FdChera. Toxic. Vol. 30, No. II, pp. 967-971, 1992 Printed in Great Britain. All rights reserved
0278-6915/92 $5.00 + 0.00 Copyright © 1992 Pergamon Press Ltd
EFFECT OF EXTRANEOUS SUPPLEMENTATION OF ASCORBIC ACID ON THE BIO-DISPOSITION OF B E N Z A N T H R O N E IN G U I N E A PIGS K. GARG, S. K. KHANNA*, M. D^S and G. B. SINGH Dyes and Food Adulterant Toxicology Laboratory, Industrial Toxicology Research Centre, PO Box 80, M. G. Marg, Lucknow-226001 (UP), India (Accepted 23 July 1992)
Abstract--The bio-elimination and organ retention of orally administered [~4C]benzanthrone, an anthraquinone dye intermediate, were determined in control and ascorbic acid-supplemented guinea pigs. Urinary excretion of benzanthrone in control and ascorbic acid-treated animals during 96 hr was 27.9 and 30.5*/,, respectively, with peak elimination at 48 hr. Faecal elimination in control and supplemented animals during 96 hr was 24.5 and 38.8%, respectively, with a peak at 48 hr. The organ retention of radiolabelled benzanthrone at the end of 96 hr was of the order of 39% in control animals (gastrointestinal tract 16°/,; liver 22%; testis 1.2°/.); aseorbic acid supplementation reduced benzanthrone retention to 19.5"/, (gastro-intestinal tract 12.7°/,; liver 6.8%). Overall, pretreatment of guinea pigs with aseorbic acid caused a 320 enhancement in the clearance of radiolabelled benzanthrone through the urine and faeces, while organ retention was reduced by about 50%. A prophylactic dose of ascorbic acid may prevent benzanthrone-induced toxic symptoms in exposed workers.
INTRODUCTION
As described in a previous paper (Garg et al., 1992), benzanthrone [7H-benz(de)anthracen-7-one], an anthraquinone dye intermediate and an oxygenated polycyclic aromatic hydrocarbon, has been reported to present a health threat to workers in dye manufacturing units during the synthesis of a number of vat and disperse dyes (NIOSH, 1979; Singh et al., 1990) and an occupational hazard to defence and navy personnel coming in contact with benzanthronederived coloured-smoke devices (Chin and Borer, 1982; NIOSH, 1979). This compound also constitutes an environmental pollutant causing a health risk to exposed populations as it has been detected in urban ambient air particulates originating from wood and coal combustion gases, municipal refuse, incineration waste and diesel/gasoline vehicle exhausts, etc. (Handa et al., 1984; Koenig et al., 1982; Ramdahl, 1983). Clinical symptoms in benzanthrone-exposed workers include a burning sensation, erythema and hyperpigrnentation of the skin, loss of appetite, intolerance to fatty foods, fatigue, weakness, weight loss, liver function impairment, gastritis and decreased sexual potency (Horakova and Merhaut, 1966; Kleiner et al., 1979; Trivedi and Niyogi, 1968). Toxicological studies in laboratory animals have suggested that the target sites are the skin, lungs, liver, urinary bladder and testis (Das et aL, 1991b; Singh, 1971; Singh et al., 1967; Singh and Khanna, 1976; Singh and Tripathi, 1973; Srivastava et al., *To whom correspondence should be addressed.
1990). Recently, it has been suggested that benzanthrone acts as a type-I substrate for cytochrome P-450 and causes the destruction of this enzyme and its dependent monooxygenases and glutathione Stransferase (Das et al., 1989 and 1991a); however, the biometabolic fate of benzanthrone, its elimination kinetics and its organ retention have not yet been elucidated. An earlier report revealed that benzanthrone exposure caused significant depletion of ascorbic acid levels in blood, adrenals and liver of exposed guinea pigs (Pandya et al., 1970); a moderate depletion was also observed in exposed rats and mice (Garg, 1989). It is not known to what extent this depletion affects bio-elimination of benzanthrone and whether ascorbic acid supplementation is advantageous. Our previous studies have shown that guinea pigs are more susceptible to benzanthrone toxicity as its bioelimination is slow and organ retention is high in this species compared with rats and mice (Garg et al., 1992). An attempt has therefore been made to study the bio-elimination and organ retention of benzanthrone in guinea pigs with and without pretreatment with ascorbic acid, as reported in this paper. MATERIALS AND METHODS
Chemicals. Anthraquinone was a product of Aldrich Chemical Co. (Milwaukee, WI, USA). [U14C]glycerol (sp. act. 171 mCi/mmol) was purchased from Amersham International pie (Bucks, UK). Neutral alumina, electrolytic iron powder and ascorbic acid were the respective products of BDH Glaxo Laboratories (India), Ranbaxy Laboratories Ltd
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Table I. Bio-eliminationof [14C]benzanthronein urineand faeces of control and ascorbicacid-fedguineapigs Control* Ascorbic acid-treated* Time (hr) Urinary Faecal Total Urinary Faecal Total 12 3.61 -t-0.33 1.17+0.16 4.78 3.17_+0.73 2.53+0.36* 5.7 24 8.08 _+0.35 6.78 4-0.36 14.86 I 1.40-+0.34* 15.61 + 0.70* 27.01 48 11.34-+ 1.02 10.92+ 0.60 22.26 12.84+ 0.93 17.40__.0.79* 30.24 72 3.56-+0.44 4.78 + 0.09 8.34 2.98 + 0.39 2.75 -+0.79 5.73 96 1.40 _+0.29 0.88 _4-0.04 2.28 0.09 + 0.01 0.51 -+0.31 0.60 Total recovery 27.99 24.53 52.52 30.48 38.80 69.28 *Values (percentagesof total dose administered)are means+ SEM of four animals. *P < 0.001. (India) and Sisco Research Laboratory (India). Hyamine hydroxide x 10, 2,5-diphenyloxazol and 1,4-bis[2-(5-phenyloxazolyl)] benzene were obtained from Packard Instrument Co. Inc. (Downers Grove, IL, USA). All other chemicals used were of analytical reagent grade. Syntheses o f [14C]-labelled benzanthrone. Radiolabelled benzanthrone was synthesized using [U-t4C]glycerol and anthraquinone as starting materials, using the basic method of Reisz (1951) with some modifications, as described previously (Garg et al., 1992). Purification of[14C]benzanthrone. The crystals were redissolved in a small volume of toluene and allowed to recrystallize. For further purification, column chromatographic resolution on neutral alumina was undertaken according to the method of Joshi et al. (1984), as described previously (Garg et al., 1992). The purity of the [14C]benzanthrone synthesized was confirmed by measuring its physicochemical characteristics (Indian Standard Specification, 1969) and by carbon and hydrogen analyses and infra-red, nuclear magnetic resonance and mass-spectroscopic studies. The specific activity of the synthesized [t4C]benzanthrone sample was calculated to be 0.52 pCi/mmol. Treatment o f animals. Adult male albino guinea pigs (250 + 25 g), obtained from the animal-breeding colony of the Industrial Toxicology Research Centre were given a commercial pellet diet (Hindustan Lever, Bombay, India) and water ad lib. under standard laboratory conditions. Animals were divided in two groups of eight. Four animals of the first group were given a single oral dose of 25 mg [~4C]benzanthrone (125,000 dpm) dissolved in groundnut oil, while the remaining four received the vehicle alone to serve as controls. Animals of the second group were given oral supplements of 50mg ascorbic acid/kg body weight for 3 days; four animals were then given a single oral dose of [14C]benzanthrone while the remaining four, given groundnut oil alone, served as controls. Ascorbic acid administration was continued until the termination of the experiment in all animals in the second group. Animals were housed individually in all-glass metabolic cages. Urine and faeces were collected at 12, 24, 48, 72 and 96 hr and kept frozen until further analysis. All the animals were killed after 96 hr and the liver, kidneys, spleen, testis and gastro-intestinal tract were removed, weighed and stored frozen.
