Reduction of the enterohepatic circulation of norethisterone by antibiotics in the rat: Correlation with changes in the gut flora

ofSreroid Biochemistry, Vol.13,pp.95to 100 Petgamon Press Ltd1980. Printed inGreatBritain

Journal

REDUCTION OF THE ENTEROHEPATIC CIRCULATION OF NORETHISTERONE BY ANTIBIO~CS IN THE RAT: CORRELATION WITH CHANGES IN THE GUT FLORA D. J. BACK, A. M. BRECKENRIDGE,FRANCESCA E. CRAWFORD, J. CROSS, ML’E. ORME, A. PERCIVAL* and P. H. ROWE

KAREN

The Departments of Pharmacology and Therapeutics and *Medical Microbiology, University of Liverpool, P.O. Box 147, Liverpool, L69 3BX, England (Received 22 March 1979) SUMMARY

When bile containing tritiated conjugates of norethisterone, collected from donor rats, was infused either into the duodenum or caecum of recipient rats, 23.5% and 42.4% of the dose was excreted in the bile respectively. Pretreatment of recipient rats with ampicillin or neomycin (2OOmg/kg/day for 4 days) or neomycin + lincomycin (IM) + 100 mg/kg/day for 4 days) significantly reduced (P < 0.001) the amount of steroid appearing in the bile of recipient rats following intracaecal administration of conjugates. There was a correlation between the reduction in enterohepatic circulation (EHC) and suppression of the gut flora. Ampicil~n treatment caused a reduction in the anaerobic element and partial suppression of aerobes. Neomycin pretreatment caused a great reduction in the aerobic flora, but only a small reduction in anaerobes. Following neomycin + hncomycin treatment both the aerobic and anaerobic flora were dramatically reduced. Rifampicin treatment (200 mg/kg/day for 4 days) did not cause a reduction in the EHC of norethisterone, or in the concentration of aerobic or anaerobic flora in the caecum. However, the aerobic flora were rifampicin resistant. To investigate the onset of this resistance recipient rats were treated with rifampicin for 1, 2 or 3 days and a graded effect both on the EHC of the steroid and the incidence of resistant organisms was evident. Following ampicillin administration (4 days) both the EHC and the gut flora were normalized 14 days after the final drug treatment.

INTRODUCTION

vehicle under nitrogen the steroid was redissolved in a mixture of 0.9% saline:ethanol (6:1, v/v) to a final con~ntration of 10 &i/ml.

We have previously reported that antibiotics interfere with the enterohepatic circulation (EHC) of the synthetic steroids norethisterone and ethinyloestradiol [l]. Using a linked rat preparation in which the bile duct cannula from a “donor” rat was inserted into the duodenum of a “recipient” rat, it was established that ampicillin and neomycin gave a similar significant reduction in the EHC. It has also been shown that neomycin reduces the EHC of mestranol, a synthetic oestrogen [2]. In this study it was demonstrated that the ability of rat caecal micro-organisms to hydrolyse conjugates of mestranol in vitro was considerably inhibited in the presence of neomycin. The aim of the present work was to examine whether or not there was a correlation between the antibiotic induced reduction in the EHC of one of the steroids, norethisterone, and changes in the gut microflora.

Animals

Male rats of the Wistar strain (220-35Og) were housed in groups in cages in well-ventilated rooms at a temperature of approx. 24°C. Collection of “donor”

MATERIALS AND METHODS Radioactive

material

[6,7(n)-3H]-Norethisterone (C3H]-N; 21.6 Ci/mmol) was obtained from the Radiochemical Centre, Amersham, England. After evaporation of the organic S.B. 13/1--c

biZe

Rats were anaesthetized with urethane (14% w/v in 0.9% saline; 10.0 ml/kg; i.p.). Polyethylene catheters (PE 50) were inserted into a femoral vein and the common bile duct. E3H]-N (lO~Ci/mI; lO,uCi/kg; 125 pg,%g) was injected iv. and bile collected over a period of 4 h. Bile was pooled from several rats and subsequently divided into 1 ml aliquots. Each aliquot was shaken with ether (4 ml) for 20 min on a rotary shaker. After centrifuging (1000 g for 1 min) the bile layer was frozen in a bath of meth~ol~ardi~. The ether layer, containing ether-extractable, “free” steroids was discarded. The “donor” bile was repooled, heated in a water bath at 37°C to remove any residual ether and then diluted with 0.9% saline to give a radioactive content 95

96

D. J. BACK et al. (c) Effect of pretreatment with rijiipicin for 1, 2, 3, or 4 days. In other studies, “recipient” rats were

of approx. lo6 d.p.m./ml. The “donor” bile prepared in this way was deemed to contain only conjugates of norethisterone.

