Document jmK5pJGyk6rRaVvveEBV9beZ

Environmental Health Perspectives Vol. 21, pp. 1977. COMPARATIVE MAMMALIAN METABOLISM OF VINYL AND VINYLIDEKE CHLORIDES IN RELATION TO ONCOGENIC POTENTIAL4 * by David E Hathway Elucidation of the role of vinyl chloride metabolites in the various reaction sequences which comprise the metabolic pathway, including the interaction of reactive metabolites with some purine and pyrimidine residues of target-organ DNA, provides some explanation for the (oncogenic) properties associated with the original substance. Comparative investigation of the biological fate of vinylidene chloride reveals (a) an agent of low oncogenic potential which is likely to be damaging only under special circumstances, and (b) species differences which suggest that the mouse is more susceptible than the rat towards vinylidene chloride oncogenicity. * Imperial Chemical Industries Limited, Central Toxicology Laboratory, Alderley Park, Cheshire SK10 4TJ, England. + Lecture delivered in the Symposium on Comparative Metabolism and Toxicity of Vinyl Chloride Related Compounds (NIEHS), held at Bethesda, Maryland, on May 2-4, 1977. RSV 0012148 2 The research work with which this communication is concerned is based on the idea that knowledge of the biology of the reactive metabolites of chemical carcinogens in the mammal, including the precise nature of the chemical changes to the DNA of the nucleus, ought to give an insight into the (oncogenic) properties of the parent compounds. In tracer studies, N>-acetyl-S-(2-hydroxymethyl)cysteine was shown to be a major vinyl chloride metabolite in rats, but according to the method of protective esterification that was used so a derivative either of N-acetyl-S- (2-chloroethyl)cysteine or of N-acetyl--(2-hydroxyethyl)cysteine was isolated from body fluids (1, 2). Thus, by Fischer-Speier methylation, H-acetyl-'fi-(2-chloroethyl)cysteine was obtained, and with diazomethane, N^acetyl-S-(2-hydroxyethyl)cysteine. It might be stated en passant that throughout the investigations described, mass spectrometry, involving E.X. and chemical ionisation sources and multiple-ion detection and all combinations of these facilities, was used extensively both for product identification and analysis and for the purposes of detection. Treatment Fig. 1 of the O-oethyl ester of Il-acetyl-S-CZ-hydroxyethyDcysteine (a) (Fig. 1) near here with the methanol-HCL reagent gave a mixture of N^acetyl-S^(2-chloroethyl) cysteine (b), and S^(2-chloroethyl)cysteine, and conversely, the O-mechyl ester of N>-acetyl-S-(2-chloroethyl)cysteine (b) was hydrolysed rapidly by water to that of N-acetyl-<S-(2-hydroxyethyl)cysteine (c) (2). Hence, the reversible reaction processes connecting the two substances would seem to be modulated through the intermediacy of episulphonium ion (c) and formation of this ion would in fact be rate-limiting in respect of the hydrolysis of N^acetyl-S-(2-chloroethyl)cysteine. Nucleophilic attack of OH on the episulphonium ion would be expected to give olefin (3), and in fact, N_-acetyl-S-vinylcysteine (d) was recovered from the urine of vinvl RSV 0012149 J chloride-treated animals whenever diazomethane esterification was used to protect ^-containing metabolites. Surprisingly, N-acetyl-^-(2-hydroxyethyl)cysteine -methyl ester was methylated with neutral methanol, and the O^methyl esters of N^acetyl-S-(2-methoxy[^C]ethy1)cysteine plus N-acetyl-S-[*^C]vinyl-cysteine degrade to give the volatile [^C]S-(2-methoxyethyl) (prop-1 or 2-enyl) sulphide. Although the mechanism of formation was not investigated, we felt that acetaldehyde, a known dissociation product of S.