Acta pharmacol. et toxicol. 1976, 39, 433-441.
From the Department of Pharmacology, University of Bergen, Bergen, Norway’, and the Institute of Toxicology, National Food Institute, SGborg, Denmark2
The Metabolism of Biphenyl. 111. Phenolic Metabolites in the Pig BY Trygve MeyerL3, John Chr. Larsenz, Ernst V. Hansenz and Ronald R. Schelinel (Received February 6, 1976; Accepted March 1, 1976)
Abstract: The phenolic metabolites of biphenyl in both male and female pigs were qualitatively and quantitatively analysed as trimethylsilyl (TMS) ethers by combined gas chromatography/mass spectrometry and gas chromatography, respectively. This xenobiotic was metabolized mainly to mono-hydroxylated biphenyl, and in small amounts to di- and tri-hydroxylated biphenyls. Urinary excretion was the only route of elimination of these compounds from the body. The total urinary recovery in male pigs was 44.8 % of the dose 96 hrs after administration of the drug, and the corresponding value in female pigs was 27.6 %. The main part of the urinary metabolites was excreted during the first 24 hrs after dosing in both sexes.
Key-words: Biphenyl - metabolism
-
phenols - pig.
The first metabolic experiment with biphenyl showed that the compound was converted to its 4-hydroxy derivative in the dog (KLINGENBERG 1891). The metabolic conversion of biphenyl into phenols has also been investigated in the rabbit. Thus, STROUD(1940) found 4-hydroxybiphenyl in rabbit urine after administration of the parent compound. This observation was confirmed by BLOCK& CORNISH (1959). BERNINGER et al. (1968) were able to demonstrate 2-hydroxy-, 4-hydroxy- and 4,4’-dihydroxy-biphenyl as urinary metabolites of biphenyl in the same species. WEST et al. (1953, 1955 & 1956) isolated 4-hydroxy-, 3,4-dihydroxy- and 4,4’-dihydroxy-biphenyl from the urine of biphenyl-treated rats. In mice, the only phenols detected foIlowing biphenyI administration were 2-hydroxy- and 4-hydroxy-biphenyl (CREAVEN & PARKE1966). Present address: National Institute of Forensic Toxicology, Oslo, Norway.
434
TRYGVE MEYER ET AL.
The use of more sensitive and specific analytical methods has revealed an increasing number of phenolic biphenyl metabolites in different animal species. Thus, RAIG& AMMON(1970 & 1972), by gas chromatographic analysis of the trimethylsilyl ethers of hydroxybiphenyls, were able to both identify and quantify the 2-hydroxy, 3-hydroxy, 4-hydroxy, 3,4-dihydroxy, 4,4‘-dihydroxy derivatives of biphenyl and the two monomethyl ethers of 3,4-dihydroxy-biphenyl in the urine of rabbits fed biphenyl. MEYERet al. (1976) found both urinary phenols and acids in the urine of rats given 14C-biphenyl, and subsequently MEYER& SCHELINE (1976) identified the mixture of urinary hydroxylated biphenyls. Thus, 2-hydroxy-, 3-hydroxy-, 4-hydroxy-, 3,4-dihydroxy-, 3,4’-dihydroxy-, 4,4’-dihydroxy- and 3,4,4’-trihydroxy-biphenyl including two monomethyl ethers of each of the two catecholic biphenyl derivatives were qualitatively and quantitatively analysed as T M S ethers by combined gas chromatography/mass spectrometry and gas chromatography, respectively. The metabolic conversion of biphenyl into its hydroxylated derivatives is carried out by liver microsomes, and liver preparations from 11 the animal species investigated have been studied by CREAVEN et al. (1965). They reported that the metabolic conversion was present in all 11 the species examined, but the extent varied with the species. The presence of enzyme systems in pig liver microsomes able to form hydroxylated derivatives from biphenyl has not been previously demonstrated. However, SAFEet al. (1975), were able to show in the pig that 4-chlorobiphenyl in vivo gives rise to its 4‘-hydroxy and 3,4’-dihydroxy derivatives. The intention of the present investigation was to study the phenolic metabolites of biphenyl both qualitatively and quantitatively in the pig.
Materials and Methods Chemicals. Biphenyl and substituted biphenyls were obtained as described previously (MEYER & SCHELINE1976). Animal experitnetits. The experiments were carried out using male and female pigs weighing 31-35 kg (Danish Landrace, obtained from a SPF breeding colony). The housing and diet has been described by OLSEN et al. (1973). During the experiments the pigs were placed in metabolism cages and allowed to eat and drink twice every day. In one experiment the animal was given a dose of 3 p neomycin sulphate 3 times daily 4 days before the administration of the test compound and during the whole experimental period. The test compound was given orally either as a solution in soya oil or in propylene glycol. The dose was 100 mg/kg. Urine and faeces were collected separately for 24 hrs periods four days after dosing and stored in containers kept at 0”. Serial 2 hrs bile samples were collected from one
PHENOLIC BIPHENYL METABOLITES IN PIG
435
pig by means of a plastic tube inserted in the common bile duct. The pig was kept under general anaesthesia with mebumal during the whole sampling period of 24 hrs. Extraction of urinary and biliary metabolites. Portions of 25 ml of the urine samples and portions of 10 ml of each 2 hrs bile samples were analysed as described previously (MEYER& SCHELINE1976). Extraction o f faecal metabolites. Aliquots (10 g) of each 24 hrs faecal samples were ground to dryness with NaCl in a mortar and thereafter quantitatively placed in extraction thimbles for continuous ether extraction overnight. The ether extracts were shaken five times with the original sample volume of 5 96 aqueous NaHCO, and once with the same volume of 0.1 M-HCl before drying over anhydrous Na,SO, and finally evaporating to dryness. The residues were dissolved in 1 ml Tri-Sil (Pierce Chemical Co., U.S. A.) to give the TMSderivatives of the phenolic fraction of the faeces. Quantitative dctermiriation of metabolites. The quantitative extraction of phenols from pig urine was found to be better than 95 70.The quantitation was performed as described previously (MEYER& SCHELINE 1976). Gas chromatography and mass spectrometry. Gas chromatography and mass spectrometry were performed as described by MEYER & SCHELINE (1976).
