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In vitro metabolism of estrogens by isolated intestinal micro-organisms and by human faecal microflora
Journal 01 Steroid Biochumnrry. Vol. 13. pp. 345 to 349 Pergamon Press Ltd 1980. Printed in Great Britain
IN VZTRO METABOLISM OF ESTROGENS ISOLATED INTESTINAL MICRO-ORGANISMS BY HUMAN FAECAL MICROFLORA
BY AND
PAULAJ&VENP& T. KOSUNEN,T. FOTW and H. ADLERCREUTZ Departments of Clinical Chemistry and Bacteriology and Immunology, University of Helsinki, Helsinki, Finland (Received 9 May 1979)
SUMMARY The effect of intestinal micro-organisms on estrogen metabolism has been studied by in oitro experiments. Estrone, estradiol and 16a-hydroxyestrone were incubated with isolated intestinal microorganisms and with human faecal flora. 16-oxoestradiol, 15a-hydroxyestrone and estriol were also used as substrates in incubations with human faecal flora. After a simple purification step the identification of the metaboiites was carried out by capillary gas chromatography and mass spectrometry. Akaligenesfaecalis, Pseudomonas aeruginosa and Staphylococcus aureus were able to convert estrone to estradiol and oice versa. The anaerobe Bacteroides fragilis could only reduce estrone to estradiol, Streptococcusfaecalis performed the reverse reaction but both were able to convert estrone to 16a-hydroxyestrone. Mixed human faecal flora was able to interconvert estrone and estradiol both in aerobic and anaerobic conditions. In addition it reduced 16a-hydroxyestrone to estriol, 16-oxoestradiol to 16-epiestriol and 15a-hydroxyestrone to 15a-hydroxyestradioi. The significance of these results is discussed in the light of present knowledge of estrogen metabolism.
INTRODUCTION That intestinal bacteria may p!ay a significant role in estrogen metabolism was first demonstrated by Engel[l] who found that treatment with antibiotics reduced the excretion of radioactive metabolites of estradiol in urine in human subjects. Since then numerous studies have demonstrated that when the intestinal microflora is altered with antibiotics and sulphonamides, the metabolism of neutral steroids [2-71 and estrogens [7-141 is significantly changed. It has also been directly or indirectly demonstrated that certain interconversions of estrogens [13, l>i7] or of neutral steroids to estrogens [l&19] may take place in the intestinal tract. Especially the great differences observed in the quality and quantity of various estrogens in pregnancy bile and faeces [ 13, 171 and the conversion of orally administered 16a-hydroxyestrone to many metabolites identified in portal venous blood emphasizes the active role of the intestine and probably the microbial flora in the metabolism of biliary estrogens. On the basis of the above findings we studied the in vitro metabolism of some estrogens of special interest when incubated with various strains of normal human faecal micro-organisms and with human faecal flora. The aim of the study was to find out the quantitatively most important conversions facilitated by normal human lower gastrointestinal tract microorganisms in both aerobic and anaerobic culture. EXPERIMENTAL Steroids
The following
steroids
were used: Estrone, estra345
diol, estriol, 16cc-hydroxyestrone, 16-oxoestradiol, 1Su-hydroxyestrone, 1See-hydroxyestradiol, 16-epiestriol, 17-epiestriol, 16,17_epiestrioL All steroids were obtained from Ikapharm (Rhamat-Gan, Israel) except 16-epiestriol which was obtained from Steraloids Inc. (Pawling, N.Y., U.S.A.). Bacteria
and incubation
conditions
Microbial cultures were carried out in metalcapped test tubes containing 10ml of broth (Lab. Lemco Broth, Oxoid, England). The hormone was first added in a volume of 1SO-600 ~1 methanol. After careful mixing the tubes were inoculated with a loopful of bacterial culture or human faeces obtained from healthy adults. The cultures were incubated both aerobically and anaerobically at 37°C with various steroids as summarized in Tables 1 and 2. Control
experiments
All steroids using the same bacteria were trolled by gas
were also incubated without bacteria conditions. Plain broth and broth with incubated in the same way and conchromatography (GC).
