- Email: [email protected]
Toxicologic implications of biotransformation by intestinal microflora
TOXICOLOGY
AND
APPLtED
Toxicologic
23,769-781(1972)
PHARMACOLOGY
Implications Intestinal
of Biotransformation Microflora
by
R. T. WILLIAMS Department of Biochemistry, St. Mary’s Hospital Medical School, London W.2, U.K. Received August 8,1972
The role of the gut microflora in the metabolism of foreign compounds has only been given serious consideration relatively recently. However, references to their probable involvement in the production of certain urinary components and in the destruction of certain ingested compounds have been made in the literature at various times for about a century. Thus in the last quarter of the nineteenth century reference is made to the ethereal sulfates which occur in the urine. These were believed to arise as a result of the intestinal putrefaction of aromatic substances present in food material. Putrefaction produced phenol, cresol, catechol, indole and skatole which were absorbed from the intestine and then converted to ethereal sulfates in the liver. An account of this is given with references to the original papers in a textbook of the last century by Halliburton (1891). Urinary indican (potassium indoxyl sulfate), for example, was thought to be derived from indole which was produced in the intestine by bacteria, since its excretion and that of other ethereal sulfates could be suppressed in dogs by inanition plus the administration of large doses of calomel or iodoform. The source of indole, however, was not found until the beginning of this century when tryptophan was discovered by Hopkins and Cole (1901, 1903) who also showed that indole could be produced from tryptophan by a putrefying pancreas preparation. Ellinger (1903) suggested that tryptophan was the precursor of indole in the intestine and later several workers (Woods, 1936; Happold and Hoyle, 1935; Dawes et al., 1947) showed that E. coli, an organism which is found in the lower intestinal tract, could convert tryptophan to indole through the enzyme system, tryptophanase. The formation of urinary indican would therefore involve metabolism by the gut flora and the tissues, particularly the liver, thus : Jp CH,CHCOOH gut ITmat tryptophan
H indole
H indoxyl Copyright All rights
0 1972 by Academic Press, Inc. of reproduction in any form reserved
769
i-i indican
770
WILLIAMS
The destruction of ingested compounds worth, 1916) and (+)-tartaric acid (Finkle, some earlier papers. Tartaric acid is mainly injection, but largely destroyed when given THE INTESTINAL
such as mesoinositol (Anderson and Bos1933) by gut bacteria is also suggested in excreted unchanged when given to man by by mouth.
MICROFLORA
AND THEIR
DISTRIBUTION
The dominant organisms in the intestine are anaerobic, and therefore many of the reactions which occur in the intestine are likely to be of a reductive nature. The common organisms which may be found in the intestines of man or animals are shown in Table 1. TABLE 1 THE FLORA
OF THE GASTROINTES~NAL
Enterobacteria Enterococci “Viridans” streptococci Staphylococci Yeasts aDrasar et
TRACT”
Lactobacilli Bacteroides Bifidobacteria Clostridia Veillonella
al. (1970).
It is important, however, to appreciate that the location of these organisms along the gastrointestinal tract varies with species (Table 2). In man and the rabbit, few if any organisms occur in the stomach and proximal small intestine. In the guinea pig there are TABLE 2 LOCATION
OF THE GUT
FLORA
IN VARIOUS
SPECIES’* b
Location
Man
Rabbit
Guinea pig
Rat
Mouse
Stomach Proximal small intestine Distal small intestine Large intestine Rectum and feces
o-5 O-5 67 7-10 10-11
O-6 o-5 6-7 8-9 9-10
5-6 5-6 6-7 8-9 9-10
7-9 6-8 7-8 8-9 9-10
7-9 7-9 7-8 8-9 9-10
a Drasar et cd., 1970. * Bacterial counts in various parts of the gastrointestinal tract; log,, number of viable organisms per gram wet weight. small numbers in the stomach and proximal small intestine, but in the rat and mouse, bacteria occur in large numbers in the stomach and proximal small intestine. The main locations of the intestinal flora in man and the rabbit are the large intestine and rectum, whereas in the rat and mouse they occur in large numbers all along the gastrointestinal tract from stomach to rectum.
BIOTRANSFORMATION
BY INTESTINAL
771
MICROFLORA
An interesting consequence of this distribution is seen in the fate of the drug carbenoxolone in man and the rat (Iveson et al., 1971). Carbenoxolone is the hydrogen succinate of glycyrrhetic acid, and its disodium salt, I, (Biogastrone R and Duogastrone R) is used in the treatment of gastric and duodenal ulcer.
COONa
=R’
’ EOCH2CH2E00Na
I
*kOCH2CH2EOONa Carbenoxolone I
In the rat, the 14C-succinate labeled drug is mainly hydrolyzed by the stomach microflora to glycyrrhetic acid (II) and succinate (III). The succinate is then oxidized to CO, which is detected in the expired air by its 14C-label, while the glycyrrhetic acid portion is found in the bile of the rat as the 30- and 3-glucuronides and the 3-sulfate ester. In man, however, the drug is absorbed from the stomach as such and excreted mainly as the 30-glucuronide of carbenoxolone (IV) :
R
,COOH ‘;OCH
I
rat (stomach flora) 2 CH
2EOOH
-
CH,;OOH +
R ‘H
I
I
CHICOOH
II
man (tissues)
. -
* co2
III
tissues
I
COOC6H906
30-glucuronide,
R’ ‘?!0CH2C~2E00~
3-O-glucuronide, 3-O-sulfate
IV
REACTIONS
,COOH
CARRIED
OUT
BY THE
GUT
MICROFLORA
The gut microflora can carry out many reactions involving drugs and other foreign compounds, particularly reactions of a hydrolytic or reductive nature. Most of these reactions have been described in a review by Scheline (1968a), and they include the hydrolysis of glucuronides and glucosides, dehydroxylation of certain catechols, decarboxylation, dealkylation, aromatization, dehalogenation, deamination, reductions of various kinds, ring fission and so on. A few examples of these reactions will suffice to illustrate the versatility of the gut flora.
772
WILLIAMS
Dehydroxylation. Homoprotocatechuic rats and rabbits to 3-hydroxyphenylacetic
acid is dehydroxylated by the gut flora of acid (Dacre et al., 1968).
CH&OOH
CHPOOH
Decarboxylation. 4-Hydroxybenzoic acid, but not 2- or 3-hydroxybenzoic decarboxylated to phenol (Scheline, 1966)
acid, is
0-0 COOH
Dealkylation. Vanillic acid is 0-demethylated by rat gut flora (Scheline, 1966), and methamphetamine is N-demethylated by guinea pig gut flora (J. Caldwell and G. M. Hawksworth, 1972, unpublished data).
Vanillic
acid
CHz
CH2
CHCH,
{HCH,
NHCH,
NH2
Methamphetamine
Aromatization. Quinic acid is aromatized by the gut flora especially in man, to benzoic acid which is excreted as hippuric acid (Adamson et al., 1970). HO gut
flora
in man+
HO Quinic
Dehalogenation.
acid
DDT is dehalogenated to DDD (Scheline, 1968a).
