J.A. Brignone

Structural requirements for the action of steroids as inhibitors of electron transfer

Abstract A series of C21, C19, and C18-steroids (including hormonal steroids and derivatives) have been tested as inhibitors of NADH-oxidase and succinate oxidase activities of heart-muscle sarcosomal fragments (Keilin-Hartree preparation). NADH oxidation is much more sensitive to steroids than succinate oxidation, in accordance with electron transfer inhibition in the vicinity of the NADH-flavoprotein site. Steroid activity on NADH-oxidase has been titrated and the 50% inhibitory concentration (I50) of each steroid has been determined. Among the steroids tested, the following were the more effective inhibitors, having I50 values ≦ 1.0 μM 19-norethynyltestosterone acetate, 5-pregnen-3β,20α-diol, 5α-pregnan-3β,20α-diol, 5-pregnen-3β,20β-diol, 5-pregnen-3β,17α,20α-triol, testosterone acetate, testosterone enol diacetate, testosterone benzoate, androstanolone acetate, and 17β-ethynylestradiol benzoate. Comparison I50 values for steroids differing in a single group (or structure) shows that the following structural features promote the inhibitory potency: (a) a trans A B junction; (b) an alcohol or ketone group at C-3 and/or C-17; (c) an acetyl chain at C-17; (d) 17α-methyl or ethynyl groups at C-17 of 17β-hydroxylated steroids; (e) acetylated or benzoylated hydroxyl groups at C-3 or C-17; (f) α configuration of 3-OH groups in 5α-androstane derivatives and β-OH configuration in 5β-pregnane derivatives; (g) α configuration of hydroxyl groups at C-17; (h) an angular β-methyl group at C-10; (i) a hydroxyl group at C-20 of 5α-pregnane and 5-pregnene derivatives. Decreased inhibitory potency is determined by the following structural features: (j) a cis A B junction; (k) Δ1 and Δ4 double bonds; (l) ionizable (hydrophylic) groups at C-3 or C-17; (m) hydroxyl groups at C-17 or C-21 of C21-steroids; (n) relatively large substituents at C-3 or C-17 (hexahydrobenzoate and propionate derivatives); (o) substituents at the skeleton of the steroid molecule (C-2, C-4, C-6, C-11, C-16) irrespective of group-polarity and configuration. Accordingly, the most effective steroid structure to inhibit electron transfer is a planar and narrow hydrocarbon framework with relatively small, adequately orientated polar end-groups and a large intermediate hydrophobic area. These structural characteristics are consistent with the interaction of steroids with the phospholipid components of the electron transfer chain at the NADH-flavoprotein site. Many of the structural requirements for inhibition of electron transfer resemble those for androgenicity.

Modified oscillation behavior and decreased d-3-hydroxybutyrate dehydrogenase activity in diabetic rat liver mitochondria☆

Abstract One month after induction of diabetes in adult white rats with streptozotocin or 4–10 months after its induction by pancreatectomy (in every case glycemia was over 3 g/liter), the following alterations were observed in liver mitochondria: (a) a decrease of amplitude and an increase of the damping factor of volume oscillations induced by potassium ions and valinomycin; (b) a 50% decrease of d -3-hydroxybutyrate dehydrogenase (HBD) activity in mitochondria disrupted by repeated freeze-thawing; (c) a similar decrease in the rate of d -3-hydroxybutyrate oxidation by intact mitochondria; (d) a significant increase of cytochrome oxidase activity and cytochrome aa 3 content. Measurement of succinate dehydrogenase and NADH dehydrogenase activity, the cytochrome b , c 1 , and c content, and the P:O ratio for mitochondria oxidizing d -3-hydroxybutyrate did not reveal significant differences between control and diabetic rat mitochondria. In the streptozotocin-injected rats, the variation of HBD activity and the modification of the mitochondrial oscillation pattern were time-dependent phenomena, both effects reaching their maximal expression about 1 month after the onset of diabetes. The variation of HBD activity followed a biphasic course, since it rose to above the control level during the first 2 weeks of diabetes, then fell progressively to about half the control value after the third week. Treatment of diabetic rats with NPH insulin (5 IU twice daily, for 3 days, reinforced by the same dose 45 min before sacrifice) restored the mitochondrial oscillation pattern, HBD activity, and rate of d -3-hydroxybutyrate oxidation by intact mitochondria to their normal values.

Effect of ovarian hormones upon liver mitochondrial function in diabetic rats

In the present study it is shown that streptozotocin (SZ)-induced chronic diabetes of female albino rats produced significant alterations in liver mitochondrial function after 30-35 days of diabetes. The disturbances were as follows: (1) a significant fall of the mean values of the respiratory control ratio and of state 3 of respiration using three substrates, 3-hydroxybutyrate, malate-glutamate and succinate, and (2) a significant increase of the mean damping factor of the oscillatory osmotic variations (with valinomycin as K+ ionophore and succinate as substrate). The same mitochondrial function parameters were analyzed for comparison in control non-diabetic rats (group N) and in the following groups of female rats with chronic diabetes: intact (group I), oophorectomized (6 days after the injection of SZ) (group O), and oophorectomized with restitution therapy of 17 beta-estradiol (from the operation until the day before killing) (group O + Eol). The O group showed significantly higher values of the respiratory control ratio and of state 3 of respiration and significantly lower damping factors than group I. The restitution treatment in the O + Eol group restored the mitochondrial functions assayed to values similar to those of group I. These data provide strong evidence that estrogens exert a negative effect at the molecular level upon impaired liver mitochondrial functions in SZ-induced diabetes.

Inhibition of succinic dehydrogenase by polysulfonated compounds.

Abstract 1. 1. Among several sulfonated aromatic ureides or diazo compounds, suramin was found to be the most powerful inhibitor of succinoxidase. 2. 2. Suramin inhibits succinoxidase at the level of succinic dehydrogenase, fulfilling the requirements of the theory for competitive inhibition. One molecule of suramin inhibits one enzyme active center. 3. 3. The suramin component 1-naphthylamine-4,6,8-trisulfonate inhibits succinic dehydrogenase competitively, but is less active than suramin. One molecule of sulfonic acid inhibits one enzyme active center. The inhibition diminishes in the presence of phosphate ions. 4. 4. Succinic dehydrogenase inhibition by trivalent arsenicals, and by alkylators and oxidants of thiol groups, is prevented by suramin, hydrolyzed suramin, and naphthylaminetrisulfonic acids. 5. 5. Suramin scarcely affects succinic dehydrogenase inhibition by p -chloromercuribenzoate, p -chloromercuriphenol, and mercuric ions. Succinate, malonate, and oxalacetate are also less effective in the protection of succinic dehydrogenase against mercurials. 6. 6. Suramin slightly diminishes succinoxidase inactivation by 2,3-dimercaptopropanol (BAL). The effect is not significant when compared with the protection of succinic dehydrogenase.

Factors affecting succinoxidase sensitivity toward oxidation with BAL

Abstract The sensitivity of succinoxidase toward oxidation with BAL is exaggerated by antimycin A and diminished by phenylurethan, ethylenediamine tetraacetate, and phosphate ions. None of these agents affect the rate of BAL oxidation. Ethylenediamine tetraacetate and phosphate ions interfere with succinoxidase inhibition by antimycin A. After treatment with BAL, succinoxidase becomes more sensitive toward phenylurethan and antimycin A.