Extractions
After centrifugation the supernatant was sterilized by filtration through a Millipore filter (0.224 The supernatant was extracted three times with an equal volume of distilled diethylether. The combined ether fractions were evaporated to dryness under nitrogen. 1 ml methanol was added to the dry samples before storage at -20°C until analysed by GC. The microbial mass of some of the bacterial and all faecal cultures were also studied. They were extracted with
aureus
Staphylococcus
24
24 24 24 24
+ + + + +
sscherichia
'roteus mirabilis
'rotcus vulq,lris
(lcbsiella
(2 strains)
pneumoniae
coli
alblcans
faccalis
Enterobacter.cloacae
Candida
Streptococcus
24
72
24
24
24
24
72
72
24
24
24
24
72
24
time,
+
Anaerobic
24
(4 strains)
Aerobic
+
fragilis
smegmatis
Mycobacterium
Bactcroides
aureus
aeruqinosa
Staphylococcus
Pseudomonas
smegmatis
fraqilis
Bacteroides
Mycobacterium
faecalis
faecalis
Streptococcus
Alcaliqencs
aureus
aeruginosa
smegmatis
faecalis
Staphylococcus
Pseudomonas
Mycobacterium
Alcaligenes
Microbe h
Incubation
II
* estradiol
+ estrone
by GC-MS
,I
II
No conversions
Estl-one + 16a-hydrosyestrone
* estriol
identified
16a-Hydroxyestrone
Estrone
Estradiol
Conversion
Table1.Bacterial enzyme-induced conversions ofestrone, estradiol and 16a-hydroxyestrone; Substrate concentrations were45 and 90&m]
341
hecal metabolism of estrogens
10 ml of methanol and the methanol extract stored at - 20°C. Deriuatization
Portions of the microbial mass and supernatant extracts were taken for analysis. All samples were converted to their trimethylsilyl ethers (TMS) as described previously [20,21]. The cisglycolic estrogen triols were converted to their acetonide-TMSderivatives [22,23] to distinguish them from the transglycolic estrogen triols. Gas-liquid (GC-MS)
chromatography-mass
spectrometry
Preliminary identification was carried out by gas chromatography (Carlo Erba Strumentazione model 2300) using open tubular glass capillary columns (OV-101) about 25m in length. The final identification of metabolites seen in the gas chromatograms was done using GC-MS (LKB 9OC@) [24]. Retention times and mass spectra were compared with those obtained for authentic standards.
scribed. The main reactions observed were oxidations and reductions. A Streptococcus faecalis strain converted estrone to I6a-hydroxyestrone (24 h incubation in aerobic conditions) and in another experiment where Bacteroides fragilis was incubated fora longer time(72h) in anaerobic conditions the same reaction was observed, but no 16u-hydroxyestrone was found after only 24 h incubation. Therefore the incubations with mixed human faecal flora were carried out for 72 h. Incubation with human faecal flora
Human faecal flora was only able to ,accomplish oxidations and reductions using the experimental conditions described and all conversions were also below 1% as in the single strain bacteria incubations. There was no significant difference in results between experiments in aerobic and anaerobic conditions. As expected, estriol was found to be very resistant and no conversions were observed. The results are shown in Table 2. DISCUSSION
RESULTS
Incubation with single microbial strains
Single microbial strains were used in incubations with estrone, estradiol and 16a-hydroxyestrone. These steroids were chosen because it was expected that they could be metabolized by intestinal bacteria. Despite the simple purification of the steroid metabolites the gas chromatogram showed few impurities and it was easy to recognize the metabolite peaks when comparing the gas chromatogram of the control samples with those obtained after the incubation experiments. The results are presented in Table 1. A great number of negative results were observed in these experiments partly because the incubation time was too short. The proportion converted may also have been so small that the metabolites could not be detected with the procedure used. All observed conversions were less than 1% under the conditions de-
In previous studies it has been demonstrated that the metabolism of hormonal steroids by intestinal bacteria is dominated by reductive pathways [25-331. Recently Lombardi et a/.[161 demonstrated in vitro that high faecal concentrations in the incubate resulted in reductive pathways but low concentrations in oxidative reactions. Estrone was converted to estradiol and 16cc-hydroxyestrone to estriol when high faecal concentrations were used and estradiol was oxidized to estrone when low concentrations were utilized. In our studies Alcaligenes faecalis, Pseudomonas aeruginosa and Staphylococcus uureus were able to convert estradiol to estrone and vice oersa. Also Mycobacterium smegmaris, which is not a faecal bacterium, was able both to reduce and oxidize estrogens at C-17. Some strains of Streptococcus faecalis were able to oxidize estradiol to estrone and one strain
Table 2. Metabolites formed upon incubation with human faecal flora in aerobit and anaerobic conditions; Incubation time was 72 h Substrate
Metabolite
Estrone
Estradiol
Estradiol
Estrone
16a-Hydroxyestrone
Estriol
16-Oxoestradiol
16-Epiestriol
15a-Hydroxyestrone
lsa-Hydroxyestradiol
Estriol
No metabolites
348
PAULA JRRVENPU ef al.