DDT
DDD
BIOTRANSFORMATION
BY INTESTINAL
773
MICROFLORA
Reduction. There are many of these reactions, including the reduction of nitro (NO,) and azo (-N=N-) to NH, groups, of carbon double bonds (-CH=CH-) to the corresponding saturated carbon chain (-CH, *CH,--) and of sulfoxides (>SO) and N-oxides (-NO) to the corresponding sulfides (>S) and tertiary amines ON), respectively. Examples of these reactions are : 1. Chloramphenicol
(Thompson et al., 1954) rat gut flora
CHOHCHCHzOH NHCOCHClz
2. Neoprontosil
CHOHCHCHzOH I NHCOCHC12
(Gingell et al., 1971)
3. Cinnamic acid CH=CHCOOH
Phenylpropionic
4. Chlorpromazine
acid
5-oxide
GX)dWH& Chlorpromazine
Ring fission. This occurs especially with certain heterocyclics; a good example is the conversion of coumarin to mellilotic acid, which includes the intermediate reduction of a double bond before ring fission (Scheline, 1968b).
a0 - a0 - QOOH Coumarin
Dihydrocoumarin
Mellilotic
acid
WILLIAMS
THE
CONSEQUENCES
OF BIOTRANSFORMATION
BY THE
GUT
FLORA
Having described briefly the organisms, their location in the gastrointestinal tract in various species and some of the reactions which they are able to carry out, let us now consider the possible consequences of their activity and how this bears on problems in toxicology and drug metabolism. The enterofloral metabolism of foreign compounds could result in the gastrointestinal microflora being involved in (1) enterohepatic circulation, (2) the production of toxic metabolites, (3) carcinogenesis, (4) detoxication, (5) the production of pharmacologically active metabolites, (6) species variations in drug metabolism and toxicity, (7) individual variations in drug metabolism and toxicity and (8) the production of metabolites not formed in the tissues. Enterohepatic circulation. Compounds or their phase I metabolites (Williams, 1967) which are excreted in the bile as conjugates, particularly glucuronides, could undergo enterohepatic circulation if the gut flora are capable of deconjugating the conjugated metabolites. The gut flora are known to be able to split glucuronides (Scheline, 1968~). This means that the presence of a compound in the body can be prolonged by reabsorption from the intestine after biliary excretion. Therefore the chances of its producing toxic effects are increased. The enterohepatic circulation of stilboestrol, for example, has been shown to involve the gut flora. This compound in the rat is excreted to a considerable extent in the bile as stilbestrol glucuronide. In the gut this glucuronide is split to give free stilbestrol, which is then reabsorbed into the circulation. This reabsorption can be inhibited by antibiotics and by the /3-glucuronidase inhibitor, saccharo-1,4-lactone, since the former suppress the bacteria and the latter inhibits the bacterial /?-glucuronidase (Clark et al., 1969). The production of toxic metabolites by the gutflora. Goodman and Gilman (1955) in their well-known textbook say: “The oral route is the most ancient method of drug administration. It is also the safest, most convenient and most economical.” On injection a drug may be more effective and more certain in its effects, but in general more toxic than by mouth. However, the reverse situation as far as toxicity is concerned is possible if the compound is converted into a toxic agent in the gut but not in the body tissues. This could occur if the compound is metabolized to a toxic agent by the gut flora. The well-known glycoside amygdalin is more toxic to mice when given by mouth than when injected ip, its po LD50 is about 300 mg/kg, whereas on ip injection the LD50 is >5000 mg/kg. It appears that amygdalin is hydrolyzed by the gut flora to mandelonitrile, which is unstable and yields cyanide. Hydrolysis of the glycoside does not occur to any great extent when it is injected. C~%CH(C~‘)OCI~H~~I~
gut flora -
C6HSCH(CN)OH
spontaneous
-
Cd-bCHO
+ HCN
Toxicity could also be the result of the excretion of a compound in the bile followed by metabolism to a toxic agent by the gut flora. A possible example of this is chloramphenicol. This drug is reported to be goitrogenic in rats (Thompson et al., 1954), and this is believed to be due to its conversion by the gut flora of the rat into goitrogenic aromatic amines. In the rat, there is a considerable biliary excretion of chloramphenicol
BIOTRANSFORMATION
BY INTESTINAL
775
MICROFLORA
glucuronide, which could then be deconjugated and reduced to the corresponding amine by the gut flora. The process can be envisaged as follows :
CHoHCHCH20H
CHOHCHCH20.CsH906
NHCOCHClz
NHCOCHCI,
CHoHFHCH20H NHCOCHC12
In man, chloramphenicol is largely excreted as its glucuronide in the urine, and little if any reduction of the nitro group occurs. Curcinogenesis and the gutflora. The possibility that cancer of the large bowel could be due to the conversion of bile salts or steroids of the diet into carcinogens by the gut flora has been discussed by Hill et al. (1971a). A similar suggestion regarding breast cancer has been put forward by Hill et al. (1971 b). Evidence is also mounting that nitrite can be converted into the carcinogenic dialkylnitrosamines by intestinal organisms (Hawksworth and Hill, 1971). That the gut bacteria can produce a carcinogen has been shown in the case of cycasin or methylazoxymethyl P-D-glucoside, which is found in cycad nuts. This compound is carcinogenic in rats when given po, but not when given by injection. The aglycon of cycasin, methylazoxymethanol, is carcinogenic by any route, and it appears that cycasin when given by mouth is hydrolyzed by gut bacteria to the carcinogenic agent thus : CH3N(0):NCH20C6H,,0s
g”t’ora*
CH3N(0):NCH20H
-t C6H,106
This is supported by the finding that when cycasin is given po to germfree rats it is not carcinogenic, although the free aglycon produces cancer in these animals (Laqueur, 1968). However, it is interesting to note that cycasin is carcinogenic on SCinjection into newborn rats, since these animals possess /3-glucosidase in their skins until they are 25 days old but none later (Hirono et al., 1968). The production of active drugs by the gut bacteria. One of the greatest advances in chemotherapy in the first half of this century was the discovery by Domagk of the antibacterial activity of Prontosil. This dyestuff and the later Neoprontosil showed antibacterial activity in vivo but little such activity in vitro. It is now known that these azo dyes were metabolized in the body by the splitting of their azo links to yield sulfanilamide, the actual antibacterial agent.
776
WILLIAMS
Prontosil
Neoprontosil
Sulfanilamide
The splitting of the azo link does in fact occur in the liver by means of the enzyme azoreductase. Like many of the water-soluble sulfonated azo food colors, however, Neoprontosil is poorly absorbed from the intestine; in the past it was often given for therapeutic purposes by injection. Recently it has been shown (Gingell et al., 1971) that in the rat, at least, much of the conversion of Prontosil and Neoprontosil to sulfanilamide is carried out by the gut bacteria. Even if given by injection in the rat, a considerable amount of these dyes, especially Neoprontosil, is excreted in the bile with the azo link intact, so that splitting of the azo link to give sulfanilamide must have taken place to a considerable extent in the gastrointestinal tract. Treatment of the rats with antibiotics to suppress their gut flora considerably reduces the production of sulfanilamide from the dyes. The production of active drugs from inactive precursors by the gut flora is a field that has hardly been investigated. It is possible that such drugs as succinylsulfathiazole and phthalylsulfathiazole which were used for the treatment of dysentery owed their activity to the release of sulfathiazole in the intestine through the activity of the gut flora. TABLE 3 AROMATIZATION OF QUINIC ACID IN VARIOUS SPECIES’
Primates
% aromatized
Man 64 Old World monkeys Rhesusmonkey 43 Baboon 40 Green monkey 45 New World monkeys Spidermonkey 5 Squirrel monkey 0 Capuchin 0 Prosimii Bushbaby 2 Slow loris 0 Tree shrew 0 nAdamsonet al. (1970).