Determination o f radioactivity in excreta and tissues. This procedure was identical to that previously described (Garg et al., 1992). RESULTS Growth rates of all groups of animals were normal. Ascorbic acid (50 mg/kg body weight) neither caused diuresis nor affected the pH of the urine in either control or benzanthrone-treated animals (data not shown). The bioelimination data of [14C]benzanthrone in control and ascorbic acid-treated guinea pigs are shown in Table 1. The radioactivity eliminated in the urine of control and ascorbic acid-treated guinea pigs after 96 hr amounted to 27.9 and 30.5%, respectively, of the dose administered. The urinary excretion of radioactivity increased gradually for 48 hr and declined thereafter (3.6, 8.1 and 11.3% in control and 3.2, ii.4 and 12.8% in ascorbic acid-fed animals at 12, 24 and 48 hr, respectively). There was an appreciable reduction of radioactivity in 72-hr (3.0-3.6%) and 96-hr (0.09-1.4%) urine samples in both groups of animals (Table I). Over the 96-hr collection period, the respective total faecal radioactivity in control and ascorbic acid-fed guinea pigs represented 24.5 and 38.8% of the administered dose of benzanthrone. The first 12-hr faecal sample showed an elimination of only 1.2% of the total radioactivity, followed by 6.8% at 24 hr and 10.9% at 48 hr in control guinea pigs. In ascorbic acid-fed animals the respective percentages were 2.5, 15.6 and 17.4 at 12, 24 and 48hr (Table 1). In both groups of animals the 72-hr and 96-hr faecal samples showed an elimination of 2.8-4.8% and 0.5-0.9% of the total radioactivity, respectively. Most of the elimination of radioactivity in urine and faeces occurred in the first 48 hr, represented approximately 42% of the dose administered (the final total recovery being 52.5%) in control animals, and 63% of the dose administered (final recovery 69.3%) in ascorbic acid-fed animals (Table !). The organ retention of radioactive benzanthrone in control and ascorbic acid-fed guinea pigs is shown in Table 2. The total organ retention of [t4C]benzanthrone at the end of the 96-hr experimental period was of the order of 39% in control animals, 16% being retained in the gastro-intestinal tract, 22% in the liver, and !.2% in the testis. However, organ
Disposition of benzanthrone Table 2. Retention of l~4C]benzanthronein organs of control and ascorbic acid-fedguinea pigs Organ Gastro-intestinal tract Kidney Liver Spleen Testis
Controlt
Ascorbic acidtreatedt
16.05 + 0.35 0 21.85 __+1.27 0 1.25 -t- 0.11
12.70 + 0.30* 0 6.81 + 0.76* 0 0*
Total recovery
39.15
19.51
tValues (percentages of total dose administered) are means + SEM of four animals. *P < 0.001.
Table 3. Effectof ascorbicacid supplementationon bio-elimination and organ retention of {~4C]benzanthronein guinea pigs Effect
Control
Elimination Urine Faeces
DISCUSSION
Benzanthrone exposure has been shown to cause the depletion of body levels of ascorbic acid (Garg, 1989; Pandya et al., 1970); guinea pigs will be particularly vulnerable to this effect since they are totally
-'-" 15 -~ _ ~ •~
10
.~
5
Urine ~ e ~
Faeces -- 40
-
--
30
-- 20 ~
-- !0 0 24
48
72
96
24
48
72
96
0
Time (hr) Fig. 1. Effect of ascorbic acid supplementation on urinary and faecal bio-elimination of [14C]benzanthrone in guinea pigs. ( 0 ) Control; (©) ascorbic acid supplementation.
Aseorbic acid- Clearance treated (%)
27.99 24.53
30.48 38.80
Total
52.52
69.28
Organ retention Gastro-intestinal tract Liver Testis
16.05 21.85 1.25
12.70 6.8 I 0.0
39.15 91.67
19.51 88.79
Total G r a n d
retention in the ascorbic acid-supplemented guinea pigs was only 19.5% (12.7% in the gastro-intestinal tract and 6.8% in the liver), with no radioactivity detectable in the testes. In neither the control nor the ascorbic acid-fed animals did the spleen and kidney show any radioactivity (Table 2). The time-dependent bio-elimination of [14C]-benzanthrone in the urine and faeces of control and ascorbic acid-supplemented guinea pigs is shown in Fig. 1. In the controls, peak elimination of radioactivity in the urine occurred at 24 (8.1%) and 48 hr (11.3%); ascorbic acid supplementation increased these values to ! 1.4% and 12.8% respectively (Fig. 1). Similarly, excretion of the radioactive compound in the faeces was more rapid in ascorbic-acid treated animals (15.6 and 17.4% at 24 and 48 hr) than in those given [~4C]-benzanthrone alone (6.8 and 10.9% at 24 and 48 hr). The overall effect of ascorbic acid supplementation on the bio-elimination and clearance of [~4C]benzanthrone in guinea pigs is shown in Table 3. Ascorbic acid-treated animals showed an increased rate of bio-elimination through urine and faeces (31.9% higher than the control animals). Similarly, ascorbic acid supplementation caused a 50% increase in clearance of [~4C]benzanthrone from the liver, testis and gastro-intestinal tract (Table 3).