further convert estrone to 16a-hydroxyestrone. The anaerobic Bacteroides fragiiis could only reduce estrone to estradiol, but was also able to convert estrone to 16a-hydroxyestrone. However, no formation of estriol from estradiol or estrbne was detected in experiments with single strains. However, Staphylococcus aureus was able to reduce 16a-hydroxyestrone to estriol. In the studies with mixed faecal flora the results were rather surprising. Both in aerobic and anaerobic conditions oxidoreduction was found at C-17 and 16a-hydroxyestrone was reduced to estriol, 16-oxoestradiol to 16-epiestriol and 1Sa-hydroxyestrone to 15a-hydroxyestradiol (Table 2). The aerobic and anaerobic environment seemed not to have any obvious influence on the metabolism. Thus, intestinal bacteria are able to convert estradiol to estrone and estrone via 16a-hydroxyestrone to estriol. The previous in vivo experiments [ 131 in which 16x-hydroxyestrone was administered orally and portal and peripheral vein blood collected in small portions during 2 l/2 h was analyzed as to their content of estrogens, suggested that very little, if any, estriol is formed in the intestinal tract from this quantitatively important biliary estrogen because estriol was detected in the peripheral vein blood before it could be observed in the portal vein blood. On the other hand indirect evidence indicates that biliary estrogen metabolites later on are partly converted to estriol during the enterohepatic circulation [34]. The likely site of this estriol formation is the liver but some conversion of biliary estrogen metabolites to estriol in the gut is possible. The in viva conversion of 16-oxoestradiol to 16-epiestriol has been shown to occur in the human organism [35,36] and some 16-epiestriol was detected in portal vein blood after oral administration of 16a-hydroxyestrone [13]. In the present study 16-oxoestradiol was converted to 16-epiestriol with human faecal flora. It is therefore possible that some of the 16-epiestriol excreted in urine results from enzymatic activity of intestinal bacteria. Rather much 15a-hydroxyestrone is excreted in bile in pregnancy, but in faeces the amount is lower, but 1Sa-hydroxyestradiol are amounts of high found [13,37]. The present study shows that the conversion of 1Sa-hydroxyestrone to 1Sa-hydroxyestradiol is facilitated by intestinal bacteria and would explain the high amounts of the reduced estrogen in faeces. Comparisons of estrogen excretion in pregnancy and non-pregnancy bile and faeces and the results of estrogen assays in faeces after administration of ampicillin and sulphonamides [ 13,381 suggest that estradiol, biologically the most active estrogen, can be formed in the intestinal tract. This may take place by the reduction of estrone, by 16-dehydroxylation followed by reduction of 16a-hydroxyestrone [13] or by aromatization of neutral steroids [19]. In the present and previous [16] studies no dehydroxylation of 16a-hydroxyestrone could be found but the results
indicate that the reduction of estrone to estradiol is probably one of the ways by which estradiol may be formed in the gut. Lombardi et a[.[161 did not find any aromatization of neutral steroids to estrogens and this interesting pathway seems therefore to need further investigation. The quantitative results obtained for biliary and faecal estrogens in nonpregnant female subjects suggest that some biliary estrone may have been reduced to estradiol excreted in faeces [38]. However, it must be emphasized that mass fragmentography was used for the assay of biliary estrogens and radioimmunoassay for faecal estrogens and the results may therefore not be completely comparable. On the other hand it was found that administration of sulphonamides to a fertile woman reduced unconjugated estrone and estradiol excretion in faeces but the administration of erythromycin to a male subject caused a huge increase in the excretion of faecal unconjugated estrone and estradiol. If estradiol is formed in the gut, it may therefore only occur in significant amounts in female subjects [38]. This finding is interesting because recently it was observed that vegetarian women excrete much more estrogen in faeces than omnivorous women [39] and it has been shown that there is a strong correlation between large bowel and breast cancer and a high dietary intake of fat and meat, and inverse relationship with high dietary fibre [N-43]. Further studies are in progress in order to find out whether these dbservations have any common denominator. Acknowledgements-The authors wish to thank Mrs Maire Laakso for assistance with the incubation experiments and Mrs Sirkka Adlercreutz for carrying out some of the CC-MS work. This work was supported by the Ford Foundation, New York, and by contract CB 74104 from the National Cancer Institute through the Breast Cancer Task Force Committee. REFERENCES 1. Engel L. L.: Recent studies on estrogen metabolism. In Biological
Activities
of Steroids
in Relation
to Cancer
(Edited by G. Pincus and E. Vollmer). Academic Press, New York (1960) pp. 11l-123. 2. JInne 0. A., Laatikainen T. J. and Vihko R. K.: Effect of reduction of the intestinal microflora on the excretion of neutral steroids in human faeces and urine. Eur. J. Biochem. 20, (1971) 12&123. 3. Eriksson H., Gustafsson J.-A., SjGvall J. and SjGvall K.: Excretion of neutral steroids in urine and faeces of women with intrahepatic cholestasis of pregnancy. Steroids
Lipids Res. 3 (1972) 3M8.