Nonprimates
% Aromatized
Dog
1
Cat Ferret Rabbit
0 0 3
Rat
5
Mouse Guinea pig Hamster Lemming Pigeon Fruit bat Hedgehog
0 0 0 0 2 0 0
BIOTRANSFORMATION
BY INTESTINAL
MICROFLORA
777
Gut bacteria and species variation in drug metabolism. Species variations in the metabolism of foreign compounds are largely due to variations in the tissue enzymes which carry out the reactions (Williams, 1967). However, if compounds are given by mouth or if there is a considerable biliary excretion of injected compounds, variations in metabolism could in some cases depend upon the gut flora. A striking example of a species variation in the metabolism of a foreign compound dependent upon the gut flora has been found with quinic acid which occurs widely in our food (tea, coffee, fruits and vegetables). When given po to man and the Old World monkeys this compound is extensively aromatized and excreted in the urine as hippuric acid (Adamson et al., 1970). In the rhesus monkey it is aromatized when given po but not when injected, and furthermore the aromatization of the po administered acid is suppressed if the monkey is given po antibiotics. Shikimic acid, an intermediate in the aromatization of quinic acid, behaves similarly. In animals lower on the evolutionary scale than Old World monkeys, quinic acid is not converted to benzoic acid to any great extent (Table 3).
Quinic acid
Benzoic acid
Shikimic acid
Individual variations in drug metabolism and the gut flora. It is possible that, under some circumstances which are not at present clear, one individual may metabolize a compound differently from another, and in some way this difference depends upon the gut flora. A good example of this is the well known sweetening agent, cyclamate. This compound is highly polar (pK, 1.9) and is therefore not readily absorbed when taken po. Tts poor absorption allows it to reach the locations in the intestinal tract where bacteria are abundant. In humans and animals which have not received cyclamate previously, the compound is excreted unchanged in the urine and feces when given po and in the urine when given by injection (Renwick and Williams, 1972a). However, if human subjects are given cyclamate daily, some subjects develop the ability to metabolize it to cyclohexylamine and its metabolites (Renwick and Williams, 1972b). This capacity to metabolize po administered cyclamate is not acquired by the body tissues but by the gut flora (Drasar et al., 1972), since injected cyclamate is not metabolized and the feces of active convertors, man or rats, of cyclamate to cyclohexylamine are also able to convert cyclamate to cyclohexylamine. The intestinal organisms which acquire the ability to split cyclamate in man appear to be enterococci. In rats the organisms are clostridia and in rabbits enterobacteria. Why some human subjects develop the ability to split cyclamate by means of their intestinal microflora better than others is at present obscure. Cyclohexylamine has been implicated in the suspected carcinogenicity of cyclamate, and it would appear that cyclamate could be more hazardous to some subjects than others because their gut flora for some obscure reason are more readily trained to split cyclamate. The strong base, cyclohexylamine, is not itself readily metabolized, but a small percentage of it is converted by body tissues to several minor
778
WILLIAMS
metabolites which include cyclohexanol, cyclohexan-trans-I ,2-diol and several aminocyclohexanols.
NHSO,H
trained
-
gut flora
body
tissues
NHz
cyclamate
+
several minor metabolites
cyclohexylamine
Metabolites formed by gutflora but not by body tissues. Many of the reactions carried out by the intestinal flora are similar to those carried out by the tissues of the body. For example, the reduction of the prontosils referred to earlier can be carried out by both the liver and the intestinal flora. It is now clear, however, that the gut bacteria can carry out reactions which do not occur to any great extent in the body tissues. Examples of these reactions have already been given, e.g., the aromatization of quinic acid and the splitting of cyclamate to cyclohexylamine. The toxicologic implications of some of these reactions are quite significant as in the cases of cycasin, amygdalin and cyclamate. There is another reaction of considerable interest which appears to occur in the gut flora but not in the tissues, namely aromatic dehydroxylation. One of the most intensely studied of the reactions of foreign compounds in the liver is aromatic hydroxylation, but the reverse of this reaction, that is the dehydroxylation of an administered phenolic compound, does not appear to occur to any great extent in the tissues. The dehydroxylation of phenolic compounds is known to occur in the body, but evidence is now accumulating which suggests that this is a reaction of the gut microflora. A good example is the dehydroxylation of 3,4-dihydroxyphenylacetic acid (Dacre et al., 1968). In rats and rabbits, this compound gives rise to 3- and 4-hydroxyphenylacetic acid; the major dehydroxylation product is the 3-compound. OH OH CHzCOOH
gutflora+
ooH CH$OOH
+
0 CHzCOOH
The dehydroxylation, but not the other metabolic reactions of 3,4-dihydroxyphenylacetic acid such as 0-methylation, can be suppressed by giving the animals antibiotics. 3-Hydroxyphenolic acids have been found as normal constituents of human urine, e.g., 3-hydroxybenzoic acid, 3-hydroxyphenylacetic acid, 3-hydroxyphenyllactic acid. These could be derived as such from the diet or by metabolism from compounds such as phenylalanine. However, the aromatic hydroxylation of compounds of this type in the liver would be expected to yield 2- or 4-, but not 3-, hydroxy derivatives. It is possible that at least part of the 3-hydroxy acids of the urine are formed by dehydroxylation of catechol acids by the gut flora thus :
BIOTRANSFORMATION
BY INTESTINAL
MICROFLORA
779
An interesting example of a reaction carried out by the gut flora but not by the tissues of the rat is the 0-demethylation of 3-0-methyldopa (L-4-hydroxy-3-methoxyphenylalanine) (Chalmers et al., 197 1). This compound is not demethylated by liver microsomal enzymes in vitro even after pretreatment of the rats with phenobarbitone to increase the activity of the microsomal enzymes. On ip administration to the whole animals, however, the compound, labeled with 14C in the methyl group, is partially demethylated as shown by the elimination of radioactive CO, in the expired air. Apparently the injected compound is partly eliminated in the bile as a conjugate and then demethylated in the gut.