-vinylcysteinederived jx-vinylcysceine~-oxide (4) might undergo concerted condensation with N-acetyl--(2-methoxyethyl)cysteine leading to elimination of thermodynamically stable glyoxylate. [There is an analogy for such a concerted condensation reaction in the work of Dabritz & Virtanen (4) on the tear-producing volatile components of the onion.] The half-mustard ^-containing metabolites of vinyl chloride did not behave as mutagens in the Ames test (2). Thiodiglycollic acid is another major vinyl chloride metabolite (1). RSV 0012150 Fig. 2 near here In order to determine whether vinyl chloride yielded chloroethylene oxide vivo, the biogenesis of several vinyl chloride metabolites and related compounds were investigated in rats (2). S^^-Hydroxyethyl)cysteine gave 0.5Z of the authentic thiodiglycollic acid, and this result was seen to be highly significant, because of the instability (v. supra) of the starting material under'exceedingly mild conditions of reaction. Th metabolic pathway concerned (Fig. 2) appears to include ena-group oxiaation, amino-acid transamination and oxidative decarboxylation, and the results of the animal feeding experiments suggest that chloroacetaldehyde (g) (Fig. 3X 4 Fig. 3 chloroacetic acid (h), and S-(2-carboxymethyl)cysteine (i) might lie on a near here conxnon pathway connecting vinyl chloride (e) with thiodiglycollic acid (j). However, other evidence implies that chloroacetic acid (h) does not belong to this metabolic pathway (e-j) (Fig.3). Thus, (i) 40.1Z has even been detected in the body fluids of any of our vinyl chloride-treated animals. Either there is a high rate of turn-over or this compound is not a major vinyl chloride metabolite. The latter possibility seems more likely, since relatively large amounts are produced in vinylidene chloride metabolism, and in those animals, thiodiglycollic acid accounts for an even greater proportion of the dose than in parallel experiments with vinyl chloride. (i) A feasible metabolic pathway for thiodiglycollic acid from chloroacetic acid and involving cysteine desulphhydrase is unacceptable. Experiments with unlabelled vinyl chloride in rats in which the cysteine- cystine pools had been labelled adequately with gave [^Cjthiodiglycollic acid, showing that a part of the O-skeleton must be derived in fact from cysteine. (iii) In rats treated with chloroacetaldehyde, the presence of thiodiglycollic acid and N-acetyl-5>-(2-hydroxyethyl)cysteine, but not of chloroacetic acid, amongst the urinary metabolites was established by mass fragmentometry. Thus, it is probable that in vivo chloroethylene oxide (f) (Fig.3) was formed (5) from vinyl chloride (e) and transformed spontaneously (6) into chloroacetaldehyde (g); there is supporting evidence (7--10) for vinyl chloride epoxidation in vitro. This supposition is supported by the fact (A) that chloroacetaldehyde affords both N-acetyl-S^(2-hydroxyethyl) cysteine and thiodiglycollic acid vivo and (B) that _S-(2-carboxymethyl) cysteine has been identified by mass fragmentometry amongst the hydrolytic products of an hepatic extract prepared from vinyl chloride-treated animals. RSV 0012151 5 Since chloroacetaldehyde and chloroethylene oxide are mutagenic in the Ames teat (11-13) and in Chinese hamster V79 cells (14), they may be relevant to vinyl chloride carcinogenicity. Respective formation of 3B-D-ribofuranosylimidazo-[2,l-i]purine or 6B-D-ribofuranosyl-5-oxo-56-dihydroimidazo-[l,2-c]pyrimidine from adenosine or cytidine by reaction with chloroacetaldehyde (15) or chloroethylene oxide was readily confirmed. The presence of these two imidazo-nucleoside derivatives has now been established by mass fragxoentometry (16) in the enzymic hydrolysate of modified rat-liver DNA, prepared from rats, which had been exposed chronically to vinyl chloride Fig. 4 (250 ppm in their drinking water) for 1 year (Fig. 4). A smaller near here proportion of the 30-D-ribofuranosylimidazo-[2,l-i]purinef than would have been expected to have been formed, was found both in the animal experiments with vinyl chloride and in model reactions between chloroacetaldehyde and calf-thymus DNA (16). This observation is consistent with some degree of DNA depurination brought about by the reaction of vinyl chloride, and in our model experiments, we have found evidence for the presence of the detached purine. Viz. imidazo-[2,l-i]purine. Hence, the alkylation that produces imidazo-derivative formation (with DNA) labilizes the purine 6-glycoside linkage, which leads to depurination. The gap so produced