Results
The retention times and the relative abundance of M' and the most prominent fragments of biphenyl, reference compounds and metabolites 1976). as TMS-ethers have been listed previously (MEYER& SCHELINE Urinary metabolites. During these experiments no phenolic metabolites of biphenyl origin were detected in the free fraction of the pig urines. Hence, the urine samples were enzyme hydrolyzed (P-glucuronidase sulphatase) immediately before analysis. The metabolites detected in the urines from two female pigs given biphenyl in soya oil are shown in table 1, from which it can be seen that the total urinary excretion of phenols in 4 days was 27.6olo of the dose given. Most of this (19 %) consisted of 4-hydroxybiphenyl, most of which was excreted within 24 hrs. Of the other metabolites, 2-hydroxybiphenyl and 4,4'-dihydroxybiphenyl made up to 2.7 and 2.0 %, respectively, of the dose whereas each of the remaining compounds comprised less than 1'30. Table 2 shows the urinary excretion values from two male pigs given biphenyl in propylene glycol. The total 96 hrs excretion of phenols in this experiment was 44.8 olo and the major part was detected in the first
+
a)
a) Combined quantitation bl Not detected
Total
3,4,4'-Trihydrox ybiphenyl
3,4’-Dihydroxy-4-methoxybiphenyl) 4,4’-Dihydroxy-3-rnethoxybiphenyl) a)
4,4’-Dihydroxybiphenyl
3,4’-Dihydroxybiphenyl
3,CDihydroxybiphenyl
3-Hydroxy-4-methoxybiphenyl ) 4-Hydroxy-3-methoxybiphenyl )
4-Hydroxybiphenyl
3-H y droxybipheny l
2-Hy droxybiphenyl
Biphenyl
Metabolite
~~
1.2
G7
(0.3)
(0.6)
(0.2)
(0.2)
8.1 (7.2)
0.2
0.3
0.7
0.1
0.2
(0.3)
(4.5)
(1.0)
b)
0.3
0.4
0.2
0.1
0.2
3.9
0.9
G7
6.3
0.2
2. day
12.6 22.4 (17.5)
0.5
0.3
0.9
0.7
0.4
0.3
5.1
1.0
G5
~~
(0.2)
(0.3)
(1.2)
(0.4)
(0.4)
(0.3)
0.3
17.8 (13.6)
(0.9)
b)
1.day
(0.4)
0.2
0.2
1.4
0.1
0.3
0.2
9.4
0.5
G5
~
1.5
0.3
0.3
0.5
0.4
G5
(2.0)
(0.2)
(0.1)
(0.2)
(0.1)
(0.1)
(0.6)
(0.6)
b)
0.2
0.8
0.7
G7
2.4
0.1
0.2
0.1
0.2
3. day
0.6
0.1
0.1
0.2
0.2
G5
(1.0)
(0.1)
(0.1)
b)
(0.3)
(0.3)
(0.3)
b)
4.day
~~
1.3
0.6
0.3
0.4
G7
~
(2.7)
3.2
G7
~~
22.8
0.8
0.2
0.5
0.5
0.8
1.1
0.5
32.4 (27.6)
(0.8)
(0.7)
(0.5)
(0.9)
1.3
15.2 22.8 (19.0)
2.1
G5
Total
Urinary excretion of phenolic metabolites in two female pigs. Values given are % of dose, with mean values in parenthesis. Urine collected daily for four days after oral administration of 100 mglkg biphenyl in soya oil.
Table 1.