3-0-Methyldopa
DOPA
It is clear from this brief review that the potential of the intestinal microflora for metabolizing foreign compounds can be quite considerable and that as a result of this metabolism, pharmacologically active, toxic and carcinogenic products may be produced. However, when considering the role of the gut flora, it is important to take into account (a) the distribution of the organisms along the gastrointestinal tract in different species and (b) certain characteristics of the compound being investigated such as absorbability from the intestine and the extent of its excretion in the bile. Since the rat has abundant microorganisms all along the gastrointestinal tract from the stomach to the rectum, whereas in man the organisms are located mainly in the large intestine and rectum, the chances of a compound being metabolized by the gut flora in the rat are much greater than in man. In the human, a compound may be entirely absorbed before it can make contact with the microflora. The nature of the compound is important from two aspects, viz., its absorbability from the intestine and the extent of its excretion in the bile, as such or as metabolites, after absorption. Compounds which are highly polar (e.g., cyclamate) are usually not readily absorbed, and they could be exposed to action of the microflora for a longer time than less polar and more absorbable compounds. Compounds or their metabolites of moderately high meolcular weight (i.e., 400-500 or more) tend to be extensively excreted in the bile (Hirom et al., 1972), and such compounds therefore have a greater opportunity of meeting the gut flora and of being metabolized by them than those poorly excreted in bile. REFERENCES
ADAMSON,R. H., BRIDGES, J. W., EVANS,M. E. and WILLIAMS, R. T. (1970).Species differences in the aromatization of quinic acid in virw and the role of gut bacteria. Biochem. J. 116, 437-443. ANDERSON, R. J. and BOSWORTH, A. W. (1916).The utilization of inositein the animal organism. The effect of inositeupon the metabolismof man.J. Biol. Chem.25, 39947. CHALMERS, J.P., DRAFFAN,G. H., REID, J.L., THORGEIRSSON, S.S.and DAVIES,D. S. (1971). Demethylation of 3-O-methyldopain the rat. Life Sci. 10 (f), 1243-1251.
780
WILLIAMS
A. G., FISCHER, L. G., MILLBURN, P., SMITH, R. L. and WILLIAMS, R. T. (1969).The role of gut flora in the enterohepaticcirculation of stiibestrolin the rat. Biochem. J. 112,17P. DACRE, J. C., SCHELINE, R. R. and WILLIAMS, R. T. (1968). The role of the tissuesand gut flora in the metabolismof [‘“Clhomoprotocatechuic acid in the rat and rabbit. J. Pharm. CLARK,
Pharmacol. 20,619-625. DAWES, A. E., DAWSON, J. and HAPPOLD, F. C. (1947). The tryptophan-tryptophanase reaction. 8. The modeof formation of indole. Biochem. J. 41, 426-431. DRASAR, B. S., HILL, M. J. and WILLIAMS, R. E. 0. (1970).The significanceof the gut flora in the safety testingof food additives.Metabolic Aspects of Food Safety. Chapter 10,245-255,
S. J. C. Roe, Blockwell Scientific Publication, Oxford and Edinburgh. DRASAR, B. S., RENWICK, A. G. and WILLIAMS, R. T. (1972).The role of the gut flora in the metabolismof cyclamate.Biochem. J. 129,881-890. ELLINGER, A. (1903).Die Indolbildung undIndicanausscheidung beimhungemdenKaninchen. Hoppe-Seyler’s Z. Physiol. Chem. 39, 44-54. FINKLE, P. (1933).The fate of tartaric acid in the human body. J. Biol. Chem. 100, 349-355. GINGELL, R., BRIDGES, J. W. and WILLIAMS, R. T. (1971). The role of the gut flora in the metabolismof prontosil and neoprontosilin the rat. Xenobiotica 1, 143-156. GOODMAN, L. S. and GILMAN, A. (1955).The Pharmacological Basis of Therapeutics, 2nd ed,
p. 6. Macmillan, New York. HALLIBURTON, W. D. (1891). Textbook
of Chemical Physiology and Pathology, p. 740. Longman’sGreen, London. HAPPOLD, F. C. and HOYLE,L. (1935).The coli-tryptophan-indole reaction 1. Enzyme preparations and their action on tryptophan and someindole derivatives. Biochem. J. 29, 1918-1926. HAWKSWORTH, G. M. and HILL, M. J. (1971). Bacteria and the N-nitrosation of secondary amines.Brit. J. Cancer 25, 520-526. HILL, M. J., CROWTHER, J. S., DRASAR, B. S., HAWKSWORTH, G., ARIES, V. and WILLIAMS, R. E. 0. (1971a).Bacteria and aetiology of cancerof the large bowel. Lancet. 1, 95-100. HILL, M. J., GODDARD, P. and WILLIAMS, R. E. 0. (1971b).Gut bacteria and aetiology of cancerof the breast.Lancer 2,472-473. HIROM, P. C., MILLBURN, P., SMITH,R. L. and WILLIAMS,R. T. (1972).Speciesvariations in the thresholdmolecularweight for the biliary excretion of anions.Biochem. J. 129,1071-1077. HIRONO, I., LAQUEUR, G. L. and SPATZ,M. (1968).Tumor induction in Fischerand OsborneMendel Rats by a SingleAdministration of Cycasin.J. Natl. Cancer Inst. 40, 1003-1010. HOPKINS,F. G., and COLE,S. W. (1901).A contribution to the chemistry of proteids Part I. A preliminary study of a hitherto undescribedproduct of tryptic digestion.J. Physiol. (London) 27,418-428. HOPKINS, F. G. and COLE, S. W. (1903).A contribution to the chemistry of proteids. Part II. The constitution of tryptophane and the action of bacteria upon it. J. Physiol. (London)
29,451-466. IVESON,P., LINDUP, W. E., PARKE,D. V. and WILLIAMS,R. T. (1971). The metabolismof carbenoxolonein the rat. Xenobiotica 1, 79-95. LAQUEUR, G. L. (1968).Toxicology of cyasin. Food Cosmet. Toxicol. 6, 577-578. RENWICK, A. G. and WILLIAMS, R. T. (1972a).The fate of cyclamatein man and other species. Biochem. J. 129,869-879. RENWICK, A. G. and WILLIAMS,R. T. (1972b).Metabolites of cyclohexylaminein man and certain animals.Biochem. J. 129, 857-867.
SCHELINE, R. R. (1966). Decarboxylation and demethylation of somephenolic benzoic acid derivatives by rat caecalcontents.J. Pharm. Pharmacol. 18, 664-669. SCHELINE, R. R. (1968a).Drug metabolismby intestinal microorganisms.J. Pharm. Sci. 57, 2021-2037. SCHELINE, R. R. (1968b).Studieson the role of the intestinalmicroflora in the metabolismof coumarin in rats. Acta Pharmacol. Toxicol. 26, 325-331. SCHELINE, R. R. (1968~).The metabolismof drugs and other organic compoundsby the intestinal microflora. Acta Pharmacol. Toxicol. 26, 332-342.
BIOTRANSFORMATION
BY INTESTINAL
MICROFLORA
781
R. Q., STURTEVANT, M., BIRD, 0. D. and GLAZKO, A. J. (1954). The effect of metabolitesof chloramphenicol(chloromycetin) on the thyroid of the rat. Endocrinology
THOMPSON,
55,665-68 WILLIAMS,
1. R. T. (1967).
Comparative
patterns
of drug
metabolism.
Fed. Proc., Fed. Amer.
Sot. Exp. Biol. 26, 1029-1039.
D. D. (1935). Indole formation of Bacterium coli. 1. The breakdownof tryptophan by washedsuspensions of Bacterium coli. Biochem. J. 29,640-648.
