Acta pharmacol. et toxicol. 1976, 39,412-418.

From thc Department of Pharmacology, University of Bergen, MFH-Bygget, 5016 Haukeland sykehus, Bergen, Norway

The Metabolism of Biphenyl. I. Metabolic Disposition of 14C-Biphenylin the Rat BY

Trygve Meyerl, Jarle Aarbakke and Ronald R. Scheline (Received February 6, 1976; Accepted March 1, 1976)

Abstract: The metabolic disposition of 14C-biphenyl in the rat was studied by liquid scintillation counting. The rats were given an oral dose of 14Gbiphenyl (100 mg/kg, 0.7-1.0 yci) and the total excretion of radioactivity after 96 hrs was 92.2 % of the dose. Urinary excretion accounted for 84.8 % and faecal excretion for 7.3 % of the dose. Most of this radioactivity, 75.8 % and 5.8 % respectively, was excreted within 24 hrs. Only trace amounts of W O , were detected in the expired air and 0.6 % of the dose was found to be still present in the rats 96 hrs after biphenyl administration. Extraction and fractionation of the 24 hrs urine samples showed that the largest fraction (nearly 30 % of the dose) consisted of conjugated phenolic metabolites. Acidic metabolites accounted for a quarter of the dose and the low levels of expired W O , indicated that these werc not products resulting from extensive degradation and decarboxylation. Key-words: Biphenyl - metabolism - radioactivity

- rat.

Biphenyl has been used as a heat transfer agent and also as a fungistat for preserving citrus fruit during transport and storage. It is mainly for its latter use that the biological and toxicological properties of biphenyl are of interest. Despite its long use, data on the toxicology of biphenyl have not been extensive. The lack of data especially in humans has given the impression that its toxicity is fairly low (Hygienic guide series: Diphenyl 1964). However, toxic effects were shown in laboratory animals (DEICHMANN et al. 1947; AMBROSE et al. 1960; Boom et al. 1961), and later also in man (WEE et al. 1965). The first clinical report of serious human biphenyl poisoning et al. (1973) who reported the death of a worker was given by HAKKINEN and eight cases of severe poisoning occurring in the Finnish paper industry. 1

Present address: National Institute of Forensic Toxicology, Oslo, Norway.

BIPHENYL METABOLISM

413

The increasing interest in the toxicological properties of biphenyl has concomitantly resulted in an effort being made to ascertain its metabolic fate in animals. The first metabolic study of biphenyl in animals was performed by KLINGENBERG (1891) who showed that 4-hydroxybiphenyl was a metabolite in dogs. This metabolite was also found in the urine of rabbits given biphenyl (STROUD1940). Findings by WEST(1940) indicated that biphenyl is converted in rats to a mercapturic acid derivative. Thus the hydrocarbon may produce a deficiency in the sulphur-containing amino acids available to the organism, resulting in inhibition of growth. Reversal of this inhibition was seen when L-cysteine or DL-methionine was administered. Growth inhibition has also been reported by DEICHMA”et al. (1947) who gave biphenyl to mice, rats and rabbits. These results strongly suggest a possible role of these amino acids in the metabolic transformation of biphenyl and N-acetyl-5-biphenyl-L-cysteinehas been isolated from the urine of rats fed biphenyl (WEST& MATHURA 1954). Phenolic metabolites of biphenyl have been isolated by WEST et al. (1953, 1955 & 1956) from the urine of rats fed biphenyl. Later, several new phenolic metabolites were demonstrated both qualitatively and quantitatively after oral administration of biphenyl to rabbits (RAIG& AMMON1970 & 1972) and to rats (MEYER& SCHELINE 1976). The purpose of the present investigation was to study the nature of the metabolites of biphenyl in the rat and the main routes of excretion. Materials and Methods Compounds.

l4C-Bipheny1, uniformly labelled in the phenyl rings, was purchased from New England Nuclear C o p , U. S. A. It was shown by autoradiography to have a radiochemical purity greater than 99 %. Animal experiments.