969
total
31.9 I
50.17
Values are percentages of total dose administered.
dependent on extraneous ascorbic acid. This assumption is supported by the comparatively slower bioelimination and higher organ retention of benzanthrone in control guinea pigs than in those given ascorbic acid supplements. Although the dose of 50mg ascorbic acid/kg body weight in the study described here may be considered to be high, no symptoms of ascorbic acid toxicity were detected; such a high dose may be necessary to counteract the depletion brought about by benzanthrone. A dose of 50mg/kg body weight has been used by several investigators to study the protective effects of ascorbic acid against xenobiotic toxicity in laboratory animals (Kamm et al., 1973; Singh and Zaidi, 1969; Susick et al., 1986). Decreased oxidative metabolism has been observed in ascorbic acid-deficient guinea pigs (Zannoni and Lynch, 1973). The lowered metabolic activity has, in turn, been associated with qualitative and quantitative alterations in the content of cytochrome P-450 and its associated enzymes (Gundermann et al., 1973; Sato and Zannoni, 1976; Zannoni et al., 1974). Reintroduction of ascorbic acid resulted in a return to basal enzyme activities (Sato and Zannoni, 1976; Zannoni and Sato, 1975) and has been considered to be essential for the normal synthesis of the haem component of cytochrome P-450 (Rikans et al., 1977). Thus, the impairment by benzanthrone of cytochrome P-450 and its dependent xenobioticmetabolizing enzymes (Das et al., 1991b) may be the result of depletion of ascorbic acid levels (Garg, 1989; Pandya et al., 1970). Supplementation with ascorbic acid in the present study caused enhanced urinary and faecal elimination and reduced benzanthrone retention in the liver of guinea pigs. It was earlier reported that ascorbic acid deficiency delays the clearance of some xenobiotics from the body (Zannoni et al., 1982 and 1984). Furthermore, ascorbic acid deficiency has been reported to reduce the detoxification potential of liver, a condition that is reversible on ascorbic acid supplementation (Beyer, 1943; Kamm et al., 1973). It is possible that its ketone component may enable benzanthrone to bind covalently with liver proteins. Such binding may be enhanced under conditions of
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ascorbic acid depletion, whereas the presence of ascorbic acid may reduce the ketone group and thereby prevent it from binding to macromolecules. Ascorbic acid has earlier been shown to decrease covalent binding of xenobiotics to hepatic nucleic acid fractions (Shah and Bhattacharya, 1982; Smart and Zannoni, 1986). The retention of benzanthrone in the liver, noted in the present study, may also explain the intolerance of fatty food in workers exposed to benzanthrone in the dye-manufacturing industries (Marhoid, 1983; Singh et al., 1990). Regular prophylactic ascorbic acid ingestion may be indicated in such workers. In some of the earlier reports the hepatotoxic effect of hydrazine was found to be weakened by ascorbic acid and enhanced by ascorbic acid deficiency (Beyer, 1943) and similar findings have been reported for the hepatotoxic effects of other xenobiotics (Cardesa et aL, 1974; Kamm et al., 1973). Next to the liver, the gastro-intestinal tract shows the greatest degree of [~4C]benzanthrone retention, possibly because of unabsorbed compound in the lumen. Ascorbic acid supplementation led to a slight but significant decrease in the amount of [~4C]benzanthrone detected in the gastro-intestinal tract. The slight accumulation of radiolabelled benzanthrone that occurred in guinea pig testes was completely cleared by supplements of ascorbic acid. The mechanism by which ascorbic acid enhances benzanthrone elimination has not yet been elucidated, but the following possibilities are suggested. First, ascorbic acid may act as a reducing agent, thereby reducing the ketone moiety present in the benzanthrone molecule; alternatively, it may help in the hydroxylation of benzanthrone as ascorbic acid has been shown to encourage hydroxylation reactions (Peterkofsky, 1972; Staudinger et al., 1961; Udenfriend et al., 1954), thus increasing the polarity of benzanthrone metabolites and facilitating elimination. Secondly, ascorbic acid may act as a scavenger of free radicals, converting the reactive radicals of benzanthrone metabolites to polar metabolites and resulting in higher elimination. However, these putative mechanisms need further elucidation. In conclusion, the results of this study suggest that ascorbic acid supplements may encourage urinary and faecal clearance of benzanthrone by preventing its organ retention, and thereby reducing its toxicity. Acknowledgements--The authors are grateful to Dr P. K. Ray, Director of the Industrial Toxicology Research Centre, for his keen interest in this study. Financial assistance provided by the Indian Council of Medical Research, New Delhi, is gratefully acknowledged. Thanks are due to Mr K. G. Thomas and Mr Umesh Prasad for typographical and computer assistance, respectively. REFERENCES
Beyer K. H. (1943) Protective action of vitamin C against experimental hepatic damage. Archives of Internal Medicine 71, 315-324.
