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Sequential Alterations in Mitochondrial Inner and Outer Membrane Electron Transport and in Respiratory Control during Feeding of Amino Azo Dyes; Stability of Phosphorylation. Correlation with Swelling-Contraction Changes and Tumorigenesis Threshold¹

Seamen's Memorial Research Laboratory, USPHS Hospital, New Orleans, Louisiana 70118, and the Department of Medicine (Biochemistry), Tulane University School of Medicine, New Orleans, Louisiana 70112

In relation to preceding investigations on the swelling and contraction properties of rat liver mitochondria during the feeding of amino azo dyes, the respiratory rate, the P:O ratio, the respiratory control index, as well as the NADH₂ cytochrome c reductase and diaphorase activities were studied in rat liver mitochondria during the time-course of feeding 0.06% 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) and 2-methyl-4-dimethylaminoazobenzene (2-Me-DAB), and in mitochondria from 3'-Me-DAB-induced hepatoma. It was found that the P:O ratio remains at the normal values throughout 16 weeks of feeding 3'-Me-DAB, using pyruvate, -ketoglutarate, or glutamate as substrates; the ratio is very slightly decreased throughout the feeding of 2-Me-DAB for 10 weeks. Mitochondria from 3'-Me-DAB-induced hepatomas give a scattering of P:O ratio values with all three substrates indiocating partial or total uncoupling depending on the individual tumor.

Between 0 and 7 weeks (with a maximum at 3 weeks) the Q_O2 notably increases during feeding of 3'-Me-DAB; the increase is the largest with -ketoglutarate, less with glutamate, and the smallest with pyruvate. This increase is due to temporary release of the respiratory control. Determination of the respiratory control indexes showed, in fact, considerable minima at 3 weeks during feeding of 3'-Me-DAB, with either of the three substrates or with ß-hydroxybutyrate. The Q_O2 values of tumor mitochondria are lower than the Q_O2 of normal controls, and the decrease is statistically significant with pyruvate and glutamate. Unlike 3'-Me-DAB, 2-Me-DAB causes a large decrease of respiration with glutamate or pyruvate from the very onset and throughout the 10-week feeding period; in contrast, with -ketoglutarate there is a gradual, moderate increase with a peak at 6 weeks.

Of the two outer membrane-localized electron transport segments, diaphorase activity shows a sharp minimum at 4 weeks during feeding of 3'-Me-DAB. This correlates with the minima of swelling and "contraction" and with the onset of irreversibility of tumor induction with this dye. With NADH₂ cytochrome c reductase the 4-week decrease is not seen; however, immediately following this period, from 5 weeks on, a temporary, over fourfold, rise of activity is observed with a maximum at 7 weeks. The high NADH₂ cytochrome c reductase activity at 7 weeks is largely observed in hepatoma mitochondria. These changes of NADH₂ oxidation are either absent or entirely different during administration of 2-Me-DAB. The successive changes observed in mitochondrial membrane-linked functions probably represent steps of progression in 3'-Me-DAB carcinogenesis.

¹ Supported by Research Grant CA-05431 from the National Cancer Institute, USPHS. Presented in part at the Ninth International Cancer Congress, Tokyo, October 1966, Abstract S0318.

² Present address: Section of Neurosurgery, Department of Surgery, University of Michigan Medical School, Ann Arbor, Michigan.

Received 9/ 5/68. Accepted 1/30/69.