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Studies on Hepatic Protein-bound Dye Formation in Rats Given Single Large Doses of 3'-Methyl-4-Dimethylaminoazobenzene^*

H. V. Gelboin, J. A. Miller and E. C. Miller

( McArdle Memorial Laboratory for Cancer Research, The Medical School, University of Wisconsin, Madison 6, Wis.)

1. Protein-bound dye was found in the liver within a few hours after intraperitoneal or oral administration of single 36–54-mg. doses of 3'-methyl-4-dimethylaminoazobenzene (3'-methyl-DAB) to rats. Within 18–24 hours the level was as high as those obtained after 1 or more weeks of administration of 4–6 mg. daily in the diet. The protein-bound dyes formed under both conditions were similar with regard to their specificity for the liver, their intracellular distribution within the liver, their absence from the induced liver tumors, their half-lives, and the spectral and solubility properties of the dye released by alkaline hydrolysis.

2. Five hours after the injection of 36 mg. of 3'-methyl-DAB, there was 50 per cent more bound dye per mg. of microsomal liver protein than per mg. of supernatant liver protein. By 24 hours, these concentrations were equal.

3. Administration of 50 mg. of ethionine simultaneously with 36 mg. of 3'-methyl-DAB depressed the formation of protein-bound dye to 50 per cent of normal; with larger doses, inhibitions of 75 per cent were obtained. This inhibition was prevented by an equimolar amount of methionine. A large inhibition in the formation of protein-bound dye was also noted in hypophysectomized rats.

4. Rats maintained on a 50 per cent casein diet for 8 days formed 1.6 times as much hepatic protein-bound dye from 50 mg. of 3'-methyl-DAB as rats maintained on a protein-free diet. Protein-depleted rats given dye 12–36 hours after transfer to the 50 per cent casein diet bound larger amounts. A maximum increase to 55 per cent above that of the high protein controls occurred in the rats given dye 36 hours after transfer. By 80 hours, when the liver protein had been largely replaced, bound-dye formation decreased to the normal range.

5. The above data suggest that, in these experiments, dye is bound to certain liver proteins during their synthesis rather than to protein formed previously.

6. Administration of 0.2 per cent of DL-ethionine with 0.04 per cent of 3'-methyl-DAB inhibited tumor induction by the dye in only one of three experiments. Protein-bound dye formation was inhibited to only a small extent, as compared with controls restricted to the same food intake.

7. The hepatocarcinogenic activity of ethionine for rats, previously reported by Popper et al. and by Farber, was confirmed.

^* This investigation was supported in part by grant C355 of the National Cancer Institute, U.S. Public Health Service, Institutional Grant 71 from the American Cancer Society, and the Alexander and Margaret Stewart Trust Fund. A preliminary account of some of these data has been presented (5). We are indebted to Mrs. Bette Wabers and Mrs. Ivanne Jones for technical assistance.

Public Health Research Fellow of the National Cancer Institute.

Received 2/ 3/58.