Male albino rats weighing 230-270 g were used. The main routes of excretion were investigated using a glass metabolic cage (“Metabowl”, Jencons Scientific Ltd., England) ventilated with air from which water vapour and CO, had been removed. The animals were maintained on a commercial pellet diet (Vestlandske KjZpelag, Norway) both before and during the experiments and they were given the diet and drinking water ad libiturn. Urine and faeces were collected separately in 24 hrs samples for four days after oral administration of the dose of 14C-biphenyl (100 mg/kg, 0.7-1.0 pci). Carbon dioxide was trapped in a solution of 20% ethanolamine in ethanol in two traps connected to the air outlet. After 96 hr of the experimental period, the animals were killed by decapitation and tissue samples were immediately removed, weighed and kept frozen at -20“ until analysis. Analysis of radioactivity. After addition of 10 ml of the scintillation liquid Triton X-100 the samples were

414

T. MEYER, J . AARBAKKE AND R. R. SCHELINE

kept in the dark and cold before the radioactivity was determined in a Packard Liquid Scintillation Spectrometer (Mod, 3320). Counting efficiency was determined by the internal standard method (W-toluene) and acceptable values were obtained with samples of tissues, CO,, urine and faeces. Preparation of samples. Samples of lung, hearth, kidney, brain, spleen, liver, skeletal muscles, peritoneal fat, genital tract, gastrointestinal tract included its contents and faeces were homogenized in distilled water to give a suspension from which approximately 100 mg was placed in a counting vial, dissolved in 2 ml Soluene (Packard) and decolorized with 0.5 ml 30 % H,O, at 50" for 30 min. before analysis. The radioactivity in the urine was determined in 0.5 ml aliquots of each 24 hrs portion. The radioactivity in the blood after the 96 hrs experimental period was determined in 0.2 ml aliquots of the sample. The radioactivity in the expired air was determined in 1 ml aliquots of the ethanolamine solution from each of the traps. For investigation of the nature of the radioactivity in the 24 hrs urines, 5 ml aliquots of each sample were adjusted to pH 2 and extracted five times with 5 ml portions of ether. The aqueous residue was retained. The combined ether extracts were shaken three times with the original sample volume of 5 % aq. NaHCO, and then once with 5 ml 0.1 N-HCI. The final p H values of these aqueous solutions were found to he approximately 8 and 1, respectively. The exact volumes of the aqueous urinary residues and the NaHCO, and HC1 fractions were determined before the radioactivity in 0.1 ml aliquots of each was determined. The amounts of radioactivity in the ether extracts were calculated as the difference between the total amounts and the contents of the three aqueous solutions. The aqueous urinary residues after the ether extractions were then adjusted to pH 5 with 1 N-NaOH and acetate buffer and hydrolyzed with a preparation of flglucuronidase and sulphatase (Glusulase, Endo Lab., u. S. A.) according to the method used by BAKKE& SCHELINE(1969) before being extracted again as described above. This procedure gave the free phenolic and acidic fractions before enzyme hydrolysis and the corresponding conjugated fraction after hydrolysis had taken place.

Results

Approximately three fourths of the radioactivity of the administered dose of biphenyl was excreted in the urine during the first 24 hrs (table 1). The extent of excretion of radioactivity in the urine diminished greatly after 48 hrs and the mean total excretion during the 96 hrs period was 84.8 ?fo of the dose. The mean total 96 hrs faecal excretion of radioactivity was 7.3 % of which 5.8 70could be detected in the first 24 hrs portions of faeces. Only trace amounts of radioactivity were detected in the 96 hrs expired air. After the four day experimental period very small amounts of radioactivity were found in the tissue samples. The total amount remaining in the animals was 0.6 %, of which 0.1 % could be found in peritoneal fat, 0.3 TOin the gastrointestinal tract including its contents, 0.1 9" in skeletal muscles and 0.1 % in the genital tract.

415

BIPHENYL METABOLISM

Table 1. Excretion of radioactivity in three rats given (14C)-biphenyl (100 mg/kg, 0.7-1.0 ,xi) by stomach tube. Route of excretion

0-24 hrs

24-48 hrs

48-96 hrs

Total 0-96 hrs

Urine

75.8 (74.4-76.4-76.7)

7.1 (5.7-7.7-7.8)

1.9 (1.4-1.4-1.5)

84.8 (84.0-84.1-86.2)

Faeces

5.8 (4.1-6.2-7.1)

1.3 (0.7-0.8-2.4)

0.2 (0-0-0.7)

7.3 (5.6-7.8-8.5)

"1

")

0.1 (0.01-0.1-0.2)

8.4

2.1

92.2

Expired air

Total recovery

YA

,

81.6

Values are given as mean percentages of animal radioactive dose with the values from each animal in parenthesis.

* Not analyzed until after 96 hrs.