WOODS,
AND
APPLtED
Toxicologic
23,769-781(1972)
PHARMACOLOGY
Implications Intestinal
of Biotransformation Microflora
by
R. T. WILLIAMS Department of Biochemistry, St. Mary’s Hospital Medical School, London W.2, U.K. Received August 8,1972
The role of the gut microflora in the metabolism of foreign compounds has only been given serious consideration relatively recently. However, references to their probable involvement in the production of certain urinary components and in the destruction of certain ingested compounds have been made in the literature at various times for about a century. Thus in the last quarter of the nineteenth century reference is made to the ethereal sulfates which occur in the urine. These were believed to arise as a result of the intestinal putrefaction of aromatic substances present in food material. Putrefaction produced phenol, cresol, catechol, indole and skatole which were absorbed from the intestine and then converted to ethereal sulfates in the liver. An account of this is given with references to the original papers in a textbook of the last century by Halliburton (1891). Urinary indican (potassium indoxyl sulfate), for example, was thought to be derived from indole which was produced in the intestine by bacteria, since its excretion and that of other ethereal sulfates could be suppressed in dogs by inanition plus the administration of large doses of calomel or iodoform. The source of indole, however, was not found until the beginning of this century when tryptophan was discovered by Hopkins and Cole (1901, 1903) who also showed that indole could be produced from tryptophan by a putrefying pancreas preparation. Ellinger (1903) suggested that tryptophan was the precursor of indole in the intestine and later several workers (Woods, 1936; Happold and Hoyle, 1935; Dawes et al., 1947) showed that E. coli, an organism which is found in the lower intestinal tract, could convert tryptophan to indole through the enzyme system, tryptophanase. The formation of urinary indican would therefore involve metabolism by the gut flora and the tissues, particularly the liver, thus : Jp CH,CHCOOH gut ITmat tryptophan
H indole
H indoxyl Copyright All rights
0 1972 by Academic Press, Inc. of reproduction in any form reserved
769
i-i indican
770
WILLIAMS
The destruction of ingested compounds worth, 1916) and (+)-tartaric acid (Finkle, some earlier papers. Tartaric acid is mainly injection, but largely destroyed when given THE INTESTINAL
such as mesoinositol (Anderson and Bos1933) by gut bacteria is also suggested in excreted unchanged when given to man by by mouth.
MICROFLORA
AND THEIR
DISTRIBUTION
The dominant organisms in the intestine are anaerobic, and therefore many of the reactions which occur in the intestine are likely to be of a reductive nature. The common organisms which may be found in the intestines of man or animals are shown in Table 1. TABLE 1 THE FLORA
OF THE GASTROINTES~NAL
Enterobacteria Enterococci “Viridans” streptococci Staphylococci Yeasts aDrasar et
TRACT”
Lactobacilli Bacteroides Bifidobacteria Clostridia Veillonella
al. (1970).
It is important, however, to appreciate that the location of these organisms along the gastrointestinal tract varies with species (Table 2). In man and the rabbit, few if any organisms occur in the stomach and proximal small intestine. In the guinea pig there are TABLE 2 LOCATION
OF THE GUT
FLORA
IN VARIOUS
SPECIES’* b
Location
Man
Rabbit
Guinea pig
Rat
Mouse
Stomach Proximal small intestine Distal small intestine Large intestine Rectum and feces
o-5 O-5 67 7-10 10-11
O-6 o-5 6-7 8-9 9-10
5-6 5-6 6-7 8-9 9-10
7-9 6-8 7-8 8-9 9-10
7-9 7-9 7-8 8-9 9-10
a Drasar et cd., 1970. * Bacterial counts in various parts of the gastrointestinal tract; log,, number of viable organisms per gram wet weight. small numbers in the stomach and proximal small intestine, but in the rat and mouse, bacteria occur in large numbers in the stomach and proximal small intestine. The main locations of the intestinal flora in man and the rabbit are the large intestine and rectum, whereas in the rat and mouse they occur in large numbers all along the gastrointestinal tract from stomach to rectum.
BIOTRANSFORMATION
BY INTESTINAL
771
MICROFLORA
An interesting consequence of this distribution is seen in the fate of the drug carbenoxolone in man and the rat (Iveson et al., 1971). Carbenoxolone is the hydrogen succinate of glycyrrhetic acid, and its disodium salt, I, (Biogastrone R and Duogastrone R) is used in the treatment of gastric and duodenal ulcer.
COONa
=R’
’ EOCH2CH2E00Na
I
*kOCH2CH2EOONa Carbenoxolone I
In the rat, the 14C-succinate labeled drug is mainly hydrolyzed by the stomach microflora to glycyrrhetic acid (II) and succinate (III). The succinate is then oxidized to CO, which is detected in the expired air by its 14C-label, while the glycyrrhetic acid portion is found in the bile of the rat as the 30- and 3-glucuronides and the 3-sulfate ester. In man, however, the drug is absorbed from the stomach as such and excreted mainly as the 30-glucuronide of carbenoxolone (IV) :
R
,COOH ‘;OCH
I
rat (stomach flora) 2 CH
2EOOH
-
CH,;OOH +
R ‘H
I
I
CHICOOH
II
man (tissues)
. -
* co2
III
tissues
I
COOC6H906
30-glucuronide,
R’ ‘?!0CH2C~2E00~
3-O-glucuronide, 3-O-sulfate
IV
REACTIONS
,COOH
CARRIED
OUT
BY THE
GUT
MICROFLORA
The gut microflora can carry out many reactions involving drugs and other foreign compounds, particularly reactions of a hydrolytic or reductive nature. Most of these reactions have been described in a review by Scheline (1968a), and they include the hydrolysis of glucuronides and glucosides, dehydroxylation of certain catechols, decarboxylation, dealkylation, aromatization, dehalogenation, deamination, reductions of various kinds, ring fission and so on. A few examples of these reactions will suffice to illustrate the versatility of the gut flora.
772
WILLIAMS
Dehydroxylation. Homoprotocatechuic rats and rabbits to 3-hydroxyphenylacetic
acid is dehydroxylated by the gut flora of acid (Dacre et al., 1968).
CH&OOH
CHPOOH
Decarboxylation. 4-Hydroxybenzoic acid, but not 2- or 3-hydroxybenzoic decarboxylated to phenol (Scheline, 1966)
acid, is
0-0 COOH
Dealkylation. Vanillic acid is 0-demethylated by rat gut flora (Scheline, 1966), and methamphetamine is N-demethylated by guinea pig gut flora (J. Caldwell and G. M. Hawksworth, 1972, unpublished data).
Vanillic
acid
CHz
CH2
CHCH,
{HCH,
NHCH,
NH2
Methamphetamine
Aromatization. Quinic acid is aromatized by the gut flora especially in man, to benzoic acid which is excreted as hippuric acid (Adamson et al., 1970). HO gut
flora
in man+
HO Quinic
Dehalogenation.
acid
DDT is dehalogenated to DDD (Scheline, 1968a).