might then be filled by various bases, resulting in 'mispairing1 during DNA replication. These results are very important, because in general, there is excellent agreement between the severe damaging effect of depurination to DNA and mutagenicity (17-19)* Thus, in retrospect, one would suspect vinyl chloride of being mutagenic/ carcinogenic. RSV 0012152 6 On the ocher hand, vinylidene chloride (a) (Fig. 5) metabolism in Fig. 5 near here rats gave thiodiglyeollic acid (g) and an N-acetyl--cygteinyl-acetyl derivative (e) as major urinary metabolites, plus substantial amounts of chloroacetic acid (b), dithioglycollic acid (j) and thioglycollic acid (h) (20). It is probable that chloroacetic acid (b), which is a vinylidene chloride metabolite per se, lies on a major metabolic pathway for vinylidene chloride (Fig. 5), since it_affords several metabolites in common with vinylidene chloride (20). There is a strong supposition, that detoxification of chloroacetic acid (b) is effected through a glutathione S^acyl transferase catalysed reaction process and ensuing degradative sequence for the resulting carboxymethylglutathione (d), and that this represents the principal metabolic pathway for chloroacetic acid and a major one for vinylidene chloride. Thiodiglyeollic acid is the ultimate detoxification product, and previous work (2) established the biotransformation of S-(2-carboxymethyl) cysteine (f) into that substance. A feasible metabolic pathway to thiodiglyeollic acid from chloroacetic acid and involving cysteine desulphhydrase is unacceptable. In experiments (rats) with unlabelled vinylidene chloride in which the cysteine-cystine pools had been labelled with C, labelled thiodiglyeollic acid resulted, and a part of the C-skeleton of that substance must be derived in fact from cysteine (20). Formation of a small amount of [^Cjdithioglycollic acid (j) (and hence of the intermediate [^Cjthioglycollic acid) (h) is reconcilable with the action of Michaelis's (21) unspecific 0-thionase, which would lyse a small proportion of the preponderating [**C]thioglycollic acid (v. infra) RSV 0012153 Moreover, Kolbe electrolysis (22) of one molecular proportion of the [^C]thiodiglycollic acid metabolite from [l-^C]l, 1-dichloroethylene or [l-*^C]chloroacetic acid gave 1 equivalent of ^C02(23) and this evidence is consistent with the transformation of vinylidene chloride into chloroacetic acid by a mechanism involving migration of one Cl atom and the loss of the other one (20, 23). Hence, the metabolic pathway (Fig. 5), which was tentatively proposed for the biotransformation of vinylidene chloride into thiodiglycollic acid does in fact operate in rats. It is equivocal whether the very small amounts of CO2 and urea are produced by the action of epoxide hydratase on 1,1-dichloroethylene oxide or by a minor oxidative pathway for chloroacetic acid. There is a strong supposition that the N-acetyl-S^cysteinylactyl derivative (e) (Fig.5), which is a metabolite of vinylidene chloride, but not of chloroacetic acid, may be formed in fact from 1,1-dichloroethylene oxide through the agency of glutathione S^epoxide transferase to afford ^-glutathione acetyl chloride (c) and its subsequent reactions (20). This supposition is important, since the reactivity displayed by 1,1-dichloroethylene oxide (v..supra) appears to be relevant to the possible interaction of reactive vinylidene chloride metabolites with mouse-kidney Fig. 6 DNA (Fig. 6), which is a prerequisite of tumour initiation (24). Such near here interaction would be analogous to that of vinyl chloride with rat-liver DNA iii vivo, which forms imidazo derivatives with some nucleoside residues (16). Further work in progress to investigate this hypothesis. RSV 0012154 8 Fig. 7 near here Comparative studies (25) provide clues of differences between rats and mice in the processing of vinylidene chloride (Fig. 7). Thus, in mice, the production of thiodiglycollic acid is considerably reduced and the formation of the N^acetyl-S^cysteinylacetyl metabolite is increased. The higher 0~thionese activity in mice than