Table 2 shows the fractionation of the radioactivity in the 24 hrs urine from rats given biphenyl. The subsequent extractions of the ether with NaHCOs solution revealed radioactivity both in the NaHCOs fraction and in the residual ether, indicating the presence of both acidic and phenolic metabolites of biphenyl in the rat urine. Moreover, the extraction of the residual ether with dilute HC1 showed that smaller amounts of radioactivity could be detected in the HCl fraction, suggesting the presence of amphoteric metabolites of biphenyl. Discussion

Metabolic conversion of biphenyl into more polar derivatives is necessary for its elimination from the body. The present study shows that the

metabolism of biphenyl in the rat is extensive, and that 92.2 00 of the administered dose was recovered 96 hrs after dosing. Less than 1 Yo of the dose was recovered from the tissues. The major part of the metabolites was excreted in the urine during the first 24 hrs period, and a minor fraction appeared in the faeces. Only trace amounts of 14C02were detected in the expired air from the rats, suggesting that decarboxylation reactions are of minor importance in the metabolism of biphenyl in this species. The present findings indicate that biphenyl is converted through oxidative metabolic reactions in the rat to phenols and acids. The enzyme systems responsible for the biphenyl hydroxylation reactions have been localized in the liver microsomes of several rodents and other species (CREAVEN

T. MEYER, J. AARBAKKE AND R. R. SCHELINE

416

Table 2. Fractionation of the radioactivity in the first day urine from rats given an oral dose of W-biphenyl (100 mg/kg, 0.7-1.0 pci). Free

Ether fraction

Conjugated

28.6 (23.1-28.4-34.3)

Total

41.1 (25.2-41.3-56.7)

Free NaHCO, fraction

HCl fraction

12.5 (2.1-12.9-22.4)

Conjugated

18.7 (27.9-17.8-10.5) 6.8 (9.4-7.1-4.0)

Total

25.5 (37.3-24.9-14.5)

Free

(1.3-0.6-0.4)

Conjugated

0.4 (0.6-0.4-0.3)

Total

1.2 (1.9-1 .O-O.7)

Water soluble not extractable residues

8.0 (12.0-7.2-4.8)

Total

0.8

75.8 (74.4-76.8-76.7)

~

Values are given as mean percentages of radioactive dose with the values from each animal in parenthesis.

et al. 1965) and two types of these systems have been shown to be responsible for the different patterns of biphenyl hydroxylation observed in different species (BURKE& MAYER1975; BURKE& BRIDGES1975). The excretion of phenols and acids of biphenyl origin confirms the earlier findings of WEST et a!. (1956), who isolated phenols, biphenylmercapturic acid and p-(3-D-glucuronosidobiphenyl from rat urine. These metabolites accounted for 50% of the dose given. Urinary phenols have also been demonstrated in rabbits after oral administration of biphenyl (RAIG &

BIPHENYL METABOLISM

417

AMMON1970 & 1972). A report on the phenols of biphenyl origin in the rat has been given by MEYER& SCHELINE(1976), who identified and quantified twelve different phenolic metabolites by mass spectrometry and gas chromatography, respectively. The amounts of phenolic-behaving metabolites in this report (approximately 40-50 9’0) are in contrast to the findings of MEYER& SCHELINE (1976), who reported only trace amounts of phenols in the free fractions of the urines and 22.3 Yo conjugated phenols in the first 24 hrs samples. This discrepancy suggests the presence of unknown phenols or very weak acids of unknown nature in the urinary ether fractions. The radioactivity in the aqueous NaHC03 fraction of the rat urine suggests the presence of acidic metabolites of biphenyl. Furthermore, the presence of small amounts (approximately 1 9‘0)of radioactivity in the HCI fraction indicates that one or more amphoteric metabolites, e. g. biphenylmercapturic acid, may be formed. WEST et al. (1956) found that 1.7 Yo of the dose was converted to this metabolite. The acidic metabolites of biphenyl in the rat apart from biphenylmercapturic acid and the glucuronides, might originate from a catechol derivative by ring cleavage of the diol moiety, a reaction possibly produced by the intestinal microflora. Similar metabolic reactions have been shown to take place when biphenyl is incubated with Pseudomonas putida (CATELANI et al. 1971) and in pure cultures of Gram-negative bacteria isolated from tropical and temperate soils (LUNT& EVANS1970). The present report shows that 5.8Yo of the radioactive dose used is recovered in the 24 hrs faeces. The corresponding value found by gas (1976) is 4.7 Yo. This material conchromatography by MEYER& SCHELINE sisted mainly of the 4-hydroxy, 4,4’-dihydroxy- and 3,4,4’-trihydroxy derivatives of biphenyl. These results indicate that biphenyl metabolites are excreted in the bile of rats, a finding which has also been shown by MILLBURN et al. (1967), LEVINEet al. (1970) and MEYER & SCHELINE (1976). Acknowledgements The technical assistance of Mrs. E. Tepstad and Mrs. A. Hetle is greatly appreciated. REFERENCES Ambrose, A. M., A. N. Booth, F. DeEds & A. J. Cox, Jr.: A toxicological study of biphenyl, a citrus fungistat. Food Res. 1960, 25, 328-336. Bakke, 0. M. & R. R. Scheline: Analysis of simple phenols of interest in metabolism. 11. Conjugate hydrolysis and extraction methods. Anal. Biochem. 1969, 27, 451-462. Booth, A. N., A. M. Ambrose, F. DeEds & A. J. Cox, Jr.: The reversible nephrotoxic effects of biphenyl. Toxicol. Appl. Pharmacol. 1961, 3, 560-567.