l-
b
G
1
3 iri P
5 z
0
a m w
a)
a) Combined quantitation b) Not detected
Total
3,4,4’-Trihydroxybiphenyl
3,4‘-Dihydroxy-4-methoxybiphenyl ) 4,4’-Dihydroxy-3-methoxybiphenyl ) a)
4,4’-Dihydroxybiphenyl
3,4’-Dihydroxybiphenyl
3,4-DihydroxybiphenyI
3-Hydroxy-4-methoxybiphenyl) 4-Hydroxy-3-methoxybiphenyl )
4-Hydroxybiphenyl
3-Hydroxybiphenyl
2-Hy droxybiphenyl
Metabolite
(0.6)
0.3
0.2
1.5
1.1
0.2
0.2
17.3
0.5
22.4 (26.4)
(0.3)
(0.4)
(1.6)
(0.9)
(0.3)
(0.2)
(20.2)
30.3
0.2
0.6
1.7
1.6
0.4
0.2
23.0
0.6
1.1
2.0 (1.6)
G12A
G 11A
1. day *
7.8
0.2
0.2
0.3
0.2
0.2
0.1
5.4
0.2
(9.8)
(0.2)
(0.2)
(0.6)
(0.3)
(0.3)
(0.1)
(6.9)
(0.2)
11.7
0.2
0.2
0.9
0.4
0.3
0.1
8.4
0.2
1.o
1.o
(1.0)
G12A
G11A
2.day
4.5
0.1
0.1
0.2
0.1
0.1
0.1
2.7
0.2
0.9
(4.7)
(0.2)
(0.2)
(0.3)
(0.1)
(0.1)
(0.1)
(2.8)
(0.2)
4.9
0.2
0.2
0.4
0.1
0.1
0.1
2.8
0.1
0.9
G 12A
(0.9)
G11A
3. day
3.3
0.1
0.1
0.1
0.1
0.1
0.1
2.0
0.1
0.6
(4.0)
(0.1)
(0.1)
(0.3)
(0.1)
(0.1)
(0.2)
(2.2)
(0.2)
4.6
0.1
0.1
0.4
0.1
0.1
0.2
2.3
0.2
1.1
G 12A
(0.9)
G 11A
4. day
45.9
0.6
1.o
2.3
2.0
0.8
0.5
33.1
1.1
4.5
(44.8)
(0.7)
(0.9)
(2.8)
(1.9)
(0.8)
(0.6)
1.o
43.6
0.8
0.7
3.2
1.7
0.7
0.6
30.8
(32.0)
(1.1)
4.1
G12A
(4.3)
G 11A
Total
Urinary excretion of phenolic metabolites in two female pigs. Values given are % of dose, with mean values in parenthesis. Urine collected daily for four days after oral administration of 100 mglkg biphenyl in propylene-glycol.
1 able L.
4
w
P
5!
E
E 4
6
z&
z
;4
92:
G
E 0
rn
438
TRYGVE MEYER ET A L .
24 hrs urines. As found previously with female pigs, 4-hydroxybiphenyl (32 Yo) predominated and the major part was excreted during the first 24 hrs. Furthermore, the respective figures for 2-hydroxybiphenyl and 4,4'-dihydroxybiphenyl were found to be 4.3 70 and 2.8 ,% of the dose in the 96 hrs urines. The contribution from each of the other phenolic metabolites was less than 1%I of the dose except for 3,4'-dihydroxybiphenyl which was found to amount to 1.9% four days after the animals had been given biphenyl. The effect of suppresion of the intestinal microflora on the metabolism of biphenyl to phenolic compounds was studied in a single female pig given the test compound as a solution in propylene glycol. The results are shown in table 3, from which it can be seen that the qualitative metabolic pattern is similar to that of the untreated pigs. However, the amounts of each metabolite and the total amount excreted were more similar to those found for the male pigs than those for the female pigs. Faecal metabolites. No phenolic metabolites of biphenyl were detected in any of the experiments. However, unchanged biphenyl (18.4 % and 5.0 Yo of the dose) was detected in the faeces of the two female pigs (G5 and G7, respectively) and most of this was excreted during the first day. No biphenyl was detected in the faeces of the two male pigs (G11A and G12A). Biliary metabolites. No phenols of biphenyl origin were detected in the bile from the pig during the 24 hrs experimental period.
Discussion The present report deals with the formation of phenols from biphenyl in the pig. The experiments show no qualitative differences between male and female pigs, but a quantitative difference was encountered between the two sexes. The total 96 hrs recovery from male pigs was 44.8,% of the dose while the corresponding value of the female pigs was 27.6 Yo. However, some of this difference may be due to the fact that biphenyl absorption in the female pigs was not quantitative. These values reflect solely urinary excretion and no faecal excretion of phenolic metabolites was detected. This result is in keeping with the finding that these metabolites were not excreted in the bile. A similar finding was reported by SAFEet al. (1975), who were not able to find any hydroxylated derivatives of 4-chlorobiphenyl in the pig bile 2 hrs after administration of the latter compound.
439
PHENOLIC BIPHENYL METABOLITES I N PIG
Table 3. Urinary excretion of phenolic metabolites in a female pig given neomycin sulphate. The figures are % of dose and the urine was collected daily for four days after oral administration of 100 mglkg biphenyl in propylene-glycol. ~~
~
1. day
2. day
3. day
4. day
Total
1.6
0.2
0.6
0.7
3.1
27.8
2.2
3.5
1.0
34.5
0.1
b)
0.1
0.1
0.3
3,4-DihydroxybiphenyI
0.1
0.1
0.2
b)
0.4
3,4‘-Dihydroxybiphenyl
0.5
b)
0.1
b)
Metabolite
2-H ydrox ybiphenyl ~~~
~~
3-Hydroxybiphenyl 4-Hydroxybiphenyl 3-Hydroxy-4-methoxybiphenyl ) 4-Hydroxy-3-methoxybiphenyl )
a)
0.6 ~~
4,4’-Dihydroxybiphenyl
0.9
0.1
0.2
~~
0.1
1.3
0.4
~
3,4’-Dihydroxy-4-methoxybiphenyl) 4,4’-Dihydroxy-3-methoxybiphenyl) a)
b,
0.1
0.3
b)
3,4,4‘-Trihydroxybiphenyl
b)
b)
b)
b)
32.1
2.8
5.1
2.0
Total
42.0
a) Combined quantitation b) Not detected
This indicates that pigs are poor biliary excretors of hydroxylated biphenyls in comparison with the rat, a species in which biliary excretion of these et al. 1967; LEVINE et al. 1970; compounds has been established (MILLBURN MEYER& SCHELINE 1976). The absence of the entero-hepatic component in the metabolism of biphenyl in pigs explains why the urinary excretion of biphenyl metabolites in a neomycin-treated pig was not decreased as compared with that found in normal animals. The finding that 4-hydroxybiphenyl is the main metabolite from biphenyl in pigs parallels earlier results in dogs (KLINGENBERG 1891), rabbits (STROUD 1940; BLQCK& CORNISH1959; RAIG& AHMON1970 & 1972), mice (CREAVEN & PARKE1966) and rats (WESTet al. 1956). Recent experiments
440
TRYGVE MEYER ET AL.