4. Eriksson H. and Gustafsson J.-A.: Excretion of steroid hormones in adults. Steroids in faeces from adults. Eur. J. Biochem. 18(1971) 146-150. 5. Trybuchowski H.: Effect of ampicillin on the urinary output of steroidal hormones in pregnant and nonpregnant women. C/in. chim. Acta 45 (1973) 9-18. 6. Martin F., Peltonen J., Laatikainen T., Tikkanen M. and Pulkkinen M.: Excretion of unconjugated and conjugated progesterone metabolites in pregnancy urine during ampicillin administration. Clin. chim. Acfa 55 (1974) 71-80. 7. Martin F., Peltonen J., Laatikainen T.. Pulkkinen M. and Adlercreutz H.: Excretion of progesterone meta-
Faecal metabolism of estrogens bolites and estriol in faeces from pregnant women during ampicillin administration. J. steroid Biochem. 6 (1975) 1339-1346. 8. Willman K. and Pulkkinen M. 0.: Reduced maternal plasma and urinary estriol during ampicillin treatment. Am. J. Obstet. Gynecot. 109 (1971) 893-896. 9. Puikkinen M. 0. and Willman K.: Reduction of maternal estrogen excretion by neomycin. Am. J. Obstet. Gynecol. 115 (1973) 1153. 10. Tikkanen M. J., Pulkkinen M. 0. and Adlercreutz H.: Effect of ampicillin treatment on the urinary excretion of estriol conjugates in pregnancy. J. steroid Biochem. 4 (1973) 439-440. il. loehm F. H., Di Pietro D. L. and GOSS D. A.: The effect of ampicillin administration on urinary estriol and serum estradiol in the normal pregnant patient. Am. J. Obsret. Gvnec. 119 (1974) 98-103. 12. Adlercreutz H., Martin F.,’ Tikkanen M. J. and Pulkkinen M.: Effect of ampicillin administration on the excretion of twelve estrogens in pregnancy urine. Acfa ~n~ocr.Co~e~~. 80 (1975) 55 l-557. 13. Adlercreutz H.. Martin F., Pulkkinen M., Dencker H.. Rim& U., SjGberg N.-O. and Tikkanen M. J.: Intestinal metabolism of estrogens. J. clin. Endocr. Metab. 43 (1976) 497-505. 14. Adlercreutz H., Martin F., Lehtinen T., Tikkanen M. J. and Pulkkinen M. 0.: Effect of ampicillin administration on plasma conjugated and un~onjugated estrogen and progesterone levels in pregnancy. Am. J. ffbstet. Gynecoi. 128 (1977) 26627 1. 15. Diczfalusy E.. Franksson C., Lisboa B. P. and Martinsen B.: Formation of oestrone glucosiduronate by the human intestinal tract. Acta endocr. Copenh. 40 (1962) 537-551. 16. Lombardi P., Goldin B., Boutin E. and Gorbach S. L.: Metabolism of androgens and estrogens by human fecal micro-organisms. J. steroid Biockern. 9 (1978) 795-801. 17. Adlercreutz H. and Martin F.: Estrogens in human pregnancy faeces. Acta endocr. Copenh. 83 (1976) 410-419. 18. Dodson R. M. and Muir R. D.: Microbial transformations. II. The microbial aromatization of steroids. J. Am. Chem. Sot. 80 (1958) 5004-5005. 19. Goddard P. and Hill M. J.: Degradation of steroids by intestinal bacteria. IV. The aromatization of ring A. Biochim. biophys. Acta 280 (1972) 336-342. 20. Luukkainen T., Vandenheuvel W. J. A., Haahti E. 0. A. and Horning E. C.: Gas-chromatographic behaviour of trimethvlsilvlethers of steroids, Biochim. biophys. Actu 52 (lQ61j599601. 21 Gtundy S. M., Ahrens E. H. and Miettinen T. A.: Quantitative isolation and gas-liquid chromatographic analvsis of total fecal bile acids. J. Linid Res. 6 (1965) ~ I 397dlO. 22 Adlercreutz H., Laiho S. and Luukkainen T.: Preparation of steroid derivatives for gas chromatography. including studies of the gas chromatographic properties of steroidal acetonides. In Gas Ckrofl7~rogrup~~c ~e~erminution of Hormonal Steroids (Edited by F. Polvani, M. Surace and M. Luisi). Academic Press. New York (1967) pp. 69-83. 23. McCloskey J. A. and McCelland M. J.: Mass spectra of 0-isopropylidene derivatives of unsaturated fatty esters. J. Am. Chem. Sot. 87 (1965) 5090-5093. 24. Adlercreutz H., Martin F.. Wahlroos 0. and Soini E.: .Mass spectrometric and mass fragmentog~phic determination of natural and synthetic steroids in biological fluids. J. steroid Eiochem. 6 (1975) 247-259. 25. Marcus P. J. and Talalay P.: Induction and purifica-