in rats accounts for the greater conversion of thiodiglycollic acid into dithioglycollic acid via thioglycollic acid in the former species of animal. Yllner's (26) mice excreted a proportion of a dose of chloroacetic acid as unchanged starting acid. Thus, in mice, the metabolic pathway from chloroacetic acid to thiodiglycollic acid seems to be readily saturable, possibly on account of an inadequacy in the reaction catalysed by glutathione S^acyl transferase. Under these circumstances, detoxification of 1,1-dichloroethylene oxide by glutathione S-epoxide transferase and the modification of DNA by 1,1-dichloroethylene oxide or chloroacetyl chloride would be expected to be more significant in mice than in rats. This diagnosis of species susceptibility seems to accord with Maltoni's (24) discovery of vinylidene chloride oncogenicity in (the kidneys of) mice. Vinylidene chloride emerges as an agent of low, perhaps very low, oncogenic potential, which can be damaging only in a special set of biological circumstances, which ve have partially defined (v,* supra) and on which work is continuing. RSV 0012155 9 ACKNOWLEDGEMENT The author is indebted to his colleagues Messrs T Green* and B K Jones, Drs A G Salmon and P L Batten* and Mr G H Walker for their invaluable contributions and help. TVU /WTO T. tt RSV 0012156 REFERENCES 1. Green, T., and Hathvay, D.E., The biological face in rats of vinyl chloride in relation to its oncogenicity* Chen.-Biol. Interactions 11.: 545 (1975). 2. Green, T. and Hathvay, D.E., The chemistry and biogenesis of S'-containing metabolites of vinyl chloride in rats, Chem-Biol. Interactions, 7: 137 (1977). 3. Ogston, A.G. et al., The replacement reactions of SB'-dichlorodiethyl sulphide and of some analogues in aqueous solution: the isolation of 8-chloro-B*-hydroxydiethylsulphide, Trans. Faraday Soc., 44: 45 (1948). ti 4. Dabritz, E. and Virtanen, A.)I., S^-Vinyl-cystein-S^-oxyd, ein Homologes n zur Vorstufe der tranentreibenden Substanz der Zviebel, Chem. Ber., 98: 781 (1965). 5. Van Duuren, B.L., On the possible mechanism of carcinogenic action of vinyl chloride, Ann. New York Acad. Sci., 246: 258 (1975). *i 6. Gross, H., and Freiburg, J., Zur Existenz von Chlorathylenoxid, J. prakt. Chem., 311: 506 (1969). 7. Runaug, U., et al., The mutagenicity of vinyl chloride after metabolic activation, AMBIO, 3i 194 (1974). RSV 0012157 8. Barbin, A., et^ al_., Liver-microsome mediated formation of alkylating agents from vinyl bromide and vinyl chloride, Biochem. Biophys. Res. Comm., 67: 596 (1975). 11 9. Greia, H., et al., Mutagenicity in vitro and potential carcinogenicity of chlorinated ethylenes as a function of metabolic oxirane formation, Biochem. Pharmacol., 24: 2013 (1975). 10. Salmon, A.G., Cytochrome F450 and the metabolism of vinyl chloride. Cancer Lett., 2^ 109 (1976). 11. Bartsch, H., Malaveille, C., and Montesano, R., Human, rat and mouse liver-mediated mutagenicity of vinyl chloride in S, typhinuriua strains, Xnt. J. Cancer, 15: 429 (1975). 12. Malaveille C., et al.. Mutagenicity of vinyl chloride, chloroethylene oxide, chloroacetaldehyde and chloroethanol, Biochem. Biophys. Res. Coco., 65: 363 (1975). 13. McCann, J., et al.. Mutagenicity of chloroacetaldehyde, a possible metabolic product of 1,2-dichloroethane (ethylene dichloride), chloroethanol (ethylene chlorohydrin} vinyl chloride and cyclophosphamide, Froc. Nat. Acad. Sci. USA, 72: 3190 (1975). 14. Huberman, E., Bartsch, H., and Sachs, L., Mutation induction in Chinese hamster V79 cells by two vinyl chloride metabolites, chloroethylene oxide and chloroacetaldehyde, Int. J. Cancer, 15: 539 (1975). 15. Barrio, J.R., Secrist, J.A., and Leonard, N.J., Fluorescent adenosine and cytidine derivatives, Biochem. Biophys. Res. Comm., 46: 597 (1972)RSV 0012156 12 16. Green, T., and Hathvay, D.E., Interactions of vinyl chloride vith ratliver DNA in vivo, Chem.Biol, Interactions, in the press. 17. Lavley, P.D., e,t al. Inactivation of bacteriophage T7 by mono-and di-functional sulphur mustards in relation to cross-linking and depurination of bacteriophage DNA, J. Mol. Biol., 39: 181 (1969). 