Cardesa A., Mirvish S. S., Haven G. and Shubik P. (1974) Inhibitory effect of ascorbic acid on the acute toxicity of dimethylamine. Proceedings of the Society for Experimental Biology and Medicine 145, 124-128. Chin A. and Borer L. (1982) Investigations of the effluents produced during the functioning of Naval colored smoke devices. Proceedings on Industrial Pyrotechnology Seminar. Vol 8. 129-148. Das M., Garg K., Joshi A., Singh G. B. and Khanna S. K. (1991a) Interaction of benzanthrone with cytochrome P-450: altered patterns of hepatic xenobiotic metabolism in rats. Journal of Biochemical Toxicology 6, 37-44. Das M., Garg K., Singh G. B. and Khanna S. K. (1989) Benzanthrone: A new substrate for hepatic microsomal cytochrome P-450. Biochemistry International 18, 1237-1244. Das M., Garg K., Singh G. B. and Khanna S. K. (1991b) Bio-elimination, organ retention profile and target tissue sites of an anthraquinone dye-intermediate, Benzanthrone. International Journal of Toxicology and Occupational Environmental Health l, 142. Garg K. (1989) Chemo-toxicological effects of benzanthrone, an anthraquinone based dye intermediate. PhD thesis, Lucknow University, India. Garg K., Khanna S. K., Das M. and Singh G. B. (1992) Comparative study of the biodisposition of benzanthrone in different rodent species. Food and Chemical Toxicology 30, 517-520. Gundermann K., Degwitz E. and Staudinger H. (1973) Mixed function oxygenation of ( + ) and ( - ) hexobarbital and spectral changes of cytochrome P-450 in liver of guinea pig fed without ascorbic acid. Hoppe-Seyler's Zeitschrift fiir Physiologische Chemie 354, 238-242. Handa T., Yamaguchi T., Sawai K., Yamamura T., Koseki Y. and Ishii I. (1984) In situ emission levels of carcinogenic and mutagenic compounds from diesel and gasoline engine vehicleson an express way. Environmental Science and Technology 18, 895-902. Horakova E. and Merhaut J. (1966) Health of workers producing benzanthrone. Pracovni Lbkaistvi 18, 78-81. ISI (1969) Indian Standard Specification for benzanthrone. ISI 5044, 1. Joshi A., Khanna S. K., Singh G. B. and Krishnamurti C. R. (1984) Mode of interaction of benzanthrone with serum proteins. Industrial Health 22, 279-293. Kamm J. J., Dashman J., Conney A. H. and Burns J. J. (1973) Protective effect of ascorbic acid on hepatotoxicity caused by sodium nitrite plus aminopyrine. Proceedings of the National Academy of Sciences of the U.S.A. 70, 747-749. Kleiner A. I., Sonkin I. S., Nestrugina Z. F., Krylova E. V., Rezenkina L. D. and Ermilova I. I. (1979) Health status of workers in the present production of benzanthrone. Gigiene Truda I Professional'nye Zabolevaniya 10, 43-45. Koenig J., Balfanz E., Funcke W. and Romanowski T. (1982) Quinone and ketone derivatives of PAH in particulate matter from ambient air. Proc. of 7th Polynuclear Aromatic Hydrocarbons. Edited by M. Cook and A. Dennis. pp. 711-720. Battelle Press, Columbus, OH. Marhold J. V. (1983) Benzanthrone. In Encyclopedia of Occupational Health and Safety. 3rd revised Ed. Edited by E. Pharmeggiani, pp. 256-257. International Labour Office, Geneva. NIOSH (1979) Toxic effects of chemical substances-Benzanthrone. US-NTIS Report US AMBRDL-TR7704. pp. 34-45. Pandya K. P., Singh G. B. and Joshi N. C. (1970) Effect of benzanthrone on the body level of ascorbic acid in guinea pigs. Acta Pharmacologica et Toxicologica 28, 499-506. Peterkofsky B. (1972) The effect of ascorbic acid on collagen polypeptide synthesis and proline hydroxylation during the growth of cultured fibroblast. Archives of Biochemistry and Biophysics 152, 318 328.
Disposition of benzanthrone Ramdahl T. (1983) Polycyclic aromatic ketones in environmental samples. Environmental Science and Technology 17, 6664570. Reisz E. (1951) Benzanthrone, Fr 973, 154, Feb. 8. (CA: 47, p. 3351 C). Rikans L.E., Smith C. R. and Zannoni V. G. (1977) Ascorbic acid and heme synthesis in deficient guinea pig liver. Biochemical Pharmacology 26, 797-799. Sato P. H. and Zannoni V. G. (1976) Ascorbic acid and hepatic drug metabolism. Journal of Pharmacology and Experimental Therapeutics 198, 295-307. Shah A. L. and Bhattacharya R. K. (1982) In vivo effect of L-ascorbic acid on benzo(a)pyrene metabolite-DNA adduct formation in rat liver. Journal of Biosciences 4, 263~68. Singh G. B. (1971) Effect of benzanthrone on lung parenchyma of experimental animals. Journal of Science of Labour 47, 423-425. Singh G. B. and Khanna S. K. (1976) Effect of benzanthrone in testis and male accessory sex glands. Environmental Research 12, 327-333. Singh G. B., Khanna S. K. and Das M. (1990) Recent studies on toxicity of benzanthrone. Journal of Scientific and Industrial Research 49, 288-296. Singh G. B., Sharma S. N. and Zaidi S. H. (1967) Effect of benzanthrone on skin of mice. Indian Journal of Medical Science 21, 727-728. Singh G. B. and Tripathi V. N. P. (1973) Effect of benzanthrone on the urinary bladder of guinea pigs. Experientia 22, 6834584. Singh G. B. and Zaidi S. H. (1969) Preliminary clinical and experimental studies on benzanthrone toxicity. Journal of the Indian Medical Association 52, 558-560. Smart R. C. and Zannoni V. G. (1986) Effect of dietary ascorbate on covalent binding of benzene to bone marrow and hepatic tissues. Biochemical Pharmacology 35, 3180-3182. Srivastava L. P., Misra R. B. and Joshi P. C. (1990) Photosensitizing potential of benzanthrone. Food and Chemical Toxicology 28, 6534558.