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Burke, M. D. & J. W. Bridges: Biphenyl hydroxylations and spectrally apparent interactions with liver microsomes from hamsters pre-treated with phenobarbitone and 3-methylcholanthrene. Xenobiotica 1975, 5, 357-376. Burke, M. D. & R. T. Mayer: Inherent specificities of purified cytochromes P-450 and P-448 toward biphenyl hydroxylation and ethoxyresorufin deethylation. Drug. Metub. D i ~ p .1975, 3, 245-253. Catelani, D., C. Sorlini & V. Treccani: The metabolism of biphenyl by Pseudomonas putida. Experientia 1971, 27, 1173-1174. 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. Biochent. 1. 1965, 96, 879-885. Deichmann, W. B., K. V. Kitzmiller, M. Dierker & S. Witherup: Observations on the effects of diphenyl, 0-and p-aminodiphenyl, 0- and p-nitrodiphenyl and dihydroxyoctachlorodiphenyl upon experimental animals, J . Ind. H y g . Tox. 1947, 29, 1-13. Hygienic guide series: Diphenyl. A m . Ind. H y g . Ass. J . 1964, 25, 522-524. Hakkinen, J., E. Siltanen, S. Hernberg, A. M. Seppalainen, P. Karli & E. Vikkula: Diphenyl poisoning in fruit paper production. A new health hazard. Arch. Environ. Hecllth 1973, 26, 70-74. Klingenberg, K.: Studien iiber die Oxydationen aromatischer Substanzen im tierischen Organismus. Juhresber. Tierchem. 1891, 21, 57-58. Levine, W. G., P. Millburn, R. L. Smith & R. T. Williams: The role of the hepatic endoplasrnic 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. Lunt, D. & W. C . Evans: The microbial metabolism of biphenyl. Biochem. J . 1970, 118, 54P-55P. Meyer, T. & R. R. Scheline: The metabolism of biphenyl. 11. Phenolic metabolites in the rat. Acta pharmacol. et toxicol. 1976, 39, 419-432. 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. 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. Stroud, S. W.: The metabolism of the parent compounds of some of the simpler synthetic oestrogenic hydrocarbons. J . Endocrinol. 1940, 2, 55-62. Weil, E., L. Kusterer & M. H. Brogard: Intolkrance a un produit d’imprkgnation antifongique des emballages d’argrumes. Arch. Mal. Prof. 1965, 26, 405-408. West, H. D.: Evidence for the detoxication of diphenyl through a sulfur mechanism. Proc. Soc. Exp. Biol. M e d . 1940, 43, 373-375. 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. & G. R. Mathura: Synthesis of some arylsubstituted L-cysteines and their fate in the animal body. J . Biol. Chem. 1954, 208, 315-318. 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, 1. H. Miller & G. R. Mathura: T h e fate of diphenyl in the rat. Arch. Biochem. Biophys. 1956, 60, 14-20.

The metabolism of biphenyl. I. Metabolic disposition of 14C-biphenyl in the rat.

Acta pharmacol. et toxicol. 1976, 39,412-418. From thc Department of Pharmacology, University of Bergen, MFH-Bygget, 5016 Haukeland sykehus, Bergen,...
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