18. Roberts, J.J., Nucleic "acid modifications and cancer, In: 'Biology of Cancer*, E. J. Ambrose and F.J.C. Roe, Eds., Halstead Press, Chichester, 2nd ed., 1975. 19. Loveless, A., Genetic and Allied Effects of Alkylating Agents, Buttervorths, London, 1966. 20. Jones, B.K. and Hathvay, D.E., The biological fate of vinylidene chloride in rats, Chem-Biol, Interactions, in the press. 21. Michaelis, L., and Schubert, M.P., The reaction of iodoacetic acid on mcrcaptans and amides, J. Biol. Cheat., 106: 331 (1934). 22. Kolbe, H., Untersuchungen uber die Elektrolyse organischer Verbindungen, Justus Liebigs Annalen der Chetrtie, 69: 257 (1849). 23. Walker, G.H. and Hathvay, D.E., Electrochemical analysis of the [carboxy-^C]aliphatic carboxylic acid metabolites resulting from tracer molecules, Biochem. J., in the press. RSV 0012159 13 24. Maltoni, C., Proceedings of Che TAPPI International Conference, held in Hamburg on January 26, 1977. 25. Jones, B.K. and Hathvay, D.E., Differences between mice and rats in Che metabolism of vinylidene chloride, Brit. J. Cancer, in the press. 26. Yllner, S., Metabolism of chloroacetate-1-14 C in the mouse, Acta Pharmacol, et toxicol., 30: 69 (1971). RSV 0012160 (a) OH Hx l/H XC-C, H^l XH SR (c) +OH -OH h2c-ch2 w s I ch. Cl -Cl CH(NHAc)C02H -H + /H >< RS^ VH (d) (b) Cl H\ I^H c-r H^l VH SR Fig. 1 Scheme for the interrelationship of some ^-containing vinyl chloride metabolites. KSV 0012161 OH I H2C-CiH2 (I) S I CHo I* CH(NH2)C02H ho2c-ch2 s I CHo (II) CH(NH,)C0oH ho2c-ch2 (III) S I CHo C-COoH II * 0 S(CH2C02 Fig. 2. Scheme suggesting the biotransforaation of S^(2-hydroxyethyl) cysteine into thiodiglycollic acid. (I) End-group oxidation; (II) transamination; (III) oxidative decarboxylation. RSV 0012162 (e) <n (9) tf1 (h) 1 C-0 chch9sch9ch9 I 2 2I 2 NH OH I C02H CHCH2SCH2CH2 NH(Ac) OH -OH +OH' CHR-S" ^CH' -H R-Sv X*CR> H/ 2 chch2sch2co2h NH I CO?H I2 CHCH2$CH2C02H (i) co9h I2 c-ch9sch9co9h II 2 2 2 0 S(CH2C02H)2 <j) Fig. 3 Scheme for the biogenesis of ^-containing vinyl chloride metabolites RSV 0012163 CtCti^HO +DNA H=dtiCl o,i >-- aful <z$sociite,tl ^tLejiiLvin&tto?!' RSV 0012164 Fig. A Scheme suggesting the model reaction of chloroacetaldehyde with (calf-thymus) DNA and the biotransformation of hepatocyte DNA by vinyl chloride ^ir^ vivo. Both reaction processes afford 6-D-ribofuranosyl-5-oxo-3,6-dihydroimidazo-[12-c3pyrimidine (left-hand side) and 3B_D-ribofuranosylimidazo--[2*1-i]purine (right-hand side). /HOgCCHCHgSCHgCOgH ' OH i S(CH2C02H)2 1 1 HSCHjCOpH 1 1 (sch2co2h)2 (e) (h) (J) Fig. 5 Scheme for vinylidene chloride metabolism in rats. RSV O0I2165 CtCHfiCCt o Fig. 6. Scheme suggesting the feasible interaction of reactive vinylidene chloride metabolites, 1,1-dichloroethylene oxide and chloroacetyl chloride, with adenosine and cytidine respectively. RSV 0012166 METABOLISM (1) of Chloroacetic acid Metabolite Rats Chloroacetic acid - Thiodiglycollic acid 90 N-Acctyl-S-(2-carboxymethyl)cysteine 2 Yllner 6-221 37 40 Mice BKJ-DEH 30-40 40 Chloroacetic acid Thiodiglycollic acid Thioglycollic acid Dithioglycollic acid N-Acctyl-S-eys teinylacetyl derivative (2) of Vinvlidcnc chloride 337 3 35 5 20 48 70 Fig. 7. The relative proportions of products resulting from the metabolism (1) of chloroacetic acid and (2) of vinylidene chloride in rats and mice. RSV 0012167 precursors of benzoic acid which occur naturally. The hippuric acid content of end-of-shift urine samples and measured toluene exposures were found to correlate well in individuals exposed in leather finishing and rubber coating operations. The air-urine correlations suggest that exposures to 200ppm of toluene would be expected to pro duce urinary hippuric acid content of about 7Gm./liter In samples collected at the end of the work shift. The hippuric acid test was found to be useful in evaluating the adequacy of cavtridge- type respirators in lank painting work. Increas ing hippuric acid excretion levels during expo sure demonstrated the limited protection afforded by the cartridge-type respirators which were