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
349
tion of a- and /3-hydroxysteroid dehydrogenases. J. biol. Chem. 218 (1956) 661-674. Schubert K., Schlegel J. and Horhold C.: Der Hydrierungsverlauf bei 17-Ketosteroiden mit Darmbakterien unter anaeroben Bedingungen. Z. Nfffur~rsc~. 17b (1962) 84-86. Schubert K., Schlegel J. and H&hold C.: Stereospezitische Hydrierung von A’ ,QAndrostadiendion-(3.17) zu A’-5/l-Androstendion(3.17) und SP_Androstanol-(3x)on-(17) mit CIostridium paruputrificum unter anaeroben Bedingungen. Hoppe Seylers 2. physiol. Chum. 332 (1963) 310-313. Schubert K., Schiegei J. and H&hold C.: Selektive Hydrierung von Steroidhormonen durch Clostridien unter anaeroben Bedingungen. Z. Nafurforsch. 18b (1963) 284286. Schubert K., Schlegel J. and Hijrhoid C.: Stereospecific hydrogenation of A“-, A’,4-. A4.‘j- and A’,4.6-3-ketosteroids by Clostridium paraputrificum. Steroids, Suppl. l(1965) 175-184. Bokkenheuser V. D.. Winter J., Dehazya P., de Leon 0. and Kelly W. G.: Formation and metabolism of tetrahydrodeoxycorticosterone by human fecal flora. J. steroid Biochem. 7 (1976) 837-843. Bokkenheuser V. D., Winter J.. Dehazya P. and Kelly W. G.: Isolation and characterization of human fecal bacteria capable of 21-dehydroxylating corticoids. Appi. ~n~~ronm. ~~erob~o~. 34 ( 1977) 57 i -577. Winter J. and Bokkenheuser V. I).: 21-Dehydroxylation of corticoids by anaerobic bacteria isolated from human fecal flora. J. steroid Biochem. 9 (1978) 379-384. Eyssen H. and Parmentier G.: Biohydrogenation of sterols and fatty acids by the intestinal microflora. Am. J. din. Nutr. 27 (I 974) 1329-I 340. Adlercreutz H.. Tikkanen M. J., Wichman K.. Svanborg A. and Anberg A.: Recurrent jaundice in pregnancy. IV. quantitative determination of urinarv and biliary estrogens, including studies in Pruritus &m_u~ damn. J. clin. Endocr. Metab. 38 (1974) 51-57. Lewitz M.. Spitzer J. R. and Twombly G. H.: Interconversion of 16-oxygenated estrogens. I. The synthesis of estriol 16-C’* and its metabolism in man. J. biol. Chem. 231 (1958) 787-797. Lewitz M., Rosen M. F. and Twombly G. H.: Interconversion of 16-oxygenated estrogens. II The metabolism of 16-ketoestradiol-I 7fi-16-C’4 in man. Arch. Biochem. 88 (1960) 212.
Adlercreutz H. and Martin F: Oestrogen in human pregnancy faeces. Acta mdocr. Cope&. 83 (1976) 410-419. 38. Adlercreutz H., Martin F., Jirvenpli P. and Fotsis T. Steroid absorption and enterohepatic recycling. Contraception 20 (1979) 201-223. 39. Adlercreutz H., Goldin B. and Gerlock S.: Estrogens in faeces: ElTect of menstrual cycle, diet and antimicrobial drugs on excretion. Paper presented at the Third European Congress in Clinical Chemistry, Brighton 3.6-8.6 1979. 40. Armstrong’B. and Doff R.: Environmental factors and cancer-incidence and mortality in different countries with special reference to dietary practices. Inr. J. Cancer 15 (1975) 617-631. 41. Howell M. A.: Diet and etiological fdctors in the development of cancer of the colon and rectum. J. chron. Dis. 28 (1975) 67-80. 42. Draser B. S. and Irving D.: Environm~n~l factors and cancer of the colon and breast. Br. J. Cnnccpr27 (1973) I67- 172. 43. Irving D. and Draser B. S.: Fibre and cancer of the colon. Br. J. Cuwer 28 (1973) 462-463. 37.
