinal permeability in the rat. Acta Physiol Scand 153, 249-254. Received 31 May 1994, accepted 20 October 1994. ISSN 0001-6772. Paediatric Gastrointestinal Unit and the Heller Institute for Medical Research, Chaim Sheba Medical Center, Tel Aviv University, Israel.

Intestinal permeability was determined in rats receiving colchicine 0.5 f 0.15 mg day-' in drinking water (30 mg L-') for periods up to 23 days. The lactulose/mannitol method was used to determine whole gut permeability before and on days 2,4, 8, 18 and 23 of colchicine administration. The 8-h urinary lactulose excretion following the test meal increased significantly in rats receiving colchicine, compared with the pretreatment value. Increased lactulose Permeability was present after 2 days and remained stable throughout the experimental period. Mannitol urinary excretion was not changed. Colchicine increases intestinal tight junction permeability by an as yet undetermined mechanism.

Key zvords: colchicine, intestinal permeability, lactulose, mannitol.

Colchicine has been in use for more than 200 years. Initially used for the treatment and prevention of gouty arthritis, the drug has been reported to benefit patients with a variety of conditions associated with chronic inflammation and fibrosis (Souder & Roudle 1986, Seidman et al. 1987, Bodenheimer et al. 1988). In familial Mediterranean fever (FMF) the drug has been proven useful both in inducing clinical remission as well as preventing renal deterioration by amyloid deposits (Zemer et al. 1974, 1986).

Despite the excellent safety profile of col- chicine in the long-standing prophylaxis of gout and FMF, a number of serious but rare side effects have been described : agranulocytosis, aplastic anaemia, myelopathy and neuropathy (Kuncl et al. 1987). The most common side effect by far relates to gastrointestinal function : nausea, vomiting, diarrhoea and abdominal pain occur in a significant percentage of patients,

Correspondence : Anita Jonas, M.D., Paediatric Gastrointestinal Unit, Chaim Sheba Medical Center, Tel Hashomer 52621, Israel.

seem to be dose related, are difficult to control and interfere with the patients' therapeutic compliance (Peters et al. 1983).

Information in humans regarding the effect of colchicine on intestinal function has been gathered from a small number of volunteers receiving the drug for 4-8 days at high doses. Increased stool nitrogen and electrolyte losses and decreased D-xylose and B12 absorption were described (Race et al. 1970). Small intestinal histology was studied in three patients and showed decreased lactase activity in all and significant light microscopic pathology in one patient. Small bowel biopsies of nine F M F patients on long-term colchicine therapy revealed a pattern of mild hyperplastic cryptvillous atrophy indicative of an increased cell turnover (Hart et al. 1993).

In the experimental animal, treatment with colchicine for brief periods of time induced structural and functional pathology which seemed to be dose related. Intraperitoneal injection of 0.1 mg 100 g-' body weight for 3 days in young rats produced marked inflam-

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250 A. Fradkin et al.

mation of the lamina propria but little villous changes (Levin 1966). In uivo perfusion and in vitro uptake studies of monosaccharides showed significantly decreased absorption of all sugar tested. However, these animals were ill and many died 72 h after the drug was initiated. Oral administration of colchicine to rats a t daily intake of 4-5 mg kg-' body for 2 weeks decreased disaccharidase activity. T h i s was associated with increased cellular renewal (Herbst et al. 1970). Severe diarrhoea and small intestinal destruction occurred at doses of 6 mg kg-' body wt.

Ultrastructural studies of colchicine induced alteration of the small bowel mature enterocytes showed redistribution of plasma membrane con- stituents from the apical to the basolateral membrane (Pavelka et al. 1983). Incorporation of labelled glycoprotein precursor into micro- villous membranes was delayed in the apical membrane and increased in the basolateral membrane as a result of microtubular alteration induced by the drug (Ellinger et al. 1983).

T h e intestinal epithelium forms a functional barrier against the permeation of antigenic components. Measuring intestinal permeability appears to provide a useful and reliable index of mucosal integrity. We studied the effect of long- term low dose colchicine administration on intestinal permeability to mannitol and lactulose in the rat.

METHODS Animals and experimental designs. Wistar rats

weighing 15@200 g were housed in individual wire bottom cages and received water and standard rat chow ad lib. The animal cages were maintained at a constant temperature with a 12-h light/dark cycle. Groups of five rats received colchicine (Sigma) dissolved in water at a concentration of 30 mg L-' for 8 days (acute experiments) and 23 days (chronic experiment). The quantity of water consumed was monitored daily and body wt weekly. Special attention was given to general appearance, activity and stool consistency. Permeability tests were performed before and on days 2, 4, 8, 18 and 23 of colchicine administration. Rats were transferred to individual metabolic cages 3 days before and during the permeability test. Food was removed 10 h prior to the test, but free access to water was allowed. Urine was collected. At 08.00 h 0.4 mL of the test solution containing 50 g L-' lactulose, 20 g L-' mannitol and 223 g L-' glucose was administered by gastric lavage and water was removed for the next 2 h. Free access to water was allowed thereafter. Urine was collected

for 8 h following the test solution administration, measured and stored at - 20 "C. Urine lactulose and mannitol concentration was determined expressed as the per cent recovery of the ingested dose and the L/M ratios calculated.