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Staudinger H., Krisch K. and Leonhauser S. (1961) Role of ascorbic acid in microsomal electron transport and the possible relation to hydroxylation reactions. Annals of the New York Academy of Sciences 92, 195 207. Susick R. L., Abrams G. D., Zurawski C. A. and Zannoni V. G. (1986) Ascorbic acid and chronic alcohol consumption in guinea pig. Toxicology and Applied Pharmacology 84, 329-335. Trivedi D. H. and Niyogi A. K. (1968) Benzanthrone hazard in dye factory. Indian Journal of Industrial Research 14, 13-22. Udenfriend S., Clark C. T., Axelrod J. and Brodie B. B. (1954) Ascorbic acid in aromatic hydroxylation. I. A model system for aromatic hydroxylation. Journal of Biological Chemistry 208, 731 739. Zannoni V. G. and Lynch M. M. (1973) The role of ascorbic acid in drug metabolism. Drug Metabolism Reviews 2, 57459. Zannoni V. G. and Sato P. H. (1975) Effects of ascorbic acid on components of liver microsomal drug metabolizing enzymes. Annals of the New York Academy of Sciences 258, 119-131. Zannoni V. G., Lynch M. M. and Sato P. H. (1974) Effect of ascorbic acid on drug metabolizing systems in the neonatal guinea pig. In Perinatal Pharmacology: Problems and Priorities. Edited by J. Dancies and J. C. Hwang. pp. 131-147. Raven Press, New York. Zannoni V. G., Holsztynska E. J. and Lau S. S. (1982) Biochemical functions of ascorbic acid in drug metabolism. In Ascorbic Acid: Chemistry, Metabolism and Uses. Edited by P. A. Seibs and B. H. Tolbert. pp. 349-368. American Chemical Society, Washington, DC. Zannoni V. G., Susick R. C. and Smart R. C. (1984) Ascorbic acid as it relates to the metabolism of drugs and environmental chemicals. In Nutrition in the 20th Century. pp. 121-135. Edited by M. Winick. Vol. 13. John Wiley & Sons, New York.
0278-6915/92 $5.00 + 0.00 Copyright © 1992 Pergamon Press Ltd
EFFECT OF EXTRANEOUS SUPPLEMENTATION OF ASCORBIC ACID ON THE BIO-DISPOSITION OF B E N Z A N T H R O N E IN G U I N E A PIGS K. GARG, S. K. KHANNA*, M. D^S and G. B. SINGH Dyes and Food Adulterant Toxicology Laboratory, Industrial Toxicology Research Centre, PO Box 80, M. G. Marg, Lucknow-226001 (UP), India (Accepted 23 July 1992)
Abstract--The bio-elimination and organ retention of orally administered [~4C]benzanthrone, an anthraquinone dye intermediate, were determined in control and ascorbic acid-supplemented guinea pigs. Urinary excretion of benzanthrone in control and ascorbic acid-treated animals during 96 hr was 27.9 and 30.5*/,, respectively, with peak elimination at 48 hr. Faecal elimination in control and supplemented animals during 96 hr was 24.5 and 38.8%, respectively, with a peak at 48 hr. The organ retention of radiolabelled benzanthrone at the end of 96 hr was of the order of 39% in control animals (gastrointestinal tract 16°/,; liver 22%; testis 1.2°/.); aseorbic acid supplementation reduced benzanthrone retention to 19.5"/, (gastro-intestinal tract 12.7°/,; liver 6.8%). Overall, pretreatment of guinea pigs with aseorbic acid caused a 320 enhancement in the clearance of radiolabelled benzanthrone through the urine and faeces, while organ retention was reduced by about 50%. A prophylactic dose of ascorbic acid may prevent benzanthrone-induced toxic symptoms in exposed workers.
INTRODUCTION
As described in a previous paper (Garg et al., 1992), benzanthrone [7H-benz(de)anthracen-7-one], an anthraquinone dye intermediate and an oxygenated polycyclic aromatic hydrocarbon, has been reported to present a health threat to workers in dye manufacturing units during the synthesis of a number of vat and disperse dyes (NIOSH, 1979; Singh et al., 1990) and an occupational hazard to defence and navy personnel coming in contact with benzanthronederived coloured-smoke devices (Chin and Borer, 1982; NIOSH, 1979). This compound also constitutes an environmental pollutant causing a health risk to exposed populations as it has been detected in urban ambient air particulates originating from wood and coal combustion gases, municipal refuse, incineration waste and diesel/gasoline vehicle exhausts, etc. (Handa et al., 1984; Koenig et al., 1982; Ramdahl, 1983). Clinical symptoms in benzanthrone-exposed workers include a burning sensation, erythema and hyperpigrnentation of the skin, loss of appetite, intolerance to fatty foods, fatigue, weakness, weight loss, liver function impairment, gastritis and decreased sexual potency (Horakova and Merhaut, 1966; Kleiner et al., 1979; Trivedi and Niyogi, 1968). Toxicological studies in laboratory animals have suggested that the target sites are the skin, lungs, liver, urinary bladder and testis (Das et aL, 1991b; Singh, 1971; Singh et al., 1967; Singh and Khanna, 1976; Singh and Tripathi, 1973; Srivastava et al., *To whom correspondence should be addressed.
1990). Recently, it has been suggested that benzanthrone acts as a type-I substrate for cytochrome P-450 and causes the destruction of this enzyme and its dependent monooxygenases and glutathione Stransferase (Das et al., 1989 and 1991a); however, the biometabolic fate of benzanthrone, its elimination kinetics and its organ retention have not yet been elucidated. An earlier report revealed that benzanthrone exposure caused significant depletion of ascorbic acid levels in blood, adrenals and liver of exposed guinea pigs (Pandya et al., 1970); a moderate depletion was also observed in exposed rats and mice (Garg, 1989). It is not known to what extent this depletion affects bio-elimination of benzanthrone and whether ascorbic acid supplementation is advantageous. Our previous studies have shown that guinea pigs are more susceptible to benzanthrone toxicity as its bioelimination is slow and organ retention is high in this species compared with rats and mice (Garg et al., 1992). An attempt has therefore been made to study the bio-elimination and organ retention of benzanthrone in guinea pigs with and without pretreatment with ascorbic acid, as reported in this paper. MATERIALS AND METHODS
Chemicals. Anthraquinone was a product of Aldrich Chemical Co. (Milwaukee, WI, USA). [U14C]glycerol (sp. act. 171 mCi/mmol) was purchased from Amersham International pie (Bucks, UK). Neutral alumina, electrolytic iron powder and ascorbic acid were the respective products of BDH Glaxo Laboratories (India), Ranbaxy Laboratories Ltd
967
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K. GARG et al.