on rats however, have revealed that 4,4'-dihydroxybiphenyl is the main metabolite of biphenyl in this species (MEYER& SCHELINE 1976). The over-all qualitative metabolic picture of biphenyl in the pig is similar to that seen in the rat (MEYER& SCHELINE 1976), but quantitative differences were found between these two species. Thus, minor amounts of di- and tri-hydroxylated biphenyls were encountered in the pig in contrast to the rat. Moreover, 2-hydroxybiphenyl was a more important metabolite of biphenyl in the pig than in the rat. Acknowledgements The authors are indebted to Norges Almenvitenskapelige ForskningsrAd and to Norsk Medisinaldepot for grants for the purchase of the G C M S system and the gas chromatograph and integrator unit used in this study. One of us (T. M.) is indebted to Norges Almenvitenskapelige Forskningsrtid for a travel grant in connection with these experiments. We also greatly appreciate the generous help in performing the experiments on the pigs offered by K. A. Kondrup and the technical assistance of Mrs. A. Hetle in analysing the samples.
REFERENCES Berninger, H., R. Ammon & I. Berninger: Spektrophotometrische Identifizierungen von Hydroxyderivaten des Biphenyls. Drug. Res. 1968, 18, 880-883. Block, W. D. & H. H. Cornish: Metabolism of biphenyl and 4-chlorobiphenyl in the rabbit. J . B i d . Chem. 1959, 234, 3301-3302. Creaven, P. J., D. V. Parke & R. T. Williams: A fluorimetric study of the hydroxylation of biphenyl in vitro by liver preparations of various species. Biochem. J . 1965, 96, 879-885. Creaven, P. J. & D. V. Parke: The stimulation of hydroxylation by carcinogenic and non-carcinogenic compounds. Biochem. Pharmacol. 1966, 15, 1-16. Klingenberg, K.: Studien uber die Oxydationen aromatischer Substanzen im tierischen Organismus. Jahresber. Tierchem. 1891, 21, 57-58. Levine, W. G., P. Millburn, R. L. Smith & R. T. Williams: The role of the hepatic endoplasmic reticulum in the biliary excretion of foreign compounds by the rat. The effect of phenobarbitone and SKF 525-A. Biochem. Pharmacol. 1970, 19, 235-244. Meyer, T., J. Aarbakke & R. R. Scheline: The metabolism of biphenyl. I. Metabolic disposition of W-biphenyl in the rat. Acta pharmacol. et toxicol. 1976, 39, 412-418. Meyer, T. & R. R. Scheline: The metabolism of biphenyl. 11. Phenolic metabolites in the rat. Acta pharmacol. et toxicol. 1976, 39, 419432. Millburn, P., R. L. Smith & R. T. Williams: Biliary excretion of foreign compounds. Biphenyl, stilboestrol and phenolphthalein in the rat: Molecular weight, polarity and metabolism as factors in biliary excretion. Biochem. J . 1967, 105, 1275-1281. Olsen, P., G. Wurtzen, E. Hansen, J. Carstensen & E. Poulsen: Short-term peroral toxicity of the food colour Orange RN in pigs. Toxicology 1973, 1, 249-260.
PHENOLIC BIPHENYL METABOLITES IN PIG
44 1
Raig, P. & R. Ammon: Gaschromatographische Analyse der phenolischen Stoffwechselprodukte des Biphenyls. Drug. Res. 1970, 20, 1266-1269. Raig, P. & R. Ammon: Nachweis einiger neuer phenolischer Stoffwechselprodukte des Biphenyls. Drug. Res. 1972, 22, 1399-1404. Safe, S., L. 0. Ruzo, D. Jones, N. S. Platonow & 0. Hutzinger: The metabolism of 4-chlorobiphenyl in the pig. Can. 1. Physiol. Pharmacol. 1975, 53, 392-396. Stroud, S. W.: The metabolism of the parent compounds of some of the simpler synthetic oestrogenic hydrocarbons. f. Endocrinol. 1940, 2, 55-62. West, H. D., G. R. Mathura, E. A. Jones, L. K. Akers & J. R. Lawson: Metabolism of diphenyl. Fed Proc. 1953, 12, 228-229. West, H. D., J. R. Lawson, I. H. Miller & G . R. Mathura: Fate of diphenyl in the rat. Fed. Proc. 1955, 14, 303-304. West, H. D., J. R. Lawson, I. H. Miller & G . R. Mathura: The fate of diphenyl in the rat. Arch. Biochem. Biophys. 1956, 60, 14-20.