Serum colchicine levels obtained at the end of the chronic experiment were determined in the Clinical Pharmacology Unit (Halkin et al. 1980).

Analytical methods. Urine mannitol was assayed by a modified spectrophotometric method, following its oxidation with periodate (Corcoran et al. 1947). Because of the presence of small amounts of en- dogenous mannitol, pretest urine samples served as blanks. The precision of the assay for mannitol recovery varied between 89 and 106y0, with a coefficient of variation between assays of i 6.5 yo.

Urine lactulose was measured enzymatically after hydrolysis by P-galactosidase in which lactulose was first converted to fructose and galactose, followed by spectrophomatical determination of equimolar products of NADPH (Behrens et al. 1984). As a result of the possible interference by the presence of free monosaccharides in urine samples and especially by the presence of lactose which serves as a substrate for the same enzyme, a series of precautions were used: blanks of urine samples in which P-galactosidase was omitted were performed and staging of the reaction by separate addition of isomerase followed by subtraction of the various spectrophotometrical readings was performed (Northrop et al. 1990). Following this procedure the precision of lactulose recovery rate was 98 to 1 0 2 ~ 0 with a co-efficient of variation between assays of 3.1 Yo

SD. Stat- istical analysis was performed using an IBM computer with STA PAK 4.11 program, Northwest Analytical, Inc., Portland, Oregon. Student's t-test was performed and values of P < 0.05 were regarded as significant.

Statistics. Data are presented as mean

RESULTS

Rats consumed daily 16.4f4.9 m L H,O during the first 8 days of colchicine administration and 19.2k3.6 m L during the chronic experiment. T h e calculated dose of drug considered per animal was 0.5 f0.15 mg day-'. Mean serum colchicine concentration in the rats completing 23 days of drug administration was 3.82

2.7 ng mL-' (range 1.0-6.7 ng mL-l). These values are comparable with those recorded in FMF patients receiving 1-2 mg day-' colchicine therapy (Halkin et al. 1980).

Food intake and weight decreased starting 48 h after colchicine administration. Mean weekly weight gain in animals housed in indi- vidual cages under normal laboratory conditions

Colchicine increases intestinal permeability 25 1

Table 1. Permeability test in five rats receiving daily colchicine 0.5 f0.15 mg P.O. in a 30 mg L-' solution for 8 days. (Values are mean f SD)

Days of colchicine administration yo Urinary recovery No. 0 2 4 8

Mannitol 5 5.2f1.9 5.8+ 1.7 4.0f 1.0 5.3 2 1.9 Lactulose 5 0.75 f 0.3 1.720.3"" 1.4+0.1*" 1.3f0.3" L / M ratio 5 0.17f0.05 0.62&0.4*"* 0.3510.08*'* 0.3f0.14***

* P < 0.05, ** P < 0.02 and ***P < 0.006 - vs. control period.

was 12.0+ 2.5 g. This decrease to 6.5 & 1.7 g for rats housed in metabolic cages and declined further to 4.5 & 1.8 g in animals receiving colchicine. Except for occasional loose stools diarrhoea was not observed. Two of the five gQE animals in the chronic experiment became severely anorectic and presented blood stained nasal discharges during the last week of drug gQa administration. They were therefore excluded 2

Intestinal permeability was investigated in the first group of five rats before and after 2, 4 and 3 8 days of colchicine administration (Table 1). zo.2 Mannitol urinary recovery was not altered by 2 colchicine administration and was stable during the whole period tested. Lactulose excretion increased significantly after colchicine ad- ministration; the highest values were observed after 48 h of treatment and remained thereafter at high values throughout the experiment. The L/M ratio paralleled the lactulose values

Lactulose recovery and the L / M ratio for the p individual animal are shown in Fig. 1. Wide indi- vidual variations were observed, but with one exception, control values are clearly separated from those obtained after colchicine administra- c> 1.0

tion (mean L / M ratio at 48 h P < 0.006, at 0 5 96 h P < 0.02 and at 192 h P < 0.05). L

In the rats receiving colchicine therapy for prolonged periods, intestinal permeability was tested after a total cumulative dose of 10.8 + 1.8 mg rat-' (18 days) and 13.6f 1.7 mg rat-' (23 days). Table 2 shows values of mannitol and lactulose recovery in these animals. Despite

a

from the study. 3 f 0.4

2.0

5 (Table 1). F

4 "'/ d

ours 6 4.8 ds lo1 significant side effects observed, the animals Fig. 1. Per ceht urinary lactulose excretion and showed normal values of mannitol absorption and urinary recovery. Lactulose excretion and the L/M ratio were increased as 'Ompared with the pre-treatment period.

lactulose/mannitol ratios of individual rats before (0) and after 1.1 483 96 and 192 Of colchicine administration at daily doses of 0.5 f0.15 mg rat-'. Bars represent means for statistics, see Table 1.

