Choline Oxidase Activity in Rat Liver During 3-Methyl-4- Dimethylaminoazobenzene Carcinogenesis

Choline Oxidase Activity in Rat Liver during 3-Methyl-4- Dimethylaminoazobenzene Carcinogenesis

GLADYSE. WOODWARD

(Biochemical Research Foundation, Newark, DelÃ .)

The oxidation of choline to betaine is necessary little loss in choline oxidase activity during 48 for the utilization of a choline methyl group in the hours' storage at 5Â°C. production of methionine in vitro (5, 11). Since the Choline oxidase activity determination.â€”Oxygen methionine content of the proteins in rat liver uptake was measured at 37Â°C. in air in the War tumors induced by azo dyes is very much lower burg manometric apparatus. Each flask contained than in liver (12), the effect of a carcinogenic diet dialyzed homogenate equivalent to 0.25 gm. of containing 3'-methyl-4-dimethylaminoazobenzene tissue and 0.5 ml. of 0.2 M phosphate buffer of the (3'-Me-DAB) on the choline oxidase activity of desired pH in a total liquid volume of 2 ml. For de liver before and during development of a tumor termination of the rate of oxygen uptake, 4 mg. of has been investigated. The incorporation of 3'-Me- choline chloride per flask was used as substrate, DAB in the diet has been found to produce an and the pH of the buffer used was 7.9. The rate early and pronounced inhibition of the liver cho was corrected for the oxygen uptake of a similar line oxidase. mixture in the absence of choline chloride. Ac tivity was calculated from the initial linear portion EXPERIMENTAL of the rate curve and expressed as c.mm. per hour Animals.â€”Male rats of the Wistar strain, which per gram of fresh tissue. had been maintained on a stock diet of Purina For determining the maximum oxygen uptake, Laboratory Chow until the weight was about 150 1 mg. of choline chloride was used as substrate, and gm., were placed on the semi-synthetic diet of oxidation was allowed to proceed until it was cer Giese, Miller, and Baumann (7) in which 0.06 per tain that the oxidation of choline had stopped. The cent of the liver carcinogen, 3'-Me-DAB, was in initial pH was measured in mixtures duplicate to corporated. Control rats of the same weight were those in the manometer flasks. fed the basal diet without the carcinogen. The Determination of end-products of choline oxida rats were allowed food and water ad libitum and tion.â€”The protein in an oxidation mixture in a were given 1 drop of halibut liver oil per week. Warburg flask was precipitated by the addition Animals were sacrificed from time to time by de of 3 ml. of 2 per cent sulfosalicylic acid. The pro capitation, and the blood was permitted to drain. tein-free supernatant fluid obtained by centrifuga - The typical changes described by Miller et al. (IO) tion was used for betaine aldehyde and choline were noted in the livers from the rats on the car determinations. The betaine aldehyde was de cinogenic diet. termined by the bisulfite method of Cliftand Cook Enzyme preparation.â€”Liver or tumor was ex (4), which Bernheim and Bernheim (l) had shown cised from the animal immediately after decapita could be used for this aldehyde. The choline was tion and cooled to 5Â°C. Samples of tissue then estimated by the periodide method (2, 9), which were blotted dry, weighed, and homogenized in the depends upon observation, under the microscope, presence of an equal part of 0.2 Mphosphate buffer, of characteristic crystals of choline periodide at pH 7.9, in a Potter-Elvehjem glass homogenizer formed in serially diluted samples. The presence which was cooled in an ice-water bath. The of betaine aldehyde and betaine in considerable homogenate was dialyzed in cellophane tubing for amounts in proportion to choline noticeably re 21 hours at 5Â°C. against 2 changes of 100 parts of tarded the formation of the choline periodide distilled water, in order to remove naturally occur crystals, even though these products themselves ring oxidizable substrates. The freshly dialyzed do not form crystals with the periodide reagent homogenate was used; however, there was but (3, 9). The betaine produced was calculated from the oxygen uptake in excess of that required for Received for publication August 10, 1951. the amount of betaine aldehyde found. 918

