George A. Blondin

Mammalian mitochondria as in vitro monitors of water quality.

To measure acute toxicity effectively, a system must provide a simple, sensitive, and rapid measurement of physiological parameters which are indicative of overall organism (bacteria) or organelle (mitochondria) viability. To detect a broad spectrum of toxicants, the parameters should be associated with a major metabolic process controlled by interdependent enzyme systems. In the bioassay reported here, phosphorylating submitochondrial particles (SMP), prepared by sonic disruption of the heavy fraction of intact bovine heart mitochondria serve as in vitro monitors of aquatic toxicity. The bioassay is based on the phenomenon of energy-coupled reverse electron transfer (RET), discovered in plant mitochondria, and later explored using SMP preparations from mammalian sources. RET responses permit rapid, simple, and sensitive measurement of acute toxicity by spectrophotometric recording of the rate of NAD reduction. The criterion of toxicity in this test is inhibition of NAD reduction in the presence of toxic substances. The RET reaction was chosen over other methods because responses are easily quantifiable and the energy-coupled RET reaction is competent in reconstituted freeze-dried submitochondrial particles. Thus, SMP - a stable biological preparation- can be distributed to other laboratories. This feature is important in selecting a routine bioassay.

The mechanism of mitochondrial swelling: II. Pseudoenergized swelling in the presence of alkali metal salts

Abstract Alkali metal salts can induce large amplitude swelling of mitochondria in absence of either electron transfer or hydrolysis of ATP. This type of swelling is referred to as pseudoenergized swelling. The requirements for pseudoenergized swelling in salt media have been systematically investigated. The ammonium, sodium, and lithium salts of weakly dissociating acids (e.g., acetate, phosphate, arsenate, phenylacetate) are competent in the induction of rapid swelling when present at 0.15 m concentration whereas the potassium, cesium, and rubidium salts of the same series of acids, at the same concentration, are not competent. The incompetent salts can become competent in presence of one or another reagent of the peptide-antibiotic family (e.g., valinomycin, gramicidin, nigericin). These reagents facilitate selectively the entry of appropriate alkali metal salts into or through the mitochondrial membranes; this facilitation overcomes the distinction between competent and incompetent alkali metal salts. The chlorides of alkali metals as a class are incompetent in inducing pseudoenergized swelling but again the presence of reagents of the peptide-antibiotic family can confer competence. Thus, competence is a function of the rate of penetration of the mitochondrial membranes by the salts in question. Gramicidin shows little or no discrimination with respect to the nature of the alkali metal ion or the nature of the anion in respect to its facilitating action; valinomycin shows some discrimination with respect to the nature of the alkali metal ion; and nigericin is specific for alkali metal salts of weak acids. Cadmium ions at 10 −4 m can duplicate the action of gramicidin in facilitating the induction of pseudoenergized swelling by alkali metal chlorides. This facilitation by cadmium does not extend to the alkali metal acetates. Calcium ions at 10 −4 m also have gramicidin-like action but this action is manifest only after the ions in question have been translocated into the mitochondrion in an energized process. Once inside the mitochondrion, calcium ions have the same capability as does cadmium for potentiating pseudoenergized swelling induced by alkali metal chlorides. Hypotonic swelling is viewed as a special case of pseudoenergized swelling in which the induction is achieved not by exogenous salt but rather by salts contained within the mitochondrion. Three factors play a paramount role in pseudoenergized swelling: (1) the Donnan effect that provides the driving force; (2) the interaction of salt with groups in the membrane that leads to the conformational changes intrinsic to swelling; and (3) the permeability of the membrane that determines which salts are competent in inducing swelling and which are not. Solutes that do not penetrate the mitochondrial membranes can suppress pseudoenergized swelling.

Assessment of chemical toxicity using mammalian mitochondrial electron transport particles

New spectrophotometric bioassay procedures have been developed for evaluating chemical toxicity, using electron transport particles isolated from bovine heart mitochondria, based on the ability of many toxic chemicals to interfere with the integrated function of electron transport enzymes. The sensitivity of the mitochondrial assays is compared to published sensitivities of otherin vivo andin vitro toxicity testing methods. Regression analysis of logarithmically transformed toxicity values for 42 chemicals, including 8 pesticides, 5 drugs, 6 metals, 8 alcohols, 5 respiratory inhibitors, 4 phenols, and 2 phthalates, indicates excellent correlation between the sensitivity of the new assays and the sensitivity of mammalian cytotoxicity studies (r2 =0.86). Data from aquatic exposure toxicity tests conducted in fish are also highly correlated with the mitochondrial assay results (r2=0.79). However, correlation of data from these methods with median lethal dose studies conducted in rats is not as good because of the inability ofin vitro and aquatic exposure analyses to account for the gastrointestinal absorption, hepatic metabolism, and excretion processes which modify toxic responses following oral administration.