Table I. Bio-eliminationof [14C]benzanthronein urineand faeces of control and ascorbicacid-fedguineapigs Control* Ascorbic acid-treated* Time (hr) Urinary Faecal Total Urinary Faecal Total 12 3.61 -t-0.33 1.17+0.16 4.78 3.17_+0.73 2.53+0.36* 5.7 24 8.08 _+0.35 6.78 4-0.36 14.86 I 1.40-+0.34* 15.61 + 0.70* 27.01 48 11.34-+ 1.02 10.92+ 0.60 22.26 12.84+ 0.93 17.40__.0.79* 30.24 72 3.56-+0.44 4.78 + 0.09 8.34 2.98 + 0.39 2.75 -+0.79 5.73 96 1.40 _+0.29 0.88 _4-0.04 2.28 0.09 + 0.01 0.51 -+0.31 0.60 Total recovery 27.99 24.53 52.52 30.48 38.80 69.28 *Values (percentagesof total dose administered)are means+ SEM of four animals. *P < 0.001. (India) and Sisco Research Laboratory (India). Hyamine hydroxide x 10, 2,5-diphenyloxazol and 1,4-bis[2-(5-phenyloxazolyl)] benzene were obtained from Packard Instrument Co. Inc. (Downers Grove, IL, USA). All other chemicals used were of analytical reagent grade. Syntheses o f [14C]-labelled benzanthrone. Radiolabelled benzanthrone was synthesized using [U-t4C]glycerol and anthraquinone as starting materials, using the basic method of Reisz (1951) with some modifications, as described previously (Garg et al., 1992). Purification of[14C]benzanthrone. The crystals were redissolved in a small volume of toluene and allowed to recrystallize. For further purification, column chromatographic resolution on neutral alumina was undertaken according to the method of Joshi et al. (1984), as described previously (Garg et al., 1992). The purity of the [14C]benzanthrone synthesized was confirmed by measuring its physicochemical characteristics (Indian Standard Specification, 1969) and by carbon and hydrogen analyses and infra-red, nuclear magnetic resonance and mass-spectroscopic studies. The specific activity of the synthesized [t4C]benzanthrone sample was calculated to be 0.52 pCi/mmol. Treatment o f animals. Adult male albino guinea pigs (250 + 25 g), obtained from the animal-breeding colony of the Industrial Toxicology Research Centre were given a commercial pellet diet (Hindustan Lever, Bombay, India) and water ad lib. under standard laboratory conditions. Animals were divided in two groups of eight. Four animals of the first group were given a single oral dose of 25 mg [~4C]benzanthrone (125,000 dpm) dissolved in groundnut oil, while the remaining four received the vehicle alone to serve as controls. Animals of the second group were given oral supplements of 50mg ascorbic acid/kg body weight for 3 days; four animals were then given a single oral dose of [14C]benzanthrone while the remaining four, given groundnut oil alone, served as controls. Ascorbic acid administration was continued until the termination of the experiment in all animals in the second group. Animals were housed individually in all-glass metabolic cages. Urine and faeces were collected at 12, 24, 48, 72 and 96 hr and kept frozen until further analysis. All the animals were killed after 96 hr and the liver, kidneys, spleen, testis and gastro-intestinal tract were removed, weighed and stored frozen.
Determination o f radioactivity in excreta and tissues. This procedure was identical to that previously described (Garg et al., 1992). RESULTS Growth rates of all groups of animals were normal. Ascorbic acid (50 mg/kg body weight) neither caused diuresis nor affected the pH of the urine in either control or benzanthrone-treated animals (data not shown). The bioelimination data of [14C]benzanthrone in control and ascorbic acid-treated guinea pigs are shown in Table 1. The radioactivity eliminated in the urine of control and ascorbic acid-treated guinea pigs after 96 hr amounted to 27.9 and 30.5%, respectively, of the dose administered. The urinary excretion of radioactivity increased gradually for 48 hr and declined thereafter (3.6, 8.1 and 11.3% in control and 3.2, ii.4 and 12.8% in ascorbic acid-fed animals at 12, 24 and 48 hr, respectively). There was an appreciable reduction of radioactivity in 72-hr (3.0-3.6%) and 96-hr (0.09-1.4%) urine samples in both groups of animals (Table I). Over the 96-hr collection period, the respective total faecal radioactivity in control and ascorbic acid-fed guinea pigs represented 24.5 and 38.8% of the administered dose of benzanthrone. The first 12-hr faecal sample showed an elimination of only 1.2% of the total radioactivity, followed by 6.8% at 24 hr and 10.9% at 48 hr in control guinea pigs. In ascorbic acid-fed animals the respective percentages were 2.5, 15.6 and 17.4 at 12, 24 and 48hr (Table 1). In both groups of animals the 72-hr and 96-hr faecal samples showed an elimination of 2.8-4.8% and 0.5-0.9% of the total radioactivity, respectively. Most of the elimination of radioactivity in urine and faeces occurred in the first 48 hr, represented approximately 42% of the dose administered (the final total recovery being 52.5%) in control animals, and 63% of the dose administered (final recovery 69.3%) in ascorbic acid-fed animals (Table !). The organ retention of radioactive benzanthrone in control and ascorbic acid-fed guinea pigs is shown in Table 2. The total organ retention of [t4C]benzanthrone at the end of the 96-hr experimental period was of the order of 39% in control animals, 16% being retained in the gastro-intestinal tract, 22% in the liver, and !.2% in the testis. However, organ
Disposition of benzanthrone Table 2. Retention of l~4C]benzanthronein organs of control and ascorbic acid-fedguinea pigs Organ Gastro-intestinal tract Kidney Liver Spleen Testis
Controlt
Ascorbic acidtreatedt
16.05 + 0.35 0 21.85 __+1.27 0 1.25 -t- 0.11
12.70 + 0.30* 0 6.81 + 0.76* 0 0*
Total recovery
39.15
19.51
tValues (percentages of total dose administered) are means + SEM of four animals. *P < 0.001.
Table 3. Effectof ascorbicacid supplementationon bio-elimination and organ retention of {~4C]benzanthronein guinea pigs Effect
Control
Elimination Urine Faeces
DISCUSSION
Benzanthrone exposure has been shown to cause the depletion of body levels of ascorbic acid (Garg, 1989; Pandya et al., 1970); guinea pigs will be particularly vulnerable to this effect since they are totally
-'-" 15 -~ _ ~ •~
10
.~
5
Urine ~ e ~
Faeces -- 40
-
--
30
-- 20 ~
-- !0 0 24
48
72
96
24
48
72
96
0
Time (hr) Fig. 1. Effect of ascorbic acid supplementation on urinary and faecal bio-elimination of [14C]benzanthrone in guinea pigs. ( 0 ) Control; (©) ascorbic acid supplementation.