252 A. Fradkin et al.

Table 2. Permeability test of three rats receiving daily colchicine 0.5f0.15 mg P.O. in a 30 mg L-' solution for 23 days. (Values are meanf SD)

Days of colchicine administration yo Urinary recovery No. 0 18 23

Mannitol 3 5.2f2.0 5.0f 1.4 4.4+ 1..3 Lactulose 3 0.85f0.3 1.64i0.7' 1.76 f 1.14"' L /M ratio 3 0.12 i 0.08 0.34 i 0.14"" 0.36 f 0.28"

* P < 0.004 and ** P < 0.03 - vs. control period.

D I S C U S S I O N

Our study demonstrates a clear-cut increase of intestinal permeability in colchicine-treated rats. This was mainly due to increased lactulose permeation. Mannitol passage remained un- changed.

Lactulose excretion in normal, untreated rats receiving a test load of 20 mg lactulose in the range 0 . 4 1 % during a 5-h urine collection, values which fall within the range of results obtained in other studies using this probe (Cobden et al. 1981, Turner et al. 1988). In the experimental animal altered permeability has been described in situations of mucosal injury associated with intestinal ischaemia-reperfusion injury (Horton & Walter 1993), infection with Ni'ppostrongylzls brasiliensis (Cobden et al. 1979) or by the use of detergents (Cobden et al. 1981).

In vivo studies of the entire gut length such as performed in our study are rare. Using this method, uraemic rats were found to have increased intestinal permeability to PEG (Magnusson et al. 1992), whereas the induction of intestinal hypersensitivity increased perme- ability to lactulose (Turner et al. 1988).

Altered intestinal permeability is considered a clear-cut predictor of intestinal pathology, although the precise mechanism or even location of events are still being investigated (Bjarnason et al. 1986).

Among the various probes used to measure intestinal permeability, the double probe method used in our study has several advantages: factors such as transit time, renal function or com- pleteness of urine collection have little effect on the recovery ratio of the two probes.

Using this method the lactulose mannitol ration was found to be increased in conditions associated with mucosal injury and inflammation

(Isolauri et al. 1989, Juby et al. 1989, Katz et al. 1989) or induced by drugs such as NSAIDS (Bjarnason et al. 1989, Krugliak et al. 1990). In all these situations, as in our study, the constant finding is enhanced lactulose permeability, whereas mannitol excretion remains unchanged or decreases.

The overall assumption is that a leaky epithelium will enhance lactulose absorption by passage through paracellular tight junction. The fact that mannitol, although of smaller molecular weight and radius, is not similarly affected has not been well explained. The concept of transcellular passage of mannitol through mem- brane pores of the enterocyte and its decrease in situations associated with reduced mucosal sur- face has not been sustained by in vitro permeability studies and the validity of this mechanism is controversial among authors.

We found lactulose permeability to be significantly enhanced in rats receiving col- chicine. We found similar results in colchicine treated FMF patients (unpublished data). Blood levels in treated animals were within the therapeutic range level observed in humans. This is especially important, given the dose related effect of the drug on other parameters of internal injury such as inflammation, cellular renewal and enterocyte enzyme deficiency.

The effect of the drug became evident after 48 h of administration and remained stable throughout the experimental period. The rap- idity of action of the drug is in keeping with previous studies following morphologic and enzymatic changes of the small intestine after a single injection of the drug (Herbst et al. '1970).

Colchicine has been shown to influence a wide range of cellular processes through its binding to microtubules. The redistribution of membrane constituents of enterocytes especially of apical

Colchicine increases intestinal permeability 253

vs. basolateral plasma membrane (Pavelka et al. 1983) can easily affect structure and function of the tight junction. Such alterations have been known to occur in hepatocytes (Rassat et al. 1982) and pancreatic acinar cells (Meldolesi et al. 1978) following treatment with antimicrotubular agents.

Conversely, increased lactulose permeability could be the result of enhanced colchicine induced cell proliferation and migration along the crypt villous (Herbst et af. 1970, Horton et al. 1993) in a way similar to that occurring in diseases like celiac disease and in some intestinal infection.

T h e relevance of these subtle functional changes of the intestinal mucosa for patients receiving the drug for long periods of time has to be viewed with caution. Although similar per- meability changes have been observed by us in patients on long-term therapy, they did not correlate with the clinical gastrointestinal side effects and are probably not responsible for the drug induced diarrhoea and abdominal pain. Nevertheless, chronic intestinal hyper- permeability may have a potentially harmful effect in the long range.

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