DDT
DDD
BIOTRANSFORMATION
BY INTESTINAL
773
MICROFLORA
Reduction. There are many of these reactions, including the reduction of nitro (NO,) and azo (-N=N-) to NH, groups, of carbon double bonds (-CH=CH-) to the corresponding saturated carbon chain (-CH, *CH,--) and of sulfoxides (>SO) and N-oxides (-NO) to the corresponding sulfides (>S) and tertiary amines ON), respectively. Examples of these reactions are : 1. Chloramphenicol
(Thompson et al., 1954) rat gut flora
CHOHCHCHzOH NHCOCHClz
2. Neoprontosil
CHOHCHCHzOH I NHCOCHC12
(Gingell et al., 1971)
3. Cinnamic acid CH=CHCOOH
Phenylpropionic
4. Chlorpromazine
acid
5-oxide
GX)dWH& Chlorpromazine
Ring fission. This occurs especially with certain heterocyclics; a good example is the conversion of coumarin to mellilotic acid, which includes the intermediate reduction of a double bond before ring fission (Scheline, 1968b).
a0 - a0 - QOOH Coumarin
Dihydrocoumarin
Mellilotic
acid
WILLIAMS
THE
CONSEQUENCES
OF BIOTRANSFORMATION
BY THE
GUT
FLORA
Having described briefly the organisms, their location in the gastrointestinal tract in various species and some of the reactions which they are able to carry out, let us now consider the possible consequences of their activity and how this bears on problems in toxicology and drug metabolism. The enterofloral metabolism of foreign compounds could result in the gastrointestinal microflora being involved in (1) enterohepatic circulation, (2) the production of toxic metabolites, (3) carcinogenesis, (4) detoxication, (5) the production of pharmacologically active metabolites, (6) species variations in drug metabolism and toxicity, (7) individual variations in drug metabolism and toxicity and (8) the production of metabolites not formed in the tissues. Enterohepatic circulation. Compounds or their phase I metabolites (Williams, 1967) which are excreted in the bile as conjugates, particularly glucuronides, could undergo enterohepatic circulation if the gut flora are capable of deconjugating the conjugated metabolites. The gut flora are known to be able to split glucuronides (Scheline, 1968~). This means that the presence of a compound in the body can be prolonged by reabsorption from the intestine after biliary excretion. Therefore the chances of its producing toxic effects are increased. The enterohepatic circulation of stilboestrol, for example, has been shown to involve the gut flora. This compound in the rat is excreted to a considerable extent in the bile as stilbestrol glucuronide. In the gut this glucuronide is split to give free stilbestrol, which is then reabsorbed into the circulation. This reabsorption can be inhibited by antibiotics and by the /3-glucuronidase inhibitor, saccharo-1,4-lactone, since the former suppress the bacteria and the latter inhibits the bacterial /?-glucuronidase (Clark et al., 1969). The production of toxic metabolites by the gutflora. Goodman and Gilman (1955) in their well-known textbook say: “The oral route is the most ancient method of drug administration. It is also the safest, most convenient and most economical.” On injection a drug may be more effective and more certain in its effects, but in general more toxic than by mouth. However, the reverse situation as far as toxicity is concerned is possible if the compound is converted into a toxic agent in the gut but not in the body tissues. This could occur if the compound is metabolized to a toxic agent by the gut flora. The well-known glycoside amygdalin is more toxic to mice when given by mouth than when injected ip, its po LD50 is about 300 mg/kg, whereas on ip injection the LD50 is >5000 mg/kg. It appears that amygdalin is hydrolyzed by the gut flora to mandelonitrile, which is unstable and yields cyanide. Hydrolysis of the glycoside does not occur to any great extent when it is injected. C~%CH(C~‘)OCI~H~~I~
gut flora -
C6HSCH(CN)OH
spontaneous
-
Cd-bCHO
+ HCN
Toxicity could also be the result of the excretion of a compound in the bile followed by metabolism to a toxic agent by the gut flora. A possible example of this is chloramphenicol. This drug is reported to be goitrogenic in rats (Thompson et al., 1954), and this is believed to be due to its conversion by the gut flora of the rat into goitrogenic aromatic amines. In the rat, there is a considerable biliary excretion of chloramphenicol
BIOTRANSFORMATION
BY INTESTINAL
775
MICROFLORA
glucuronide, which could then be deconjugated and reduced to the corresponding amine by the gut flora. The process can be envisaged as follows :
CHoHCHCH20H
CHOHCHCH20.CsH906
NHCOCHClz
NHCOCHCI,
CHoHFHCH20H NHCOCHC12
In man, chloramphenicol is largely excreted as its glucuronide in the urine, and little if any reduction of the nitro group occurs. Curcinogenesis and the gutflora. The possibility that cancer of the large bowel could be due to the conversion of bile salts or steroids of the diet into carcinogens by the gut flora has been discussed by Hill et al. (1971a). A similar suggestion regarding breast cancer has been put forward by Hill et al. (1971 b). Evidence is also mounting that nitrite can be converted into the carcinogenic dialkylnitrosamines by intestinal organisms (Hawksworth and Hill, 1971). That the gut bacteria can produce a carcinogen has been shown in the case of cycasin or methylazoxymethyl P-D-glucoside, which is found in cycad nuts. This compound is carcinogenic in rats when given po, but not when given by injection. The aglycon of cycasin, methylazoxymethanol, is carcinogenic by any route, and it appears that cycasin when given by mouth is hydrolyzed by gut bacteria to the carcinogenic agent thus : CH3N(0):NCH20C6H,,0s
g”t’ora*
CH3N(0):NCH20H
-t C6H,106
This is supported by the finding that when cycasin is given po to germfree rats it is not carcinogenic, although the free aglycon produces cancer in these animals (Laqueur, 1968). However, it is interesting to note that cycasin is carcinogenic on SCinjection into newborn rats, since these animals possess /3-glucosidase in their skins until they are 25 days old but none later (Hirono et al., 1968). The production of active drugs by the gut bacteria. One of the greatest advances in chemotherapy in the first half of this century was the discovery by Domagk of the antibacterial activity of Prontosil. This dyestuff and the later Neoprontosil showed antibacterial activity in vivo but little such activity in vitro. It is now known that these azo dyes were metabolized in the body by the splitting of their azo links to yield sulfanilamide, the actual antibacterial agent.
776
WILLIAMS
Prontosil
Neoprontosil
Sulfanilamide
The splitting of the azo link does in fact occur in the liver by means of the enzyme azoreductase. Like many of the water-soluble sulfonated azo food colors, however, Neoprontosil is poorly absorbed from the intestine; in the past it was often given for therapeutic purposes by injection. Recently it has been shown (Gingell et al., 1971) that in the rat, at least, much of the conversion of Prontosil and Neoprontosil to sulfanilamide is carried out by the gut bacteria. Even if given by injection in the rat, a considerable amount of these dyes, especially Neoprontosil, is excreted in the bile with the azo link intact, so that splitting of the azo link to give sulfanilamide must have taken place to a considerable extent in the gastrointestinal tract. Treatment of the rats with antibiotics to suppress their gut flora considerably reduces the production of sulfanilamide from the dyes. The production of active drugs from inactive precursors by the gut flora is a field that has hardly been investigated. It is possible that such drugs as succinylsulfathiazole and phthalylsulfathiazole which were used for the treatment of dysentery owed their activity to the release of sulfathiazole in the intestine through the activity of the gut flora. TABLE 3 AROMATIZATION OF QUINIC ACID IN VARIOUS SPECIES’
Primates
% aromatized
Man 64 Old World monkeys Rhesusmonkey 43 Baboon 40 Green monkey 45 New World monkeys Spidermonkey 5 Squirrel monkey 0 Capuchin 0 Prosimii Bushbaby 2 Slow loris 0 Tree shrew 0 nAdamsonet al. (1970).