Aseorbic acid- Clearance treated (%)
27.99 24.53
30.48 38.80
Total
52.52
69.28
Organ retention Gastro-intestinal tract Liver Testis
16.05 21.85 1.25
12.70 6.8 I 0.0
39.15 91.67
19.51 88.79
Total G r a n d
retention in the ascorbic acid-supplemented guinea pigs was only 19.5% (12.7% in the gastro-intestinal tract and 6.8% in the liver), with no radioactivity detectable in the testes. In neither the control nor the ascorbic acid-fed animals did the spleen and kidney show any radioactivity (Table 2). The time-dependent bio-elimination of [14C]-benzanthrone in the urine and faeces of control and ascorbic acid-supplemented guinea pigs is shown in Fig. 1. In the controls, peak elimination of radioactivity in the urine occurred at 24 (8.1%) and 48 hr (11.3%); ascorbic acid supplementation increased these values to ! 1.4% and 12.8% respectively (Fig. 1). Similarly, excretion of the radioactive compound in the faeces was more rapid in ascorbic-acid treated animals (15.6 and 17.4% at 24 and 48 hr) than in those given [~4C]-benzanthrone alone (6.8 and 10.9% at 24 and 48 hr). The overall effect of ascorbic acid supplementation on the bio-elimination and clearance of [~4C]benzanthrone in guinea pigs is shown in Table 3. Ascorbic acid-treated animals showed an increased rate of bio-elimination through urine and faeces (31.9% higher than the control animals). Similarly, ascorbic acid supplementation caused a 50% increase in clearance of [~4C]benzanthrone from the liver, testis and gastro-intestinal tract (Table 3).
969
total
31.9 I
50.17
Values are percentages of total dose administered.
dependent on extraneous ascorbic acid. This assumption is supported by the comparatively slower bioelimination and higher organ retention of benzanthrone in control guinea pigs than in those given ascorbic acid supplements. Although the dose of 50mg ascorbic acid/kg body weight in the study described here may be considered to be high, no symptoms of ascorbic acid toxicity were detected; such a high dose may be necessary to counteract the depletion brought about by benzanthrone. A dose of 50mg/kg body weight has been used by several investigators to study the protective effects of ascorbic acid against xenobiotic toxicity in laboratory animals (Kamm et al., 1973; Singh and Zaidi, 1969; Susick et al., 1986). Decreased oxidative metabolism has been observed in ascorbic acid-deficient guinea pigs (Zannoni and Lynch, 1973). The lowered metabolic activity has, in turn, been associated with qualitative and quantitative alterations in the content of cytochrome P-450 and its associated enzymes (Gundermann et al., 1973; Sato and Zannoni, 1976; Zannoni et al., 1974). Reintroduction of ascorbic acid resulted in a return to basal enzyme activities (Sato and Zannoni, 1976; Zannoni and Sato, 1975) and has been considered to be essential for the normal synthesis of the haem component of cytochrome P-450 (Rikans et al., 1977). Thus, the impairment by benzanthrone of cytochrome P-450 and its dependent xenobioticmetabolizing enzymes (Das et al., 1991b) may be the result of depletion of ascorbic acid levels (Garg, 1989; Pandya et al., 1970). Supplementation with ascorbic acid in the present study caused enhanced urinary and faecal elimination and reduced benzanthrone retention in the liver of guinea pigs. It was earlier reported that ascorbic acid deficiency delays the clearance of some xenobiotics from the body (Zannoni et al., 1982 and 1984). Furthermore, ascorbic acid deficiency has been reported to reduce the detoxification potential of liver, a condition that is reversible on ascorbic acid supplementation (Beyer, 1943; Kamm et al., 1973). It is possible that its ketone component may enable benzanthrone to bind covalently with liver proteins. Such binding may be enhanced under conditions of
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ascorbic acid depletion, whereas the presence of ascorbic acid may reduce the ketone group and thereby prevent it from binding to macromolecules. Ascorbic acid has earlier been shown to decrease covalent binding of xenobiotics to hepatic nucleic acid fractions (Shah and Bhattacharya, 1982; Smart and Zannoni, 1986). The retention of benzanthrone in the liver, noted in the present study, may also explain the intolerance of fatty food in workers exposed to benzanthrone in the dye-manufacturing industries (Marhoid, 1983; Singh et al., 1990). Regular prophylactic ascorbic acid ingestion may be indicated in such workers. In some of the earlier reports the hepatotoxic effect of hydrazine was found to be weakened by ascorbic acid and enhanced by ascorbic acid deficiency (Beyer, 1943) and similar findings have been reported for the hepatotoxic effects of other xenobiotics (Cardesa et aL, 1974; Kamm et al., 1973). Next to the liver, the gastro-intestinal tract shows the greatest degree of [~4C]benzanthrone retention, possibly because of unabsorbed compound in the lumen. Ascorbic acid supplementation led to a slight but significant decrease in the amount of [~4C]benzanthrone detected in the gastro-intestinal tract. The slight accumulation of radiolabelled benzanthrone that occurred in guinea pig testes was completely cleared by supplements of ascorbic acid. The mechanism by which ascorbic acid enhances benzanthrone elimination has not yet been elucidated, but the following possibilities are suggested. First, ascorbic acid may act as a reducing agent, thereby reducing the ketone moiety present in the benzanthrone molecule; alternatively, it may help in the hydroxylation of benzanthrone as ascorbic acid has been shown to encourage hydroxylation reactions (Peterkofsky, 1972; Staudinger et al., 1961; Udenfriend et al., 1954), thus increasing the polarity of benzanthrone metabolites and facilitating elimination. Secondly, ascorbic acid may act as a scavenger of free radicals, converting the reactive radicals of benzanthrone metabolites to polar metabolites and resulting in higher elimination. However, these putative mechanisms need further elucidation. In conclusion, the results of this study suggest that ascorbic acid supplements may encourage urinary and faecal clearance of benzanthrone by preventing its organ retention, and thereby reducing its toxicity. Acknowledgements--The authors are grateful to Dr P. K. Ray, Director of the Industrial Toxicology Research Centre, for his keen interest in this study. Financial assistance provided by the Indian Council of Medical Research, New Delhi, is gratefully acknowledged. Thanks are due to Mr K. G. Thomas and Mr Umesh Prasad for typographical and computer assistance, respectively. REFERENCES