treated with rifampicin (2OOmg/kg) for 1, 2, 3 or 4 days. Following the final drug administration rats were starved overnight and the infusion study performed. (d) Effect of various timings of studies after pretreatment with ampicillin. In further studies, “recipient” rats were pretreated with ampicillin (2OOmg/kg/day for 4 days, orally). Following the final drug treatment, rats were allowed unrestricted access to food and water for either 6, 13 or 20 days. Infusion studies were performed on the 7th, 14th or 21st days following the termination of drug treatment. In all the above studies “donor” bile was infused intracaecally to anaesthetised “recipient” rats. Bile was collected at intervals of 30 min for 4 h. The radioactive content was subsequently determined. Microbiological studies. At the termination of each in uiuo investigation, the gastrointestinal tract was removed, sectioned into duodenum, upper small intestine, lower small intestine, caecum and colon and the contents placed in sterile containers. Methods for quantitative cultures of intestinal contents were as previously reported [3]. The bacteria present were counted by the method of Drasar[4].

Infusion of steroid conjugates into “recipient” rats

(a) Different routes of infusion. “Recipient” rats were anaesthetized and bile ducts cannulated as previously described. “Donor” bile was infused either intraduodenally (id.; via a cannula tipped with a broken 23 gauge needle) or intracaecally (i.c. via a cannula tipped with a broken 23 gauge needle) at a constant rate of l.Oml/h for a period of approx. 2.0 h using a constant infusion pump (Perfusor IV, Braun, Melsungen). Bile samples were collected in preweighed glass vials at 30min intervals for 6 h. The radioactive content was determined in aliquots (2011) of bile by liquid scintillation spectrometry using a commercial scintillant (NE 260; Nuclear Enterprises; Edinburgh). (b) Eflect of pretreatment with various antibiotics. “Recipient” rats were pretreated with ampicillin sodium, neomycin sulphate or rifampicin (all 2OOmg/ kg/day for 4 days orally) or neomycin + lincomycin (100 + lOOmg/kg/day for 4 days orally). All drugs except rifampicin were administered in a volume of 0.1 ml physiological saline/100 g body weight. Rifamsaline picin was suspended in physiological (200mg/ml) and sufficient NaOH (4 M) added to achieve solution. Two control groups were used: one group received physiological saline (0.1 ml/lOOg.b.w.) the other physiological saline + NaOH and (0.1 ml/100 g.b.w.). Following the final drug administration rats were starved overnight and the infusion study performed on the 5th day.

so/ ,,,,,,,,,,,

RESULTS

D@rent

routes of administration

The biliary excretion of radioactivity following id. and i.c. infusion of “donor” bile is shown in Fig. 1.

/,//////a

1

1~’

40

0

r

I/’mtraceecal

!A’.

P

.c

I/ I . l,int
Time

I/

(h)

Fig. 1. Percentage cumulative excretion of radioactivity in bile following intracaecal (W) or intraduodenal (o---+) administration of C3H]-norethisterone conjugates. Hatched bar indicates time of conjugate infusion. Each point is mean $- SEM of four experiments.

Antibiotic-steroid

interaction

97

Fig. 2. Percentage cumulative excretion of radioactivity in bile following iatracaecal admit&ration of [%I>-norethisterone conjugates to control (M), rifampicin f&--H), neomycin (&-----A), neomycin + tincomycin (A------A) and ampicillin @ -5) treated rats. For details see text. Hatched bar indicates time of conjugate irrfusion, Each point is the mean + SEM af brir experiments, Table 1. Effect of Chronic antibiotic treatment (200 mg/kgf day for 4 days) on the EHC of norethisterone and the gut ffora

Treatment

% Excretion in bile

Contro! AmpicifXin Neomycin Neo c Linco

42,4 4 4.2*** f 136**** 4:9*** 5

Rifampic&I

2.6 0.8 3.3 0.3

47.5 & S.7

Caecal flora M.An / ; LFC 11 M.An$; LFQ ~‘1 M.AnEj; LFCt; yt M,Ant; No. LFC YP M.&t [I; LFC (IF

*+* Significantly different from controk P < 0,001. M.An-Mixed anaerobes (Cfastrkiiu, Bacteroidesl. LFC-Lactose fermenting coliforms (E. coii Strep. faemh). t < 103/ml; $ 103-H35/ml; 0 I@-107/ml;

I/ l~‘-l~~#/rnl~

f Rifampicin resistant. 23.5 + 5.0% and 42.4 &- 2.4% of radioactivity was recovered after id. and i.c infusion respectively. There was no significant difference in biliary excretion of radioactivity following i,c. infusion to rats which had received onty physiological saline or ~hysio~o~~i saline plus sodium hydroxide The controls referred to in the figures and tables are thuse which received only physiofugical saline,