From the Department of Pharmacology, University of Bergen, Bergen, Norway’, and the Institute of Toxicology, National Food Institute, SGborg, Denmark2
The Metabolism of Biphenyl. 111. Phenolic Metabolites in the Pig BY Trygve MeyerL3, John Chr. Larsenz, Ernst V. Hansenz and Ronald R. Schelinel (Received February 6, 1976; Accepted March 1, 1976)
Abstract: The phenolic metabolites of biphenyl in both male and female pigs were qualitatively and quantitatively analysed as trimethylsilyl (TMS) ethers by combined gas chromatography/mass spectrometry and gas chromatography, respectively. This xenobiotic was metabolized mainly to mono-hydroxylated biphenyl, and in small amounts to di- and tri-hydroxylated biphenyls. Urinary excretion was the only route of elimination of these compounds from the body. The total urinary recovery in male pigs was 44.8 % of the dose 96 hrs after administration of the drug, and the corresponding value in female pigs was 27.6 %. The main part of the urinary metabolites was excreted during the first 24 hrs after dosing in both sexes.
Key-words: Biphenyl - metabolism
-
phenols - pig.
The first metabolic experiment with biphenyl showed that the compound was converted to its 4-hydroxy derivative in the dog (KLINGENBERG 1891). The metabolic conversion of biphenyl into phenols has also been investigated in the rabbit. Thus, STROUD(1940) found 4-hydroxybiphenyl in rabbit urine after administration of the parent compound. This observation was confirmed by BLOCK& CORNISH (1959). BERNINGER et al. (1968) were able to demonstrate 2-hydroxy-, 4-hydroxy- and 4,4’-dihydroxy-biphenyl as urinary metabolites of biphenyl in the same species. WEST et al. (1953, 1955 & 1956) isolated 4-hydroxy-, 3,4-dihydroxy- and 4,4’-dihydroxy-biphenyl from the urine of biphenyl-treated rats. In mice, the only phenols detected foIlowing biphenyI administration were 2-hydroxy- and 4-hydroxy-biphenyl (CREAVEN & PARKE1966). Present address: National Institute of Forensic Toxicology, Oslo, Norway.
434
TRYGVE MEYER ET AL.
The use of more sensitive and specific analytical methods has revealed an increasing number of phenolic biphenyl metabolites in different animal species. Thus, RAIG& AMMON(1970 & 1972), by gas chromatographic analysis of the trimethylsilyl ethers of hydroxybiphenyls, were able to both identify and quantify the 2-hydroxy, 3-hydroxy, 4-hydroxy, 3,4-dihydroxy, 4,4‘-dihydroxy derivatives of biphenyl and the two monomethyl ethers of 3,4-dihydroxy-biphenyl in the urine of rabbits fed biphenyl. MEYERet al. (1976) found both urinary phenols and acids in the urine of rats given 14C-biphenyl, and subsequently MEYER& SCHELINE (1976) identified the mixture of urinary hydroxylated biphenyls. Thus, 2-hydroxy-, 3-hydroxy-, 4-hydroxy-, 3,4-dihydroxy-, 3,4’-dihydroxy-, 4,4’-dihydroxy- and 3,4,4’-trihydroxy-biphenyl including two monomethyl ethers of each of the two catecholic biphenyl derivatives were qualitatively and quantitatively analysed as T M S ethers by combined gas chromatography/mass spectrometry and gas chromatography, respectively. The metabolic conversion of biphenyl into its hydroxylated derivatives is carried out by liver microsomes, and liver preparations from 11 the animal species investigated have been studied by CREAVEN et al. (1965). They reported that the metabolic conversion was present in all 11 the species examined, but the extent varied with the species. The presence of enzyme systems in pig liver microsomes able to form hydroxylated derivatives from biphenyl has not been previously demonstrated. However, SAFEet al. (1975), were able to show in the pig that 4-chlorobiphenyl in vivo gives rise to its 4‘-hydroxy and 3,4’-dihydroxy derivatives. The intention of the present investigation was to study the phenolic metabolites of biphenyl both qualitatively and quantitatively in the pig.
Materials and Methods Chemicals. Biphenyl and substituted biphenyls were obtained as described previously (MEYER & SCHELINE1976). Animal experitnetits. The experiments were carried out using male and female pigs weighing 31-35 kg (Danish Landrace, obtained from a SPF breeding colony). The housing and diet has been described by OLSEN et al. (1973). During the experiments the pigs were placed in metabolism cages and allowed to eat and drink twice every day. In one experiment the animal was given a dose of 3 p neomycin sulphate 3 times daily 4 days before the administration of the test compound and during the whole experimental period. The test compound was given orally either as a solution in soya oil or in propylene glycol. The dose was 100 mg/kg. Urine and faeces were collected separately for 24 hrs periods four days after dosing and stored in containers kept at 0”. Serial 2 hrs bile samples were collected from one
PHENOLIC BIPHENYL METABOLITES IN PIG
435
pig by means of a plastic tube inserted in the common bile duct. The pig was kept under general anaesthesia with mebumal during the whole sampling period of 24 hrs. Extraction of urinary and biliary metabolites. Portions of 25 ml of the urine samples and portions of 10 ml of each 2 hrs bile samples were analysed as described previously (MEYER& SCHELINE1976). Extraction o f faecal metabolites. Aliquots (10 g) of each 24 hrs faecal samples were ground to dryness with NaCl in a mortar and thereafter quantitatively placed in extraction thimbles for continuous ether extraction overnight. The ether extracts were shaken five times with the original sample volume of 5 96 aqueous NaHCO, and once with the same volume of 0.1 M-HCl before drying over anhydrous Na,SO, and finally evaporating to dryness. The residues were dissolved in 1 ml Tri-Sil (Pierce Chemical Co., U.S. A.) to give the TMSderivatives of the phenolic fraction of the faeces. Quantitative dctermiriation of metabolites. The quantitative extraction of phenols from pig urine was found to be better than 95 70.The quantitation was performed as described previously (MEYER& SCHELINE 1976). Gas chromatography and mass spectrometry. Gas chromatography and mass spectrometry were performed as described by MEYER & SCHELINE (1976).
