Document 93N9390976XbeLJRk8Nyawwv6

Reprinted from The Journal op Biological Chemistry Vol. 127, No. 2, February, 1939 THE METABOLISM OF CHLORINATED NAPHTHALENES By ROBERT V. CLEARY, JOHN MAIER, axd GEORGE H. HITCHINGS (From the Department of Physiology, Harvard School of Public Health, Boston) (Received for publication, November 16, 1938) Chlorination products of naphthalene and related compounds are of considerable interest because of their wide industrial use and possible toxic properties. The toxicity of a number of these compounds has been worked out in detail (1), and the pathological changes which result from their action have been studied (2). The injurious effects are manifested by degenerative changes in the liver (2, 3) which are somewhat different from the changes produced by chloroform and carbon tetrachloride (2). Neverthe less, small (sublethal) doses of carbon tetrachloride produce a high percentage of fatalities when administered to animals which have been exposed to certain chloronaphthalenes and chlorinated diphenyls (1). The toxicity of the chloronaphthalenes and allied substances tends to increase as the number of chlorine atoms in the molecule is increased. A mixture of trichloronaphthalenes is relatively innocuous, whereas a mixture of penta- and hexachloronaphthalenes produces considerable liver damage when given in small amounts (1). Apparently-the mode of action of these compounds depends in some way on the presence of chlorine in the molecule, and the problem of their toxicity is closely related to the general problem of the toxicity of organic chlorine. It was interesting and im portant, therefore, to determine as far as possible the fate of these compounds in metabolism. It is the purpose of this paper to report the results of experiments which indicate the main path ways in the metabolism of one of the compounds previously studied (Compound D of Bennett, Drinker, and Warren (2)). 403 DSW 254996 STLCOPCB4060687 404 Metabolism of Chloronaphthalenes EXPERIMENTAL Material for Experiment--All experiments reported here were carried out on a mixture of polychlorinated naphthalenes which from the chlorine content appears to consist largely of pentaand hexachloro compounds. This material, used commercially for electrical insulation, has not as yet been resolved into its components. It contains 62.6 per cent chlorine. It is insoluble in water, ethanol, and acetone; very soluble (30 to 90 per cent) in xylene, benzene, and carbon tetrachloride; and moderately soluble (2 to 4 per cent) in esters, ethyl ether, oleic and palmitic acids, and olive oil. The flow point is at 121 to 125, and the low vapor pressure is 0.2 mm. at 60 and 0.8 mm. at 130. For convenience the material will be referred to hereafter as Compound D. Absorption--The compound, dissolved in olive oil, was given by stomach tube to albino rats (150 to 200 gm. of body weight) in doses varying from 1 to 15 mg. per day. The feces were collected for 3 day periods, and were dried at 80 for 4 days.1 The material then was pulverized and extracted with benzene in a continuous extractor. After the benzene was distilled off, the residue was transferred to a Carius tube with the aid of a little ether. The ether was removed by vacuum distillation; silver nitrate and nitric acid were added; the tube was sealed and heated for 2 hours at 300. The silver chloride was estimated gravimetrically by the method of Pregl (4). The chloride found in the extracts varied from 0.1 to 0.4 mg. of chlorine compared with 0.4 mg. of benzene-extractable chlorine found in the excreta of a control animal over a comparable period of time. To each of two control samples, 10.0 mg. portions of finely ground chloronaphthalene were added before drying. The chloride recovered was equiv alent to 84 and 85 per cent of the organic chlorine added. Apparently, therefore, the rat can absorb chloronaphthalene given by mouth nearly quantitatively in amounts up to 15 mg. per day. When larger amounts were given (up to 40 mg. per day) the feces became semiliquid, and impossible to collect 1 Finely ground Compound D lost less than 0.1 per cent of its weight when heated in the oven under the same conditions. DSW 254997 STLCOPCB4060688 Cleary, Maier, and Hitchings 405 separately, so no quantitative experiments were undertaken. However, qualitative tests indicated the presence of organically