IN VZTRO METABOLISM OF ESTROGENS ISOLATED INTESTINAL MICRO-ORGANISMS BY HUMAN FAECAL MICROFLORA
BY AND
PAULAJ&VENP& T. KOSUNEN,T. FOTW and H. ADLERCREUTZ Departments of Clinical Chemistry and Bacteriology and Immunology, University of Helsinki, Helsinki, Finland (Received 9 May 1979)
SUMMARY The effect of intestinal micro-organisms on estrogen metabolism has been studied by in oitro experiments. Estrone, estradiol and 16a-hydroxyestrone were incubated with isolated intestinal microorganisms and with human faecal flora. 16-oxoestradiol, 15a-hydroxyestrone and estriol were also used as substrates in incubations with human faecal flora. After a simple purification step the identification of the metaboiites was carried out by capillary gas chromatography and mass spectrometry. Akaligenesfaecalis, Pseudomonas aeruginosa and Staphylococcus aureus were able to convert estrone to estradiol and oice versa. The anaerobe Bacteroides fragilis could only reduce estrone to estradiol, Streptococcusfaecalis performed the reverse reaction but both were able to convert estrone to 16a-hydroxyestrone. Mixed human faecal flora was able to interconvert estrone and estradiol both in aerobic and anaerobic conditions. In addition it reduced 16a-hydroxyestrone to estriol, 16-oxoestradiol to 16-epiestriol and 15a-hydroxyestrone to 15a-hydroxyestradioi. The significance of these results is discussed in the light of present knowledge of estrogen metabolism.
INTRODUCTION That intestinal bacteria may p!ay a significant role in estrogen metabolism was first demonstrated by Engel[l] who found that treatment with antibiotics reduced the excretion of radioactive metabolites of estradiol in urine in human subjects. Since then numerous studies have demonstrated that when the intestinal microflora is altered with antibiotics and sulphonamides, the metabolism of neutral steroids [2-71 and estrogens [7-141 is significantly changed. It has also been directly or indirectly demonstrated that certain interconversions of estrogens [13, l>i7] or of neutral steroids to estrogens [l&19] may take place in the intestinal tract. Especially the great differences observed in the quality and quantity of various estrogens in pregnancy bile and faeces [ 13, 171 and the conversion of orally administered 16a-hydroxyestrone to many metabolites identified in portal venous blood emphasizes the active role of the intestine and probably the microbial flora in the metabolism of biliary estrogens. On the basis of the above findings we studied the in vitro metabolism of some estrogens of special interest when incubated with various strains of normal human faecal micro-organisms and with human faecal flora. The aim of the study was to find out the quantitatively most important conversions facilitated by normal human lower gastrointestinal tract microorganisms in both aerobic and anaerobic culture. EXPERIMENTAL Steroids
The following
steroids
were used: Estrone, estra345
diol, estriol, 16cc-hydroxyestrone, 16-oxoestradiol, 1Su-hydroxyestrone, 1See-hydroxyestradiol, 16-epiestriol, 17-epiestriol, 16,17_epiestrioL All steroids were obtained from Ikapharm (Rhamat-Gan, Israel) except 16-epiestriol which was obtained from Steraloids Inc. (Pawling, N.Y., U.S.A.). Bacteria
and incubation
conditions
Microbial cultures were carried out in metalcapped test tubes containing 10ml of broth (Lab. Lemco Broth, Oxoid, England). The hormone was first added in a volume of 1SO-600 ~1 methanol. After careful mixing the tubes were inoculated with a loopful of bacterial culture or human faeces obtained from healthy adults. The cultures were incubated both aerobically and anaerobically at 37°C with various steroids as summarized in Tables 1 and 2. Control
experiments
All steroids using the same bacteria were trolled by gas
were also incubated without bacteria conditions. Plain broth and broth with incubated in the same way and conchromatography (GC).