Results
The retention times and the relative abundance of M' and the most prominent fragments of biphenyl, reference compounds and metabolites 1976). as TMS-ethers have been listed previously (MEYER& SCHELINE Urinary metabolites. During these experiments no phenolic metabolites of biphenyl origin were detected in the free fraction of the pig urines. Hence, the urine samples were enzyme hydrolyzed (P-glucuronidase sulphatase) immediately before analysis. The metabolites detected in the urines from two female pigs given biphenyl in soya oil are shown in table 1, from which it can be seen that the total urinary excretion of phenols in 4 days was 27.6olo of the dose given. Most of this (19 %) consisted of 4-hydroxybiphenyl, most of which was excreted within 24 hrs. Of the other metabolites, 2-hydroxybiphenyl and 4,4'-dihydroxybiphenyl made up to 2.7 and 2.0 %, respectively, of the dose whereas each of the remaining compounds comprised less than 1'30. Table 2 shows the urinary excretion values from two male pigs given biphenyl in propylene glycol. The total 96 hrs excretion of phenols in this experiment was 44.8 olo and the major part was detected in the first
+
a)
a) Combined quantitation bl Not detected
Total
3,4,4'-Trihydrox ybiphenyl
3,4’-Dihydroxy-4-methoxybiphenyl) 4,4’-Dihydroxy-3-rnethoxybiphenyl) a)
4,4’-Dihydroxybiphenyl
3,4’-Dihydroxybiphenyl
3,CDihydroxybiphenyl
3-Hydroxy-4-methoxybiphenyl ) 4-Hydroxy-3-methoxybiphenyl )
4-Hydroxybiphenyl
3-H y droxybipheny l
2-Hy droxybiphenyl
Biphenyl
Metabolite
~~
1.2
G7
(0.3)
(0.6)
(0.2)
(0.2)
8.1 (7.2)
0.2
0.3
0.7
0.1
0.2
(0.3)
(4.5)
(1.0)
b)
0.3
0.4
0.2
0.1
0.2
3.9
0.9
G7
6.3
0.2
2. day
12.6 22.4 (17.5)
0.5
0.3
0.9
0.7
0.4
0.3
5.1
1.0
G5
~~
(0.2)
(0.3)
(1.2)
(0.4)
(0.4)
(0.3)
0.3
17.8 (13.6)
(0.9)
b)
1.day
(0.4)
0.2
0.2
1.4
0.1
0.3
0.2
9.4
0.5
G5
~
1.5
0.3
0.3
0.5
0.4
G5
(2.0)
(0.2)
(0.1)
(0.2)
(0.1)
(0.1)
(0.6)
(0.6)
b)
0.2
0.8
0.7
G7
2.4
0.1
0.2
0.1
0.2
3. day
0.6
0.1
0.1
0.2
0.2
G5
(1.0)
(0.1)
(0.1)
b)
(0.3)
(0.3)
(0.3)
b)
4.day
~~
1.3
0.6
0.3
0.4
G7
~
(2.7)
3.2
G7
~~
22.8
0.8
0.2
0.5
0.5
0.8
1.1
0.5
32.4 (27.6)
(0.8)
(0.7)
(0.5)
(0.9)
1.3
15.2 22.8 (19.0)
2.1
G5
Total
Urinary excretion of phenolic metabolites in two female pigs. Values given are % of dose, with mean values in parenthesis. Urine collected daily for four days after oral administration of 100 mglkg biphenyl in soya oil.
Table 1.