The &ect of antibiotic treatment on the biliy excretion of radioactivity in recipient rats after i.c. infusion of conjugates is shown in Fig. 2 and Table 1. Ampicillin, neomycin and neomycin + lincomycin all significantly reduced (P <: 0.001) the amount of

steroid appearing in the bile of recipient rats. Analysis of the caecat contents after ampicillin pretreatment, showed that the anaerobic element (~a~icu~~~y CIos&G&a and Strqtococcif was largely suppressed. Lactose Fermenting Coliforms (L.F.C.) were partially reduced in some rats and not at all in others; some L.F.C. were resistant to ampicillin, Neomycin pretreatment caused a great reduction in the aerobic (L.F.C.) flora but only a small reduction in anaerobes. Neomycin + lincomycin caused a dramatic reduction in both aerobes and anaerobes with replacement by pure cultures of yeasts. YSMS were also present in the caecal conterrts of ampicilhn and neomycin pretreated rats, In contrast to the above findings, rifampicin pretreatment did not cause a reductiun in the bihary excretion of steroid (Fig, 2) or in the concentration of aerobic or anaerobic flora in the caecal contents. The presence of rifampicin-resistant L.F.C. (Table 1) indicated that they had emerged during the 4 days of rifampicin treatment.

Figure 3 and Table 2 show both the graded ef%bct on the EHC of norethisterone and the onset of rifampi& resistance: following treatment for 1, 2, 3 or 4 days. After all treatments the anaerobic flora remained unchanged frum cuntrols. However, following a single dose, the aerobic flora (L.F.C.) was markedly suppressed (Table 2), and this correlated with a significant decrease (P < 0.001) in the biliary excretion of steroid. After two doses of rifampicin there was a slight increase in L.F.C. and in two of the four

98

D. J.

1

2

BACK et al.

3 Time

4

5

6

(h)

Fig. 3. Percentage cumulative excretion of radioactivity in bile following intracaecal administration of C3H]-norethisterone conjugates to control rats (t-0) and rats treated with rifampicin for 1 (D---Q, 2 (A---A), 3 (A-A) or 4 (BA) days. For details see Text. Hatched bar indicates time of conjugate infusion. Each point is the mean f SEM of four experiments.

Table 2. Effect of rifampicin treatment (200 mg/kg/day) for 1,2. 3 or 4 days on the EHC of norethisterone and the gut flora

Treatment Control Rifampicin -1 day -2 days -3 days 4 days

% Excretion in bile 42.4 + 2.6 8.7*** + 10.3*** * 22.2*** + 47.7 *

2.0 1.3 1.8 5.7

Caecal flora M.AnQ; LFC§ M.An§; M.An§; M.An$; M.An§;

LFCt LFCS 11 LFC§ (1 LFC§il

*** Significantly different from controls, P < 0.001. t < 10” ml; $ 103-105/ml; p 107-1010/ml. 11Rifampicin resistant.

rats studied the aerobes were rifampicin resistant. Three or four doses of rifampicin caused little change in the flora except that the L.F.C. were rifampicin resistant.

DISCUSSION

The hydrophilic nature of glucuronide and sulphate conjugates precludes their absorption from the gastro-intestinal tract [3,5]. In the rat, norethisterone undergoes extensive biliary excretion, almost exclusively as glucuronide and sulphate conjugates [I, 63, and enterohepatic circulation [ 1,7]. Although hydrolytic enzymes are present in mammalian gastro-intestinal tissue at all levels [S], enzymes of bacterial origin are considered to be of greater importance in the deconjugation of both natural and foreign compounds [3]. The gut microflora are therefore important determinants of both the /3-glucuronidase [3,9, lo] and sulphatase [ 1l] activity of the Table 3. The effect of ampicillin treatment (200 mg/kg/day) for 4 days followed by varying times of delay on the EHC of norethisterone and the gut flora. Studies were performed on the day following the final administration (day 0), day 7, 14 and 21

Times of study after ampicillin treatment

Treatment

The results of leaving rats, treated with ampicillin (200 mg/kg/day for 4 days) for 7, 14 or 21 days following the final drug administration are summarized in Table 3 and Fig. 4. After a period of 7 days the EHC was still significantly reduced (P < 0.001) compared to controls. The aerobic flora was ampicillin resistant. Fourteen and 21 days after the final ampicillin administration the EHC was not significantly different from controls and the flora had normalized. The majority of L.F.C. were ampicillin sensitive.