Nonprimates
% Aromatized
Dog
1
Cat Ferret Rabbit
0 0 3
Rat
5
Mouse Guinea pig Hamster Lemming Pigeon Fruit bat Hedgehog
0 0 0 0 2 0 0
BIOTRANSFORMATION
BY INTESTINAL
MICROFLORA
777
Gut bacteria and species variation in drug metabolism. Species variations in the metabolism of foreign compounds are largely due to variations in the tissue enzymes which carry out the reactions (Williams, 1967). However, if compounds are given by mouth or if there is a considerable biliary excretion of injected compounds, variations in metabolism could in some cases depend upon the gut flora. A striking example of a species variation in the metabolism of a foreign compound dependent upon the gut flora has been found with quinic acid which occurs widely in our food (tea, coffee, fruits and vegetables). When given po to man and the Old World monkeys this compound is extensively aromatized and excreted in the urine as hippuric acid (Adamson et al., 1970). In the rhesus monkey it is aromatized when given po but not when injected, and furthermore the aromatization of the po administered acid is suppressed if the monkey is given po antibiotics. Shikimic acid, an intermediate in the aromatization of quinic acid, behaves similarly. In animals lower on the evolutionary scale than Old World monkeys, quinic acid is not converted to benzoic acid to any great extent (Table 3).
Quinic acid
Benzoic acid
Shikimic acid
Individual variations in drug metabolism and the gut flora. It is possible that, under some circumstances which are not at present clear, one individual may metabolize a compound differently from another, and in some way this difference depends upon the gut flora. A good example of this is the well known sweetening agent, cyclamate. This compound is highly polar (pK, 1.9) and is therefore not readily absorbed when taken po. Tts poor absorption allows it to reach the locations in the intestinal tract where bacteria are abundant. In humans and animals which have not received cyclamate previously, the compound is excreted unchanged in the urine and feces when given po and in the urine when given by injection (Renwick and Williams, 1972a). However, if human subjects are given cyclamate daily, some subjects develop the ability to metabolize it to cyclohexylamine and its metabolites (Renwick and Williams, 1972b). This capacity to metabolize po administered cyclamate is not acquired by the body tissues but by the gut flora (Drasar et al., 1972), since injected cyclamate is not metabolized and the feces of active convertors, man or rats, of cyclamate to cyclohexylamine are also able to convert cyclamate to cyclohexylamine. The intestinal organisms which acquire the ability to split cyclamate in man appear to be enterococci. In rats the organisms are clostridia and in rabbits enterobacteria. Why some human subjects develop the ability to split cyclamate by means of their intestinal microflora better than others is at present obscure. Cyclohexylamine has been implicated in the suspected carcinogenicity of cyclamate, and it would appear that cyclamate could be more hazardous to some subjects than others because their gut flora for some obscure reason are more readily trained to split cyclamate. The strong base, cyclohexylamine, is not itself readily metabolized, but a small percentage of it is converted by body tissues to several minor
778
WILLIAMS
metabolites which include cyclohexanol, cyclohexan-trans-I ,2-diol and several aminocyclohexanols.
NHSO,H
trained
-
gut flora
body
tissues
NHz
cyclamate
+
several minor metabolites
cyclohexylamine
Metabolites formed by gutflora but not by body tissues. Many of the reactions carried out by the intestinal flora are similar to those carried out by the tissues of the body. For example, the reduction of the prontosils referred to earlier can be carried out by both the liver and the intestinal flora. It is now clear, however, that the gut bacteria can carry out reactions which do not occur to any great extent in the body tissues. Examples of these reactions have already been given, e.g., the aromatization of quinic acid and the splitting of cyclamate to cyclohexylamine. The toxicologic implications of some of these reactions are quite significant as in the cases of cycasin, amygdalin and cyclamate. There is another reaction of considerable interest which appears to occur in the gut flora but not in the tissues, namely aromatic dehydroxylation. One of the most intensely studied of the reactions of foreign compounds in the liver is aromatic hydroxylation, but the reverse of this reaction, that is the dehydroxylation of an administered phenolic compound, does not appear to occur to any great extent in the tissues. The dehydroxylation of phenolic compounds is known to occur in the body, but evidence is now accumulating which suggests that this is a reaction of the gut microflora. A good example is the dehydroxylation of 3,4-dihydroxyphenylacetic acid (Dacre et al., 1968). In rats and rabbits, this compound gives rise to 3- and 4-hydroxyphenylacetic acid; the major dehydroxylation product is the 3-compound. OH OH CHzCOOH
gutflora+
ooH CH$OOH
+
0 CHzCOOH
The dehydroxylation, but not the other metabolic reactions of 3,4-dihydroxyphenylacetic acid such as 0-methylation, can be suppressed by giving the animals antibiotics. 3-Hydroxyphenolic acids have been found as normal constituents of human urine, e.g., 3-hydroxybenzoic acid, 3-hydroxyphenylacetic acid, 3-hydroxyphenyllactic acid. These could be derived as such from the diet or by metabolism from compounds such as phenylalanine. However, the aromatic hydroxylation of compounds of this type in the liver would be expected to yield 2- or 4-, but not 3-, hydroxy derivatives. It is possible that at least part of the 3-hydroxy acids of the urine are formed by dehydroxylation of catechol acids by the gut flora thus :
BIOTRANSFORMATION
BY INTESTINAL
MICROFLORA
779
An interesting example of a reaction carried out by the gut flora but not by the tissues of the rat is the 0-demethylation of 3-0-methyldopa (L-4-hydroxy-3-methoxyphenylalanine) (Chalmers et al., 197 1). This compound is not demethylated by liver microsomal enzymes in vitro even after pretreatment of the rats with phenobarbitone to increase the activity of the microsomal enzymes. On ip administration to the whole animals, however, the compound, labeled with 14C in the methyl group, is partially demethylated as shown by the elimination of radioactive CO, in the expired air. Apparently the injected compound is partly eliminated in the bile as a conjugate and then demethylated in the gut.