l-
b
G
1
3 iri P
5 z
0
a m w
a)
a) Combined quantitation b) Not detected
Total
3,4,4’-Trihydroxybiphenyl
3,4‘-Dihydroxy-4-methoxybiphenyl ) 4,4’-Dihydroxy-3-methoxybiphenyl ) a)
4,4’-Dihydroxybiphenyl
3,4’-Dihydroxybiphenyl
3,4-DihydroxybiphenyI
3-Hydroxy-4-methoxybiphenyl) 4-Hydroxy-3-methoxybiphenyl )
4-Hydroxybiphenyl
3-Hydroxybiphenyl
2-Hy droxybiphenyl
Metabolite
(0.6)
0.3
0.2
1.5
1.1
0.2
0.2
17.3
0.5
22.4 (26.4)
(0.3)
(0.4)
(1.6)
(0.9)
(0.3)
(0.2)
(20.2)
30.3
0.2
0.6
1.7
1.6
0.4
0.2
23.0
0.6
1.1
2.0 (1.6)
G12A
G 11A
1. day *
7.8
0.2
0.2
0.3
0.2
0.2
0.1
5.4
0.2
(9.8)
(0.2)
(0.2)
(0.6)
(0.3)
(0.3)
(0.1)
(6.9)
(0.2)
11.7
0.2
0.2
0.9
0.4
0.3
0.1
8.4
0.2
1.o
1.o
(1.0)
G12A
G11A
2.day
4.5
0.1
0.1
0.2
0.1
0.1
0.1
2.7
0.2
0.9
(4.7)
(0.2)
(0.2)
(0.3)
(0.1)
(0.1)
(0.1)
(2.8)
(0.2)
4.9
0.2
0.2
0.4
0.1
0.1
0.1
2.8
0.1
0.9
G 12A
(0.9)
G11A
3. day
3.3
0.1
0.1
0.1
0.1
0.1
0.1
2.0
0.1
0.6
(4.0)
(0.1)
(0.1)
(0.3)
(0.1)
(0.1)
(0.2)
(2.2)
(0.2)
4.6
0.1
0.1
0.4
0.1
0.1
0.2
2.3
0.2
1.1
G 12A
(0.9)
G 11A
4. day
45.9
0.6
1.o
2.3
2.0
0.8
0.5
33.1
1.1
4.5
(44.8)
(0.7)
(0.9)
(2.8)
(1.9)
(0.8)
(0.6)
1.o
43.6
0.8
0.7
3.2
1.7
0.7
0.6
30.8
(32.0)
(1.1)
4.1
G12A
(4.3)
G 11A
Total
Urinary excretion of phenolic metabolites in two female pigs. Values given are % of dose, with mean values in parenthesis. Urine collected daily for four days after oral administration of 100 mglkg biphenyl in propylene-glycol.
1 able L.
4
w
P
5!
E
E 4
6
z&
z
;4
92:
G
E 0
rn
438
TRYGVE MEYER ET A L .
24 hrs urines. As found previously with female pigs, 4-hydroxybiphenyl (32 Yo) predominated and the major part was excreted during the first 24 hrs. Furthermore, the respective figures for 2-hydroxybiphenyl and 4,4'-dihydroxybiphenyl were found to be 4.3 70 and 2.8 ,% of the dose in the 96 hrs urines. The contribution from each of the other phenolic metabolites was less than 1%I of the dose except for 3,4'-dihydroxybiphenyl which was found to amount to 1.9% four days after the animals had been given biphenyl. The effect of suppresion of the intestinal microflora on the metabolism of biphenyl to phenolic compounds was studied in a single female pig given the test compound as a solution in propylene glycol. The results are shown in table 3, from which it can be seen that the qualitative metabolic pattern is similar to that of the untreated pigs. However, the amounts of each metabolite and the total amount excreted were more similar to those found for the male pigs than those for the female pigs. Faecal metabolites. No phenolic metabolites of biphenyl were detected in any of the experiments. However, unchanged biphenyl (18.4 % and 5.0 Yo of the dose) was detected in the faeces of the two female pigs (G5 and G7, respectively) and most of this was excreted during the first day. No biphenyl was detected in the faeces of the two male pigs (G11A and G12A). Biliary metabolites. No phenols of biphenyl origin were detected in the bile from the pig during the 24 hrs experimental period.
Discussion The present report deals with the formation of phenols from biphenyl in the pig. The experiments show no qualitative differences between male and female pigs, but a quantitative difference was encountered between the two sexes. The total 96 hrs recovery from male pigs was 44.8,% of the dose while the corresponding value of the female pigs was 27.6 Yo. However, some of this difference may be due to the fact that biphenyl absorption in the female pigs was not quantitative. These values reflect solely urinary excretion and no faecal excretion of phenolic metabolites was detected. This result is in keeping with the finding that these metabolites were not excreted in the bile. A similar finding was reported by SAFEet al. (1975), who were not able to find any hydroxylated derivatives of 4-chlorobiphenyl in the pig bile 2 hrs after administration of the latter compound.
439
PHENOLIC BIPHENYL METABOLITES I N PIG
Table 3. Urinary excretion of phenolic metabolites in a female pig given neomycin sulphate. The figures are % of dose and the urine was collected daily for four days after oral administration of 100 mglkg biphenyl in propylene-glycol. ~~
~
1. day
2. day
3. day
4. day
Total
1.6
0.2
0.6
0.7
3.1
27.8
2.2
3.5
1.0
34.5
0.1
b)
0.1
0.1
0.3
3,4-DihydroxybiphenyI
0.1
0.1
0.2
b)
0.4
3,4‘-Dihydroxybiphenyl
0.5
b)
0.1
b)
Metabolite
2-H ydrox ybiphenyl ~~~
~~
3-Hydroxybiphenyl 4-Hydroxybiphenyl 3-Hydroxy-4-methoxybiphenyl ) 4-Hydroxy-3-methoxybiphenyl )
a)
0.6 ~~
4,4’-Dihydroxybiphenyl
0.9
0.1
0.2
~~
0.1
1.3
0.4
~
3,4’-Dihydroxy-4-methoxybiphenyl) 4,4’-Dihydroxy-3-methoxybiphenyl) a)
b,
0.1
0.3
b)
3,4,4‘-Trihydroxybiphenyl
b)
b)
b)
b)
32.1
2.8
5.1
2.0
Total
42.0
a) Combined quantitation b) Not detected
This indicates that pigs are poor biliary excretors of hydroxylated biphenyls in comparison with the rat, a species in which biliary excretion of these et al. 1967; LEVINE et al. 1970; compounds has been established (MILLBURN MEYER& SCHELINE 1976). The absence of the entero-hepatic component in the metabolism of biphenyl in pigs explains why the urinary excretion of biphenyl metabolites in a neomycin-treated pig was not decreased as compared with that found in normal animals. The finding that 4-hydroxybiphenyl is the main metabolite from biphenyl in pigs parallels earlier results in dogs (KLINGENBERG 1891), rabbits (STROUD 1940; BLQCK& CORNISH1959; RAIG& AHMON1970 & 1972), mice (CREAVEN & PARKE1966) and rats (WESTet al. 1956). Recent experiments
440
TRYGVE MEYER ET AL.