bound chlorine. . Toxicity--Doses of 5 to 10 mg. of Compound D per day were tolerated for a period of 5 weeks, the longest period studied. Doses of 15 mg. per day or greater uniformly killed the animals in 3 weeks or less. Deposition in Tissues--The observation of Drinker, Warren, and Bennett (1), that liver degeneration in rats which had been exposed to chloronaphthalene was still demonstrable microscopi cally 2 months after removal from exposure, suggested that de position of the material in the liver might take place during the period of exposure, followed by a slow liberation of the active toxic material. Consequently, liver tissue and later other organs and tissues from exposed animals were analyzed for organically bound chlorine. The tissues and organs were dried in the oven at 80 for 1 to 4 days. The dry material then was ground in a mortar and extracted with benzene. The benzene was removed by distillation and the residue was analyzed for chlorine by the method previously described, or in later experiments the chlorine was liberated by the procedure of Rauscher (5), and estimated either gravimetrically, as above, or volumetrically ((6) pp. 177-- 179). Liver--A total of fifteen livers from animals which had died from exposure to Compound D was analyzed. In one liver from an animal fed 15 mg. per day, 0.5 mg. of benzene-soluble chlorine was found; in no other sample was more than 0.3 mg. of chlorine found, parallel determinations on normal livers gave similar results; a trace (0.1 to 0.3 mg. of chlorine) usually was found in the benzene extract. Lungs--The lungs from two exposed animals gave only traces of benzene-soluble chlorine. Skin--Two analyses of skin samples (about 18 sq. cm.) failed to reveal the presence in this tissue of any significant amount of organic chlorine. Kidney--One analysis of the kidneys from three rats gave a total of 2.7 mg. of chlorine. The results show that no significant amounts of the chloro- DSW 254998 STLCOPCB4060689 406 Metabolism of Chloronaphthalenes naphthalene are stored in the tissues examined, and indicate that the compounds must be either metabolized or excreted with considerable rapidity. Detoxication Products--Naphthalene (7, 8), monobromonaphthalene (9), naphthalene monochloride (9), and bromobenzene (10) when fed to dogs are metabolized in part at least to mercapturic acids. Consequently, a series of experiments on a dog was undertaken to determine whether chloronaphthalenes would be metabolized in a similar manner. A female dog weighing 15.4 kilos was placed in a metabolism cage and given a diet consisting of casein 240 gm., sucrose 440 gm., together with brewers' yeast 88 gm., peanut oil 100 gm., cod liver Table I Urinary Sulfur Analyses Following Chloronaphlhalene Feeding The values are given in mg. of sulfur, phenol, and chlorine respectively. Sulfur analyses Phenol Chloride Day Inorganic Neutral Ethereal SO4 S SO* Free Conjugated Inorganic Total . 10* 151 11 182 12 128 13 142 33 19 22 22 6 143 74 24 166 90 19 116 36 5 146 89 633 932 935 766 760 672 * 1 gm. of Compound D was given immediately after the collection of this sample was completed. oil 100 gm., salt mixture (Karr (11)) 11.2 gm. plus 21 gm. of tri calcium phosphate per kilo of diet, and a small amount of meat extract for flavoring. The intake was stabilized at 150 gm. per day with slight loss of body weight. 24 hour urine specimens were collected by catheterization. Chlorides were estimated gravimetrically before and after evaporation and reduction of or ganic material by the method of Rauscher (5). Total sulfur, and inorganic and ethereal sulfates were estimated by the meth ods of Fiske (12), free and conjugated phenols by Folin's proce dures ((6) pp. 217-219). The data of one such experiment are presented in Table I. Administration of 1 gm. of Compound D to the dog produced a significant increase in the ethereal sulfate output from a basal DSW 254999 STLCOPCB4060690 Cleary, Maier, and Hitchings 407 level of 5 or 6 mg. of sulfur to 24 mg. the 1st day and 19 mg. the 2nd day. However, there was no significant change in the neu tral sulfur fraction, nor was there any marked change in the urinary excretion of glucuronic acid when measured by the method of Salt- (13). The chloride analyses gave results of considerable interest. No organic chlorine was found in the urine. However, there was a significant increase in the chloride output above the basal level for the 2 days following administration of the chloronaphthalene. Table II Excretion of Urinary Chloride during Chloronaphthalene Feeding Group I Group II Day Average urinary chloride Body weight Average urinary chloride Body weight 40 mg. Compound D daily Basal diet mg. gm. mg. gm. 1 4.7 172 3.3 169 2 7.3 1.9 3 G.7 162 4.2 168 4 7.2 1.6 5 8.2 149 2.0 174 6 7.7 3.8 Average......... 