RESULTS for the uptake of the second oxygen atom might be The rate of choline oxidation in liver homoge- lacking in 3'-Me-DAB liver. nates from rats on the diet containing 3'-Me-DAB That liver choline oxidase consisted of two en was found (Table 1) to be only about 25 per cent zymes had been shown by Bernheim and Bern- of that in liver homogenates from normal and con heim (1) : the first enzyme catalyzed the oxidation trol rats. This markedly lower activity was appar of choline to betaine aldehyde with the uptake of ent even in the first animal which had been on the 1 oxygen atom; the second enzyme catalyzed the 3'-Me-DAB diet for only 14 days, and was almost oxidation of betaine aldehyde to betaine with the as great as it was after more prolonged feeding. uptake of a second oxygen atom. Since 2 atoms of The oxidase activity in homogenates of the liver oxygen were taken up at pH 7.8 but only about tumor tissue, which had been separated as cleanly 1 atom at pH 6.7, they concluded that at pH 6.7 as possible from the adjacent liver tissue, was less the oxidation of betaine aldehyde was almost com- TABLE l CHOLINEOXIDASEACTIVITYINLIVERANDTUMORHOMOGENATESATpH7.8 ofOi uptake(with 4mg.cholinechloride)(cuptake(with 1mg.cholinechloride)(c Time Weightof on diet rat(gm.)1501501282102002582273043241S1176150126191167162169167Ratemm/hr/gmtissue)1,4401,3921,9201,7281,7751,4401,4401,63248057624033628824072706MaximumOs Enzyme source Rat no. (days) mm.)*14414615415814314414892691051067733585 Normal liver N-l N-2 N-8 N-4 N-6 N-Â« Control liver C-l C-2 112 C-3 167 S'-Me-DAB liver M-lf 14 M-2 21 M-S 42 M-4 62 M-5: 104 M-8Â§ 147 S'-Me-DAB tumor M-6 127 M-7 181 M-8 147 * Theoretical Ot-uptake for ÃŽatoms per molecule of choline chloride â€”160 c.mm. t Liver appeared normal. ÃŽLivercirrhotic. I Tumor masses present in liver.

than 5 per cent of that in homogenates of normal pletely inhibited. In normal liver homogenate at and control liver and less than 20 per cent of that pH 6.7, however, it has now been found (Table 2) in homogenates of the liver of S'-Me-DAB rats, an that an oxygen uptake of approximately 1 atom observation in close agreement with the findings of per molecule of choline was not evidence that the Kensler and Langemann (8) and Viollier (13). choline was oxidized only to aldehyde, because the Since the activity in tumor tissue was so slight, it oxidation had stopped with an appreciable amount may have been due merely to contamination from of choline remaining unoxidized. Therefore, the a small amount of liver tissue. oxygen uptake was considerably more than 1 atom The low choline oxidase activity in the S'-Me- per molecule of the choline which was oxidized. DAB liver was reflected not only in the rate of Part of the oxidized choline was found as aldehyde, oxygen uptake but also in the total amount of but the oxygen uptake in excess of that required oxygen taken up (Table 1). The maximum oxygen for the aldehyde found indicated betaine forma uptake obtainable at pH 7 8 with 3'-Me-DAB liver tion also. The calculated amount of betaine, to was close to 1 atom per molecule of choline, while gether with the betaine aldehyde and unoxidized with normal or control liver the oxygen uptake choline found, accounted for 86-96 per cent of the was approximately 2 atoms. The possibility was choline added. suggested, therefore, that the enzyme responsible With 3'-Me-DAB livers Nos. 5 and 8 at pH 7.8,