on rats however, have revealed that 4,4'-dihydroxybiphenyl is the main metabolite of biphenyl in this species (MEYER& SCHELINE 1976). The over-all qualitative metabolic picture of biphenyl in the pig is similar to that seen in the rat (MEYER& SCHELINE 1976), but quantitative differences were found between these two species. Thus, minor amounts of di- and tri-hydroxylated biphenyls were encountered in the pig in contrast to the rat. Moreover, 2-hydroxybiphenyl was a more important metabolite of biphenyl in the pig than in the rat. Acknowledgements The authors are indebted to Norges Almenvitenskapelige ForskningsrAd and to Norsk Medisinaldepot for grants for the purchase of the G C M S system and the gas chromatograph and integrator unit used in this study. One of us (T. M.) is indebted to Norges Almenvitenskapelige Forskningsrtid for a travel grant in connection with these experiments. We also greatly appreciate the generous help in performing the experiments on the pigs offered by K. A. Kondrup and the technical assistance of Mrs. A. Hetle in analysing the samples.
REFERENCES Berninger, H., R. Ammon & I. Berninger: Spektrophotometrische Identifizierungen von Hydroxyderivaten des Biphenyls. Drug. Res. 1968, 18, 880-883. Block, W. D. & H. H. Cornish: Metabolism of biphenyl and 4-chlorobiphenyl in the rabbit. J . B i d . Chem. 1959, 234, 3301-3302. Creaven, P. J., D. V. Parke & R. T. Williams: A fluorimetric study of the hydroxylation of biphenyl in vitro by liver preparations of various species. Biochem. J . 1965, 96, 879-885. Creaven, P. J. & D. V. Parke: The stimulation of hydroxylation by carcinogenic and non-carcinogenic compounds. Biochem. Pharmacol. 1966, 15, 1-16. Klingenberg, K.: Studien uber die Oxydationen aromatischer Substanzen im tierischen Organismus. Jahresber. Tierchem. 1891, 21, 57-58. Levine, W. G., P. Millburn, R. L. Smith & R. T. Williams: The role of the hepatic endoplasmic reticulum in the biliary excretion of foreign compounds by the rat. The effect of phenobarbitone and SKF 525-A. Biochem. Pharmacol. 1970, 19, 235-244. Meyer, T., J. Aarbakke & R. R. Scheline: The metabolism of biphenyl. I. Metabolic disposition of W-biphenyl in the rat. Acta pharmacol. et toxicol. 1976, 39, 412-418. Meyer, T. & R. R. Scheline: The metabolism of biphenyl. 11. Phenolic metabolites in the rat. Acta pharmacol. et toxicol. 1976, 39, 419432. Millburn, P., R. L. Smith & R. T. Williams: Biliary excretion of foreign compounds. Biphenyl, stilboestrol and phenolphthalein in the rat: Molecular weight, polarity and metabolism as factors in biliary excretion. Biochem. J . 1967, 105, 1275-1281. Olsen, P., G. Wurtzen, E. Hansen, J. Carstensen & E. Poulsen: Short-term peroral toxicity of the food colour Orange RN in pigs. Toxicology 1973, 1, 249-260.
PHENOLIC BIPHENYL METABOLITES IN PIG
44 1
Raig, P. & R. Ammon: Gaschromatographische Analyse der phenolischen Stoffwechselprodukte des Biphenyls. Drug. Res. 1970, 20, 1266-1269. Raig, P. & R. Ammon: Nachweis einiger neuer phenolischer Stoffwechselprodukte des Biphenyls. Drug. Res. 1972, 22, 1399-1404. Safe, S., L. 0. Ruzo, D. Jones, N. S. Platonow & 0. Hutzinger: The metabolism of 4-chlorobiphenyl in the pig. Can. 1. Physiol. Pharmacol. 1975, 53, 392-396. Stroud, S. W.: The metabolism of the parent compounds of some of the simpler synthetic oestrogenic hydrocarbons. f. Endocrinol. 1940, 2, 55-62. West, H. D., G. R. Mathura, E. A. Jones, L. K. Akers & J. R. Lawson: Metabolism of diphenyl. Fed Proc. 1953, 12, 228-229. West, H. D., J. R. Lawson, I. H. Miller & G . R. Mathura: Fate of diphenyl in the rat. Fed. Proc. 1955, 14, 303-304. West, H. D., J. R. Lawson, I. H. Miller & G . R. Mathura: The fate of diphenyl in the rat. Arch. Biochem. Biophys. 1956, 60, 14-20.