Control Ampicillin day 0 Aay 7 +day 14 day 21

“/gExcretion in bile in 6 h

Caecal flora

42.4 + 2.6

M.An§; LFCP

4.2*** + 13.7*** + 32.0 + 36.4 +

0.8 2.0 6.4 1.7

M.Ant; M.An§; M.An§; M.An&

LFC$/I; LFCt jj LFC$? LFCSd

yt

*** Significantly different from controls P < 0.001. t < 103/ml; 1 105-lO’/ml; 5 107-10’o/ml. 11Emergence of ampicillin resistant LFC’s. 7 Flora virtually normalised. Majority of LFC ampicillin sensitive: minority ampicillin resistant.

Antibiotic-steroid

lime

99

interaction

(h)

Fig. 4. Percentage cumulative excretion of radioactivity in bile following intracaecal administration of [3Hf-norethisterone conjugates to control rats (+---+I and rats treated with ampicii~in followed by no delay (&---+I~, 7 day delay (U----U), 14 day delay (A-A) and 21 day delay (m----8). For details see Text. Hatched bar indicates time of conjugate infusion. Each point is the mean i: SEM of four experiments. lumen. Attempts to characterise the bacteria responsible for production of glucuranidase activity in the rat have revealed that E, adi, Lactobacillus intestinal

spp., 3uci~~oi~e~ spp. and ~~~~vbucte$~um spp. l&rate the enzyme.

In the present study there was a difference in the extent of EHC when norethisterone conjugates were infused into the duodenum and caecum. Such a difference could be due to a differential rate of absorption of the &conjugated steroid in the duodenum and caecum, although more probably it is the result of a differential rate of hydrolytic breakdown of the conjugates due to increased concentration of bacteria in the caecum. There is good evidence that enzyme activity is greater in the caecum and colon than in the upper small intestine [lo]. Ampicillin caused a greater reduction in the EHC than neomycin alone. However, in rats treated with neomycin in combination with lincomycin there was a reduction in EHC to a level which was not significantly different to that found in rats treated with ampicillin alone. Ampicillin preferentially reduced the anaerobic flora, whereas neomycin preferentially reduced the aerobic flora. In combination with neomycin, lincomycin was active against the mixed anerobes. There was a good correlation between the reduction in EHC and the changes in the gut flora. In contrast, to the other antibiotics, long term rifampicin treatment did not reduce the EHC. This lack of effect correlated with the presence of rifampicin resistant LFC. In order to investigate the onset of rifampicin resistant, rats were treated for 1, 2, 3 or 4 days. After 1 dose of rifampicin the reduction in EHC was compar-

able to that previously attained with ampicillin and neomycin + lincomycin. LFC were reduced (103/ml) in all animals. The graded effect of rifampicin treatment for I, 2,3 or 4 days on the EHC correlated with the onset and estabfishing of resistant organisms in the caecal Flora; such organisms being capable of normal deconjugation. The rapid emergent of resistance is in a~eement with clinical findings (see review by Binda er at,[l2]). In some cases resistance can arise during monotherapy within 24-72 h. As a resuh of studies in which there was a reduction in urinary and plasma estriol levels in pregnant women during antibiotic treatment [13-151 it was suggested [lS] that antibiotics can interfere with the normal EHC and urinary excretion of estrogens during pregnancy. The present findings give further evidence that inter~ption of the EHC is the mechanism whereby antibiotics interfere with oral contra~ptive steroid therapy [16,17]. In addition, the rapidly acquired resistance strongly suggests that rifampicin does not act as other antibiotics. Indeed there is considerable evidence that the interaction between rifampi~n and contraceptive steroids [l&-26] is the result of the enzyme inducing properties of rifampicin [27,28]. Acknowledgements-We

would like to acknowledge the financial assistance of 6. D. Searle & Co. Ltd. and the World Health organization. REFERENCES 1. Back D. J., Breckenridge A. M., Chaltiner M., Crawford F. E., Orme M. L’E., Rowe P. H. and Smith E.: The effect of antibiotics on the enterohepatic circuia-

D. J. BACK et al.

100 tion of ethinyloestradiol

and norethisterone

in the rat.

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27. Bolt H. M., Kappus H. and Bolt M.: Effect of rifampttin treatment on the metabolism of oestradiol and 17a-ethinyloestradiol by human liver microsomes. Eur. J. c/in. Pharmacol. 8 (1975) 301-307. 28. Back D. J., Breckenridge A. M., Crawford F. E., Maciver M., Orme M. LE., Park B. K., Rowe P. H. and Smith E.: The effects of rifampicin on norethisterone pharmacokinetics. Eur. J. chn. Pharmac. 15 (1979) 193-197.