7.0 Basal diet 2.8 Fasting 7 2.0 135 6.3 159 8 1.4 125 4.6 148 9 1.1 120 1.6 143 The "extra" chloride amounted to 402 mg., a little over 60 per cent of the chlorine fed. This finding suggested the possibility that one of the early steps in metabolism of the chlorinated naph thalene might be the liberation of the chloride. This possibility was tested in a series of three experiments on albino rats, the results of one of which are given in Table II. 2 There is some evidence (8) to indicate that the mercapturic acid deriva tives of hydrocarbons are excreted as unstable conjugation products of glucuronic acid. DSW 255000 ' >\ .4 1 / STLCOPCB4060691 408 Metabolism of Chloronaphthalenes The rats were given a diet of lactic acid-casein, peanut oil, and corn-starch, with cod liver oil and yeast, and Osborne and Men del's (14) salt mixture modified by substituting lactic acid for the hydrochloric acid. After a few days the chloride excretion be came stabilized at a relatively low level. Three animals then were given daily 40 mg. of Compound D dissolved in olive oil by stomach tube and the three controls were given an equal amount of olive oil. The excretion of chloride rose immediately in the chloronaphthalene-fed animals and remained at a high level as long as the material was given. The animals refused to eat after the first few days and lost considerable body weight, which sug gested that part of the "extra" chloride excreted might originate in the tissues. However, when chloronaphthalene feeding was stopped, the chloride excretion promptly fell even though the animals continued to lose weight. Moreover, when similar losses in body weight were induced in the control animals by fasting, only small amounts of "extra" chloride were excreted. DISCUSSION The metabolic dehalogenation of halogeno-aryl compounds apparently has not been observed previously. The observation that it does occur in the metabolism of polychloronaphthalenes suggests the possibility that the process may take place more generally and previously may have escaped recognition. If this is proved true, the toxic properties of such compounds might be related to the process of intracellular liberation of halogen. SUMMARY The metabolism of a mixture of polychloronaphthalenes was studied. The substance was found to be absorbed completely when given in olive oil solution to albino rats in doses up to 15 mg. per day. No significant storage of the material could be detected in lung, liver, skin, or kidney, nor was any significant amount excreted in the urine. Both the rat and dog apparently were able to remove and excrete the chloride promptly. A rise in the urinary ethereal sulfate fraction, but no significant change in the neutral sulfur excretion, was noted following chloronaphthalene feeding in the dog. OS^500, STLCOPCB4060692 Cleary, Maier, and Hitchings 409 BIBLIOGRAPHY 1. Drinker, C. K., Warren, M. F., and Bennett, G. A., J. Ind. Hyg. and Toxicol., 19, 283 (1937). 2. Bennett, G. A., Drinker, C. K., and Warren, M. F., J. Ind. Hyg. and Toxicol., 20, 97 (1938). 3. Flinn, F. B., and Jarvik, N. E., Proc. Soc. Exp. Biol, and Med., 35, 118 (1936); Am. J. Hyg., 27, 19 (1938). 4. Roth, H., Quantitative organic microanalysis of Fritz Pregl, trans lated by Daw, E. B., Philadelphia, 3rd edition, 103- 104 (1937). 5. Rauscher, W. H., Ind. and Eng. Chem., Anal. Ed., 29, 296 (1937). 6. Folin, O., Laboratory manual of biological chemistry, New York and London, 5th edition (1934). 7. Bourne, M. C., and Young, L., Biochem. J., 28, 803 (1934). 8. Stekol, J. A., J. Biol. Chem., 110, 463 (1935). 9. Baumann, E., Z. physiol. Chem., 8, 190 (1883). 10. Baumann, E., and Preusse, C., Ber. chem. Ges., 12, 806 (1879). 11. Karr, W. G., J. Biol. Chem., 44, 256 (1920). 12. Fiske, C. H., J. Biol. Chem., 47, 59 (1921). 13. Salt, H. B., Biochem. J., 29, 2705 (1935). 14. Osborne, T. B., and Mendel, L. B., J. Biol. Chem., 37, 572 (1919). DSW 255002 STLCOPCB4060693