Use of submitochondrial particles for prediction of chemical toxicity in man

The mitochondrial tests are good indicators of the relative toxicity of many xenobiotics to cells and fish. The current investigation studies the ability of these procedures to predict in vivo tissue concentrations associated with clinical illness in man

Mechanism of mitochondrial swelling: III. Two forms of energized swelling

Abstract The characteristics of energized swelling of beef heart mitochondria are described and compared with those of pseudoenergized swelling. Both processes are ultimately dependent upon ion gradients. The essential difference between these two types of swelling resides in the way the ion gradient is generated. In pseudoenergized swelling in sucrose-free media containing certain alkali metal salts (e.g., sodium acetate), the salts readily penetrate the mitochondrion by passive or facilitated diffusion and achieve an equilibrium distribution determined by the Dorman effect. As a result of this equilibration and Donnan distribution, swelling takes place. In energized swelling in a sucrose-free medium containing appropriate alkali metal salts (e.g., potassium acetate), swelling is also the result of the equilibration of salts coupled with the resulting Donnan ion distribution. However, in the latter case the penetration of the cristael membrane by salts is achieved by energy dependent translocation rather than by passive or facilitated diffusion. In energized swelling in sucrose media, the salt must be concentrated interiorily by an active transport mechanism. Translocation of ions thus leads to accumulation of sufficient salt in the osmotically active space to provide an osmotic gradient in excess of that imposed by the sucrose medium, and swelling takes place. Energized swelling in sucrose-free media can be arrested by addition of uncouplers or of reagents which inhibit the energizing process. Energized swelling in sucrose-containing media is not only arrested, but also the process is reversed by the addition of these reagents. Pseudoenergized swelling is neither arrested nor reversed by this means. However, imposition of an external osmotic gradient by addition of sucrose can reverse pseudoenergized swelling and can reverse energized swelling in a sucrose-free medium which has been arrested by addition of respiratory inhibitors.

Isolation of a divalent cation ionophore from beef heart mitochondria

Summary A divalent cation ionophore has been isolated from a partial tryptic digest of mercurial treated-lipid depleted mitochondria by extraction with butanol:acetic acid:water (4:1:5). The partially purified ionophore interacts with both calcium and magnesium ions and facilitates their transfer across the mitochondrial inner membrane or across a carbon tetrachloride phase separating two discontinuous aqueous compartments. The mitochondrial divalent cation ionophore shares many of the properties of previously reported divalent cation ionophores of microbiological origin, e.g., X537A and A23187.

Resolution of the mitochondrial N,N′-dicyclohexylcarbodiimide binding proteolipid fraction into three similar sized proteins

Summary Three chloroform-methanol soluble proteins have been resolved by reverse phase HPLC of a mitochondrial proteolipid fraction from Sephadex LH-60 containing predominantly hydrophobic proteins of less than 10,000 daltons. While all three of the purified proteins comigrate during SDS-urea gel electrophoresis, amino acid analyses of the purified components reveal major differences by which each can be easily distinguished. The three proteins occur in yields of 1.58, 0.28, and 0.21 nmoles per mg protein respectively for proteins α, β, and γ. Only hydrophobic protein α is isolatable with N,N′-dicyclohexylcarbodiimide covalently bound, whereas hydrophobic proteins β and γ bind significant levels of ADP and inorganic phosphate.

The isolation and properties of a peptide ionophore from beef heart mitochondria.

Summary An ionophoretic peptide has been obtained from beef heart mitochondria by extraction with organic solvents. The purified peptide interacts with both sodium and potassium ions and facilitates the transport of these ions across the mitochondrial inner membrane. The similarity in behavior between this endogenous ion carrier and certain depsipeptide antibiotics (e.g., gramicidin-D and valinomycin) suggests that the mechanism of monovalent cation permeation in mitochondrial membrane systems is accountable in terms of endogenous ion carriers analogous to known, microbiologically derived ionophores.

The mechanism of mitochondrial swelling: IV. Configurational changes during swelling of beef heart mitochondria

Abstract Heavy beef heart mitochondria (HBHM) will swell when energized by electron transfer or hydrolysis of ATP in media of appropriate alkali metal salts (energized swelling). Under certain conditions they will also swell in presence of appropriate salts when energized conditions are excluded (pseudoenergized swelling). The configurational changes that underlie both types of large amplitude swelling have been described in terms of several component ultrastructural stages as evidenced by electron microscopic examination. Swelling of either type is characterized by the rapid transition of the mitochondrial inner membrane from the nonenergized to the energized-twisted configuration via the energized configuration. The terminal stages of swelling involve a sequence of configurational changes by which the tubules of the energized-twisted state are expanded and eventually incorporated by coalescence into one vesicular membrane. The rupture of the outer boundary membrane which occurs during the stage of expansion leads to the discharge of the swollen vesicle(s). The sequence and nature of the ultrastructural changes in large amplitude swelling appear to be identical whether the swelling is active, i.e. energized by electron transfer or ATP, or is passive as is the case in the presence of appropriate alkali metal salts.

The mechanism of mitochondrial swelling. V. Permeability of mitochondria to alkali metal salts of strong acid anions

Mitochondria do not swell appreciably when suspended in media containing the chlorides or bromides of alkali metal or ammonium ions. On the other hand, extensive swelling takes place when mitochondria are suspended in ammonium or sodium acetate. These findings have been widely interpreted to mean that the mitochondrial membrane is impermeable to chloride and bromide ions. However, the resistance of the mitochondria to volume changes is not necessarily a valid criteria of impermeability to a given ion pair. Such a conclusion presumes the as yet untested assumptions that (1) permeability to the ion pair is pair is always the rate-limiting step in swelling, and (2) permeability to the ion pair is equivalent to the driving force for water influx. We have conducted experiments addressed to the question of mitochondrial permeability by methods (tracer exchange diffusion) which are independent of volume changes. Our findings indicate that the mitochondrial membrane is very readily penetrated by alkali metal chloride and bromide salts. Further, we have concluded that the resistance to swelling in such media is associated with a lack of driving force.