3-0-Methyldopa
DOPA
It is clear from this brief review that the potential of the intestinal microflora for metabolizing foreign compounds can be quite considerable and that as a result of this metabolism, pharmacologically active, toxic and carcinogenic products may be produced. However, when considering the role of the gut flora, it is important to take into account (a) the distribution of the organisms along the gastrointestinal tract in different species and (b) certain characteristics of the compound being investigated such as absorbability from the intestine and the extent of its excretion in the bile. Since the rat has abundant microorganisms all along the gastrointestinal tract from the stomach to the rectum, whereas in man the organisms are located mainly in the large intestine and rectum, the chances of a compound being metabolized by the gut flora in the rat are much greater than in man. In the human, a compound may be entirely absorbed before it can make contact with the microflora. The nature of the compound is important from two aspects, viz., its absorbability from the intestine and the extent of its excretion in the bile, as such or as metabolites, after absorption. Compounds which are highly polar (e.g., cyclamate) are usually not readily absorbed, and they could be exposed to action of the microflora for a longer time than less polar and more absorbable compounds. Compounds or their metabolites of moderately high meolcular weight (i.e., 400-500 or more) tend to be extensively excreted in the bile (Hirom et al., 1972), and such compounds therefore have a greater opportunity of meeting the gut flora and of being metabolized by them than those poorly excreted in bile. REFERENCES
ADAMSON,R. H., BRIDGES, J. W., EVANS,M. E. and WILLIAMS, R. T. (1970).Species differences in the aromatization of quinic acid in virw and the role of gut bacteria. Biochem. J. 116, 437-443. ANDERSON, R. J. and BOSWORTH, A. W. (1916).The utilization of inositein the animal organism. The effect of inositeupon the metabolismof man.J. Biol. Chem.25, 39947. CHALMERS, J.P., DRAFFAN,G. H., REID, J.L., THORGEIRSSON, S.S.and DAVIES,D. S. (1971). Demethylation of 3-O-methyldopain the rat. Life Sci. 10 (f), 1243-1251.
780
WILLIAMS
A. G., FISCHER, L. G., MILLBURN, P., SMITH, R. L. and WILLIAMS, R. T. (1969).The role of gut flora in the enterohepaticcirculation of stiibestrolin the rat. Biochem. J. 112,17P. DACRE, J. C., SCHELINE, R. R. and WILLIAMS, R. T. (1968). The role of the tissuesand gut flora in the metabolismof [‘“Clhomoprotocatechuic acid in the rat and rabbit. J. Pharm. CLARK,
Pharmacol. 20,619-625. DAWES, A. E., DAWSON, J. and HAPPOLD, F. C. (1947). The tryptophan-tryptophanase reaction. 8. The modeof formation of indole. Biochem. J. 41, 426-431. DRASAR, B. S., HILL, M. J. and WILLIAMS, R. E. 0. (1970).The significanceof the gut flora in the safety testingof food additives.Metabolic Aspects of Food Safety. Chapter 10,245-255,
S. J. C. Roe, Blockwell Scientific Publication, Oxford and Edinburgh. DRASAR, B. S., RENWICK, A. G. and WILLIAMS, R. T. (1972).The role of the gut flora in the metabolismof cyclamate.Biochem. J. 129,881-890. ELLINGER, A. (1903).Die Indolbildung undIndicanausscheidung beimhungemdenKaninchen. Hoppe-Seyler’s Z. Physiol. Chem. 39, 44-54. FINKLE, P. (1933).The fate of tartaric acid in the human body. J. Biol. Chem. 100, 349-355. GINGELL, R., BRIDGES, J. W. and WILLIAMS, R. T. (1971). The role of the gut flora in the metabolismof prontosil and neoprontosilin the rat. Xenobiotica 1, 143-156. GOODMAN, L. S. and GILMAN, A. (1955).The Pharmacological Basis of Therapeutics, 2nd ed,
p. 6. Macmillan, New York. HALLIBURTON, W. D. (1891). Textbook
of Chemical Physiology and Pathology, p. 740. Longman’sGreen, London. HAPPOLD, F. C. and HOYLE,L. (1935).The coli-tryptophan-indole reaction 1. Enzyme preparations and their action on tryptophan and someindole derivatives. Biochem. J. 29, 1918-1926. HAWKSWORTH, G. M. and HILL, M. J. (1971). Bacteria and the N-nitrosation of secondary amines.Brit. J. Cancer 25, 520-526. HILL, M. J., CROWTHER, J. S., DRASAR, B. S., HAWKSWORTH, G., ARIES, V. and WILLIAMS, R. E. 0. (1971a).Bacteria and aetiology of cancerof the large bowel. Lancet. 1, 95-100. HILL, M. J., GODDARD, P. and WILLIAMS, R. E. 0. (1971b).Gut bacteria and aetiology of cancerof the breast.Lancer 2,472-473. HIROM, P. C., MILLBURN, P., SMITH,R. L. and WILLIAMS,R. T. (1972).Speciesvariations in the thresholdmolecularweight for the biliary excretion of anions.Biochem. J. 129,1071-1077. HIRONO, I., LAQUEUR, G. L. and SPATZ,M. (1968).Tumor induction in Fischerand OsborneMendel Rats by a SingleAdministration of Cycasin.J. Natl. Cancer Inst. 40, 1003-1010. HOPKINS,F. G., and COLE,S. W. (1901).A contribution to the chemistry of proteids Part I. A preliminary study of a hitherto undescribedproduct of tryptic digestion.J. Physiol. (London) 27,418-428. HOPKINS, F. G. and COLE, S. W. (1903).A contribution to the chemistry of proteids. Part II. The constitution of tryptophane and the action of bacteria upon it. J. Physiol. (London)
29,451-466. IVESON,P., LINDUP, W. E., PARKE,D. V. and WILLIAMS,R. T. (1971). The metabolismof carbenoxolonein the rat. Xenobiotica 1, 79-95. LAQUEUR, G. L. (1968).Toxicology of cyasin. Food Cosmet. Toxicol. 6, 577-578. RENWICK, A. G. and WILLIAMS, R. T. (1972a).The fate of cyclamatein man and other species. Biochem. J. 129,869-879. RENWICK, A. G. and WILLIAMS,R. T. (1972b).Metabolites of cyclohexylaminein man and certain animals.Biochem. J. 129, 857-867.
SCHELINE, R. R. (1966). Decarboxylation and demethylation of somephenolic benzoic acid derivatives by rat caecalcontents.J. Pharm. Pharmacol. 18, 664-669. SCHELINE, R. R. (1968a).Drug metabolismby intestinal microorganisms.J. Pharm. Sci. 57, 2021-2037. SCHELINE, R. R. (1968b).Studieson the role of the intestinalmicroflora in the metabolismof coumarin in rats. Acta Pharmacol. Toxicol. 26, 325-331. SCHELINE, R. R. (1968~).The metabolismof drugs and other organic compoundsby the intestinal microflora. Acta Pharmacol. Toxicol. 26, 332-342.
BIOTRANSFORMATION
BY INTESTINAL
MICROFLORA
781
R. Q., STURTEVANT, M., BIRD, 0. D. and GLAZKO, A. J. (1954). The effect of metabolitesof chloramphenicol(chloromycetin) on the thyroid of the rat. Endocrinology
THOMPSON,
55,665-68 WILLIAMS,
1. R. T. (1967).
Comparative
patterns
of drug
metabolism.
Fed. Proc., Fed. Amer.
Sot. Exp. Biol. 26, 1029-1039.
D. D. (1935). Indole formation of Bacterium coli. 1. The breakdownof tryptophan by washedsuspensions of Bacterium coli. Biochem. J. 29,640-648.
WOODS,