Extractions
After centrifugation the supernatant was sterilized by filtration through a Millipore filter (0.224 The supernatant was extracted three times with an equal volume of distilled diethylether. The combined ether fractions were evaporated to dryness under nitrogen. 1 ml methanol was added to the dry samples before storage at -20°C until analysed by GC. The microbial mass of some of the bacterial and all faecal cultures were also studied. They were extracted with
aureus
Staphylococcus
24
24 24 24 24
+ + + + +
sscherichia
'roteus mirabilis
'rotcus vulq,lris
(lcbsiella
(2 strains)
pneumoniae
coli
alblcans
faccalis
Enterobacter.cloacae
Candida
Streptococcus
24
72
24
24
24
24
72
72
24
24
24
24
72
24
time,
+
Anaerobic
24
(4 strains)
Aerobic
+
fragilis
smegmatis
Mycobacterium
Bactcroides
aureus
aeruqinosa
Staphylococcus
Pseudomonas
smegmatis
fraqilis
Bacteroides
Mycobacterium
faecalis
faecalis
Streptococcus
Alcaliqencs
aureus
aeruginosa
smegmatis
faecalis
Staphylococcus
Pseudomonas
Mycobacterium
Alcaligenes
Microbe h
Incubation
II
* estradiol
+ estrone
by GC-MS
,I
II
No conversions
Estl-one + 16a-hydrosyestrone
* estriol
identified
16a-Hydroxyestrone
Estrone
Estradiol
Conversion
Table1.Bacterial enzyme-induced conversions ofestrone, estradiol and 16a-hydroxyestrone; Substrate concentrations were45 and 90&m]
341
hecal metabolism of estrogens
10 ml of methanol and the methanol extract stored at - 20°C. Deriuatization
Portions of the microbial mass and supernatant extracts were taken for analysis. All samples were converted to their trimethylsilyl ethers (TMS) as described previously [20,21]. The cisglycolic estrogen triols were converted to their acetonide-TMSderivatives [22,23] to distinguish them from the transglycolic estrogen triols. Gas-liquid (GC-MS)
chromatography-mass
spectrometry
Preliminary identification was carried out by gas chromatography (Carlo Erba Strumentazione model 2300) using open tubular glass capillary columns (OV-101) about 25m in length. The final identification of metabolites seen in the gas chromatograms was done using GC-MS (LKB 9OC@) [24]. Retention times and mass spectra were compared with those obtained for authentic standards.
scribed. The main reactions observed were oxidations and reductions. A Streptococcus faecalis strain converted estrone to I6a-hydroxyestrone (24 h incubation in aerobic conditions) and in another experiment where Bacteroides fragilis was incubated fora longer time(72h) in anaerobic conditions the same reaction was observed, but no 16u-hydroxyestrone was found after only 24 h incubation. Therefore the incubations with mixed human faecal flora were carried out for 72 h. Incubation with human faecal flora
Human faecal flora was only able to ,accomplish oxidations and reductions using the experimental conditions described and all conversions were also below 1% as in the single strain bacteria incubations. There was no significant difference in results between experiments in aerobic and anaerobic conditions. As expected, estriol was found to be very resistant and no conversions were observed. The results are shown in Table 2. DISCUSSION
RESULTS
Incubation with single microbial strains
Single microbial strains were used in incubations with estrone, estradiol and 16a-hydroxyestrone. These steroids were chosen because it was expected that they could be metabolized by intestinal bacteria. Despite the simple purification of the steroid metabolites the gas chromatogram showed few impurities and it was easy to recognize the metabolite peaks when comparing the gas chromatogram of the control samples with those obtained after the incubation experiments. The results are presented in Table 1. A great number of negative results were observed in these experiments partly because the incubation time was too short. The proportion converted may also have been so small that the metabolites could not be detected with the procedure used. All observed conversions were less than 1% under the conditions de-
In previous studies it has been demonstrated that the metabolism of hormonal steroids by intestinal bacteria is dominated by reductive pathways [25-331. Recently Lombardi et a/.[161 demonstrated in vitro that high faecal concentrations in the incubate resulted in reductive pathways but low concentrations in oxidative reactions. Estrone was converted to estradiol and 16cc-hydroxyestrone to estriol when high faecal concentrations were used and estradiol was oxidized to estrone when low concentrations were utilized. In our studies Alcaligenes faecalis, Pseudomonas aeruginosa and Staphylococcus uureus were able to convert estradiol to estrone and vice oersa. Also Mycobacterium smegmaris, which is not a faecal bacterium, was able both to reduce and oxidize estrogens at C-17. Some strains of Streptococcus faecalis were able to oxidize estradiol to estrone and one strain
Table 2. Metabolites formed upon incubation with human faecal flora in aerobit and anaerobic conditions; Incubation time was 72 h Substrate
Metabolite
Estrone
Estradiol
Estradiol
Estrone
16a-Hydroxyestrone
Estriol
16-Oxoestradiol
16-Epiestriol
15a-Hydroxyestrone
lsa-Hydroxyestradiol
Estriol
No metabolites
348
PAULA JRRVENPU ef al.