the oxidation picture (Table 3) was similar to that than at pH 7.8. At pH 6.7, in a 3'-Me-DAB liver with normal liver at pH 6.7; part of the original and a liver tumor homogenate, in both of which choline had been oxidized only as far as betaine there was considerable oxidation of choline to be aldehyde, but some choline had been oxidized taine aldehyde at the higher pH's, there was little through aldehyde to betaine. A much smaller or no oxidation of choline at all; in control liver, proportion of betaine was found with No. 8 liver, as well as in normal liver, there was considerable which was in an advanced state of tumor growth, oxidation with the formation of both betaine alde than with No. 5 liver, which had not yet developed hyde and betaine. tumor masses. In homogenates of three liver tu mors at pH 7.8 there was little or no oxidation of DISCUSSION choline beyond the betaine aldehyde stage. The The impairment in the ability of homogenates of weak oxidase activity of liver tumor and of No. 8 3'-Me-DAB liver and liver tumor to oxidize cho liver was also shown by the cessation of oxidation line is expressed by the lower rate of oxygen up while a considerable amount of unoxidized choline take, the smaller total oxygen uptake, the pre was still present. With control liver, as well as ponderance of betaine aldehyde over betaine as with normal liver, no aldehyde was found at pH end-products, and the larger amount of choline 7.8, no choline was left unoxidized, and the oxygen left unoxidized. An explanation for the cessation uptake indicated that choline was oxidized almost of oxidation before all the choline has been oxi completely to betaine. dized and for the presence of both betaine alde At pH 7.2 (Table 3) the same qualitative differ hyde and betaine as end-products may perhaps ence between the end-products of choline oxida be found in a greater instability of the enzymes of tion with 3'-Me-DAB liver and with normal or the choline oxidase system in rats after feeding of control liver was observed as at pH 7.8; the extent 3'-Me-DAB. Badie and Bernheim (6) showed that of oxidation was, however, slightly less at pH 7.2 the enzyme which oxidizes choline to aldehyde is

TABLE 2 EXTENTOFOXIDATIONOFCHOLINEINPRESENCEOFNORMALLIVERHOMOGENATEATpH 6.7 CHOLINKCHLORIDEADDED(mg.)1t4OaUPTAKEMaximumfoundTheory(1 CHO CHOLINEACCOUNTED LINEOXIDIZED(CALCULATED)(mg.)*0.761.583.29CHOLINEFOUNDUN-OXIDIZED(mg.)*0.130.140.54TOTALFOB(per(mg.)* atom)(c. mm.)9217732480160320ALDEHTDEFOUND(mg.)*0.370.952.53BETAINECALCULATED(mg.)*O.S90.630.70TOTALcent)0.89 891.72 863.83 96 * Expressed as choline chloride equivalent.

TABLE 3 EXTENTOFOXIDATIONOFCHOLINEINPRESENCEOFLIVER ANDTUMORHOMOGENATES

uptake(maximumfound)(c. un RatBO.N-6C-2C-3M-5M-8M-6M-7M-8N-6C-3M-8M-7N-5N-6C-2C-3M-8M-7pH(initial)7.87.87.87.87.87.87.87.87.27.27.27.26.76.76.76.76.76.7Osoxidized(mg.)*00000.240.540.310.89000.350.410.130.220.150.351.000.78Totalcholineaccountedfor(per Enzyme source mm.)1431441481067788Â¿8514314460459270746209Aldehydefound(mg.)*0000.520.580.380.600.10000.560.540.370.550.460.5400.11Betainecalculated(mg.)*0.890.900.930.400.190.020.0600.890.900.0900.390.160.230.0600Cholinefoundcent)89909392101Â»497998990100958998849510089 Normal liver Control liver a u S'-Me-DAB liver a it S'-Me-DAB tumor

Normal liver Control liver S'-Me-DAB liver S'-Me-DAB tumor Normal liver Â« a Control liver a a S'-Me-DAB liver S'-Me-DAB tumor * Expressed as choline chloride equivalent: 1 mg. choline chloride added initially.

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1951 American Association for Cancer Research. WOODWARDâ€”LiverCholineOxÃdasein S'-Me-DAB Caranagenesis 921 a very unstable enzyme, that the stability is at pH 7.8 or 7.2, produced predominantly more greater at pH 7.8 than at pH 6.7, and that the en betaine aldehyde than betaine as the end-products zyme is more stable in the presence of substrate of the oxidation of choline, while normal or con than in its absence. They suggested that inactiva- trol liver homogenate