Effect of Feeding Amino Azo Dyes on Mitochondrial Swelling and Contraction. Kinetic Evidence for Deletion of Membrane Regulatory Sites¹

In previous investigations on the Ca⁺⁺, Hg⁺⁺, and hypotonically induced swelling of rat liver mitochondria while feeding 3′-methyl-4-dimethylaminoazobenzene (3′-Me-DAB), a sharp minimum has been observed at 4 weeks; this 4-week minimum corresponds to the onset of irreversibility of tumor induction under the conditions of administration. The present work establishes that large minima at 4 weeks also occur when glutathione, phlorizin, phosphate, or arsenate are used, which provides support that the occurrence of the minimum at 4 weeks is not related to the nature of the swelling-inducer agent. Also at 4 weeks, impressively large minima are seen in the extent of ATP-produced absorbancy rise in 0.30 m sucrose at pH 4.0, following swelling at pH 7.4 prior to ATP addition. ATP is known to bind optimally to the mitochondrial “structural protein” at pH 4.0. On the other hand, in 0.125 m KCl (pH 7.2) test medium, the 4-week minimum of ATP-produced “contraction” is small or absent. In the latter system the extent of ATP-produced “contraction” gradually decreases beginning at 6–7 weeks and approaches zero in 3′-Me-DAB hepatoma mitochondria.

In kinetic studies the sensitivity of the mitochondrial swelling response has now been defined as the ratio of the maximum swelling velocity to the inducer concentration bringing about half-maximum velocity. This ratio gradually decreases during feeding of 3′-Me-DAB and reaches low levels or zero with mitochondria from 3′-Me-DAB hepatoma (irrespective of the nature of the swelling inducer), indicating the deletion of swelling-inducer receptor sites in the mitochondria during 3′-Me-DAB carcinogenesis. Administration of the comparatively inactive 2-methyl-4-dimethylaminoazobenzene for a 4-week period brings about ratio increases or decreases of various magnitudes depending upon the inducer used.

The level of 0.6 m KCl-extractable mitochondrial protein (hitherto regarded as the contractile entity of the membrane) was unaffected by administration of 3′-Me-DAB or 2-methyl-4-dimethylaminoazobenzene for 10 weeks. A fourfold increase was found in 3′-Me-DAB hepatoma mitochondria, which are comparatively nonresponsive in all swelling and contraction assay systems.