further convert estrone to 16a-hydroxyestrone. The anaerobic Bacteroides fragiiis could only reduce estrone to estradiol, but was also able to convert estrone to 16a-hydroxyestrone. However, no formation of estriol from estradiol or estrbne was detected in experiments with single strains. However, Staphylococcus aureus was able to reduce 16a-hydroxyestrone to estriol. In the studies with mixed faecal flora the results were rather surprising. Both in aerobic and anaerobic conditions oxidoreduction was found at C-17 and 16a-hydroxyestrone was reduced to estriol, 16-oxoestradiol to 16-epiestriol and 1Sa-hydroxyestrone to 15a-hydroxyestradiol (Table 2). The aerobic and anaerobic environment seemed not to have any obvious influence on the metabolism. Thus, intestinal bacteria are able to convert estradiol to estrone and estrone via 16a-hydroxyestrone to estriol. The previous in vivo experiments [ 131 in which 16x-hydroxyestrone was administered orally and portal and peripheral vein blood collected in small portions during 2 l/2 h was analyzed as to their content of estrogens, suggested that very little, if any, estriol is formed in the intestinal tract from this quantitatively important biliary estrogen because estriol was detected in the peripheral vein blood before it could be observed in the portal vein blood. On the other hand indirect evidence indicates that biliary estrogen metabolites later on are partly converted to estriol during the enterohepatic circulation [34]. The likely site of this estriol formation is the liver but some conversion of biliary estrogen metabolites to estriol in the gut is possible. The in viva conversion of 16-oxoestradiol to 16-epiestriol has been shown to occur in the human organism [35,36] and some 16-epiestriol was detected in portal vein blood after oral administration of 16a-hydroxyestrone [13]. In the present study 16-oxoestradiol was converted to 16-epiestriol with human faecal flora. It is therefore possible that some of the 16-epiestriol excreted in urine results from enzymatic activity of intestinal bacteria. Rather much 15a-hydroxyestrone is excreted in bile in pregnancy, but in faeces the amount is lower, but 1Sa-hydroxyestradiol are amounts of high found [13,37]. The present study shows that the conversion of 1Sa-hydroxyestrone to 1Sa-hydroxyestradiol is facilitated by intestinal bacteria and would explain the high amounts of the reduced estrogen in faeces. Comparisons of estrogen excretion in pregnancy and non-pregnancy bile and faeces and the results of estrogen assays in faeces after administration of ampicillin and sulphonamides [ 13,381 suggest that estradiol, biologically the most active estrogen, can be formed in the intestinal tract. This may take place by the reduction of estrone, by 16-dehydroxylation followed by reduction of 16a-hydroxyestrone [13] or by aromatization of neutral steroids [19]. In the present and previous [16] studies no dehydroxylation of 16a-hydroxyestrone could be found but the results
indicate that the reduction of estrone to estradiol is probably one of the ways by which estradiol may be formed in the gut. Lombardi et a[.[161 did not find any aromatization of neutral steroids to estrogens and this interesting pathway seems therefore to need further investigation. The quantitative results obtained for biliary and faecal estrogens in nonpregnant female subjects suggest that some biliary estrone may have been reduced to estradiol excreted in faeces [38]. However, it must be emphasized that mass fragmentography was used for the assay of biliary estrogens and radioimmunoassay for faecal estrogens and the results may therefore not be completely comparable. On the other hand it was found that administration of sulphonamides to a fertile woman reduced unconjugated estrone and estradiol excretion in faeces but the administration of erythromycin to a male subject caused a huge increase in the excretion of faecal unconjugated estrone and estradiol. If estradiol is formed in the gut, it may therefore only occur in significant amounts in female subjects [38]. This finding is interesting because recently it was observed that vegetarian women excrete much more estrogen in faeces than omnivorous women [39] and it has been shown that there is a strong correlation between large bowel and breast cancer and a high dietary intake of fat and meat, and inverse relationship with high dietary fibre [N-43]. Further studies are in progress in order to find out whether these dbservations have any common denominator. Acknowledgements-The authors wish to thank Mrs Maire Laakso for assistance with the incubation experiments and Mrs Sirkka Adlercreutz for carrying out some of the CC-MS work. This work was supported by the Ford Foundation, New York, and by contract CB 74104 from the National Cancer Institute through the Breast Cancer Task Force Committee. REFERENCES 1. Engel L. L.: Recent studies on estrogen metabolism. In Biological
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