worn. --W.L.S. Acro-osteolysis Oymrring in Men Engaged in the Polymerization oflVinvI Chloride. D. -K: Harris and W. G, F. Adams. British Med. J. 3:712, Sep tember 16, 1967. (Imperial Chemical Industries, Ltd., Plastics Div. Two cases of acro-osteolysis are described in men engaged in cleaning autoclaves used in the polymerization of vinyl chloride. The two men had worked in the autoclave section of the plant for six and 16 years, and were identified as a result of a roentgenographic survey of the hands of 58S men engaged in the manufacture of poly vinyl chloride of whom 150 were autoclave clean ers. Both cases had pseudoclubbing of the fingers with osteolytic changes in the terminal phalanges and Raynaud's phenomenon. One case had raised nodules in the skin around his wrist, and the other had a history of a diagnosis of scleroderma, but no present evidence of the skin condition that had existed nine years previously. It appears that the disease is self-limiting, and, despite the bone destruction, leaves the patient with little disabil ity. Private inquiries indicated that similar cases had occurred in the United States and Europe, mainly among those engaged in autoclave clean ing and not in those handling the finished prod uct. --W.L.S. DERMATOLOGY Paraben Contact Dermatitis. K. D. Wuepper, JAMA 202:127, 1967. (Div. Dermatology, Dept, of Medicine, Univ. California School of Medicine, San Francisco, Calif.) Parabens are alkyl esters of p-hydroxybenzoic acid commonly added in low concentrations to creams, lotions, and cosmetics to prevent micro bial growth. The four patients who are the sub ject of this report were originally regarded as Journal cf Occupatio7ial Medicine Abstracts having dermatoses of unknown cause. During routine closed patch tests on 91 patients, these four cases demonstrated positive reactions to alkyl esters of p-hydroxybenzoic acid. They were using topical agents containing parabens. In the present study, all four patients had a positive reaction to one or more paraben esters in a con centration of 5%, whereas only one reacted to parabens in a concentration of 0.1%. Irritant re actions to 5% concentrations of parabens are thought to be negligible. An example of paraben concentration is given for hydrophilic ointment (USP). It is reported to contain 0.0259c methyl paraben and 0.0159c propyl paraben. The prob lem of cross sensitivity among the parabens and chemically related compounds is also discussed briefly. --L.J.M. EXPERIMENTAL TOXICOLOY Hyperplastic and Early Neoplastic Lesions of the Liver in Buffalo Strain Rats of Various Ages Given Subcutaneous Carbon Tetrachloride. M. D. Reuber and E. L. Glover. J. Nat. Cancer Inst., 38:891, June 1967. (National Cancer Institute, Bethesda, Mo.) Subcutaneous injections of carbon tetrachlo ride were given to inbred male and female rats 4, 12, 24, and 52 weeks old. Preneoplastic and early neoplastic lesions of the parenchymal cells developed in the liver. The 24- and 52-week old animals of both sexes had more hyperplastic nod ules as well as an occasional small hepatocellular carcinoma. The number of hyperplastic lesions per liver and size of lesions were greater in fe males than in males. Four-day-old animals given carbon tetrachloride died with hepatic and renal necrosis. --R.E.E. Hematuria Following Administration of Ethanol. J. M. Orten; K. C. Shrivastava and M. Shih. Science 157:72, July 7, 1967. (School of Medicine, Wayne State University, Detroit, Mich.) Hematuria occurred in male albino vats given 20 and 409c ethyl alcohol in lieu of drinking water for several weeks. Erythrocytes were ob served in the urinary sediment of a number of the treated rats. The amounts of hemoglobin in the blood of the treated group were not significantly different from those of the control group, which indicates that the degree of hematuria was not sufficient to produce an anemia. Some possible explanations for the hematuria are given. Con siderable damage to the epithelial cells of the renal proximal tubule and significant renal en largement were observed. Three human patients 2G3 RSV 0012166