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Cardesa A., Mirvish S. S., Haven G. and Shubik P. (1974) Inhibitory effect of ascorbic acid on the acute toxicity of dimethylamine. Proceedings of the Society for Experimental Biology and Medicine 145, 124-128. Chin A. and Borer L. (1982) Investigations of the effluents produced during the functioning of Naval colored smoke devices. Proceedings on Industrial Pyrotechnology Seminar. Vol 8. 129-148. Das M., Garg K., Joshi A., Singh G. B. and Khanna S. K. (1991a) Interaction of benzanthrone with cytochrome P-450: altered patterns of hepatic xenobiotic metabolism in rats. Journal of Biochemical Toxicology 6, 37-44. Das M., Garg K., Singh G. B. and Khanna S. K. (1989) Benzanthrone: A new substrate for hepatic microsomal cytochrome P-450. Biochemistry International 18, 1237-1244. Das M., Garg K., Singh G. B. and Khanna S. K. (1991b) Bio-elimination, organ retention profile and target tissue sites of an anthraquinone dye-intermediate, Benzanthrone. International Journal of Toxicology and Occupational Environmental Health l, 142. Garg K. (1989) Chemo-toxicological effects of benzanthrone, an anthraquinone based dye intermediate. PhD thesis, Lucknow University, India. Garg K., Khanna S. K., Das M. and Singh G. B. (1992) Comparative study of the biodisposition of benzanthrone in different rodent species. Food and Chemical Toxicology 30, 517-520. Gundermann K., Degwitz E. and Staudinger H. (1973) Mixed function oxygenation of ( + ) and ( - ) hexobarbital and spectral changes of cytochrome P-450 in liver of guinea pig fed without ascorbic acid. Hoppe-Seyler's Zeitschrift fiir Physiologische Chemie 354, 238-242. Handa T., Yamaguchi T., Sawai K., Yamamura T., Koseki Y. and Ishii I. (1984) In situ emission levels of carcinogenic and mutagenic compounds from diesel and gasoline engine vehicleson an express way. Environmental Science and Technology 18, 895-902. Horakova E. and Merhaut J. (1966) Health of workers producing benzanthrone. Pracovni Lbkaistvi 18, 78-81. ISI (1969) Indian Standard Specification for benzanthrone. ISI 5044, 1. Joshi A., Khanna S. K., Singh G. B. and Krishnamurti C. R. (1984) Mode of interaction of benzanthrone with serum proteins. Industrial Health 22, 279-293. Kamm J. J., Dashman J., Conney A. H. and Burns J. J. (1973) Protective effect of ascorbic acid on hepatotoxicity caused by sodium nitrite plus aminopyrine. Proceedings of the National Academy of Sciences of the U.S.A. 70, 747-749. Kleiner A. I., Sonkin I. S., Nestrugina Z. F., Krylova E. V., Rezenkina L. D. and Ermilova I. I. (1979) Health status of workers in the present production of benzanthrone. Gigiene Truda I Professional'nye Zabolevaniya 10, 43-45. Koenig J., Balfanz E., Funcke W. and Romanowski T. (1982) Quinone and ketone derivatives of PAH in particulate matter from ambient air. Proc. of 7th Polynuclear Aromatic Hydrocarbons. Edited by M. Cook and A. Dennis. pp. 711-720. Battelle Press, Columbus, OH. Marhold J. V. (1983) Benzanthrone. In Encyclopedia of Occupational Health and Safety. 3rd revised Ed. Edited by E. Pharmeggiani, pp. 256-257. International Labour Office, Geneva. NIOSH (1979) Toxic effects of chemical substances-Benzanthrone. US-NTIS Report US AMBRDL-TR7704. pp. 34-45. Pandya K. P., Singh G. B. and Joshi N. C. (1970) Effect of benzanthrone on the body level of ascorbic acid in guinea pigs. Acta Pharmacologica et Toxicologica 28, 499-506. Peterkofsky B. (1972) The effect of ascorbic acid on collagen polypeptide synthesis and proline hydroxylation during the growth of cultured fibroblast. Archives of Biochemistry and Biophysics 152, 318 328.
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Staudinger H., Krisch K. and Leonhauser S. (1961) Role of ascorbic acid in microsomal electron transport and the possible relation to hydroxylation reactions. Annals of the New York Academy of Sciences 92, 195 207. Susick R. L., Abrams G. D., Zurawski C. A. and Zannoni V. G. (1986) Ascorbic acid and chronic alcohol consumption in guinea pig. Toxicology and Applied Pharmacology 84, 329-335. Trivedi D. H. and Niyogi A. K. (1968) Benzanthrone hazard in dye factory. Indian Journal of Industrial Research 14, 13-22. Udenfriend S., Clark C. T., Axelrod J. and Brodie B. B. (1954) Ascorbic acid in aromatic hydroxylation. I. A model system for aromatic hydroxylation. Journal of Biological Chemistry 208, 731 739. Zannoni V. G. and Lynch M. M. (1973) The role of ascorbic acid in drug metabolism. Drug Metabolism Reviews 2, 57459. Zannoni V. G. and Sato P. H. (1975) Effects of ascorbic acid on components of liver microsomal drug metabolizing enzymes. Annals of the New York Academy of Sciences 258, 119-131. Zannoni V. G., Lynch M. M. and Sato P. H. (1974) Effect of ascorbic acid on drug metabolizing systems in the neonatal guinea pig. In Perinatal Pharmacology: Problems and Priorities. Edited by J. Dancies and J. C. Hwang. pp. 131-147. Raven Press, New York. Zannoni V. G., Holsztynska E. J. and Lau S. S. (1982) Biochemical functions of ascorbic acid in drug metabolism. In Ascorbic Acid: Chemistry, Metabolism and Uses. Edited by P. A. Seibs and B. H. Tolbert. pp. 349-368. American Chemical Society, Washington, DC. Zannoni V. G., Susick R. C. and Smart R. C. (1984) Ascorbic acid as it relates to the metabolism of drugs and environmental chemicals. In Nutrition in the 20th Century. pp. 121-135. Edited by M. Winick. Vol. 13. John Wiley & Sons, New York.
