Joseph C. Arcos

Repressible and inducible enzymic forms of dimethylnitrosamine-demethylase

Two enzymic forms, with different kinetic characteristics and responding in opposite ways to in vivo “enzyme inducer” pretreatment, underlie hepatic dimethylnitrosamine(DMN)-demethylase activity. Determination of the Hofstee plot of DMN-demethylase using a DMN substrate concentration range of 0.5 to 200 mM yields three intersecting line segments from which widely different K m and V max values may be calculated. Identically patterned Hofstee plots are obtained with rat and mouse postmitochondrial supernatant fractions, as well as with the isolated microsomes, yielding for the respective segments, similar K m values. The low-substrate-range line segment (0–4 mM) and the high-substrate-range line segment (50–200 mM) correspond, in both the rat and the mouse, to two different enzymic forms of DMN-demethylase (DMN-demethylase I and II, respectively) which have different regulatory characteristics: (A) Pretreatment of rats and mice with the polychlorinated biphenyl, Aroclor 1254, brings about repression of DMN-demethylase I (determined at 4 mM DMN) and induction of DMN-demethylase II (determined at 200 mM DMN); both responses are stronger in the rat than in the mouse. Kinetic experiments show that the differential responses of the two DMN-demethylases to Aroclor pretreatment are paralleled by corresponding changes in the V max values. (B) Pretreatment with phenobarbital represses enzyme I in both species; however, it induces enzyme II only in the rat

Dimethylnitrosamine-demethylase: Absence of increased enzyme catabolism and multiplicity of effector sites in repression. Hemoprotein involvement☆

Evidence is presented that the previously observed decrease of the Vmax of hepatic microsomal demethylation of dimethylnitrosamine (DMN), following pretreatment of rats with 3-methylcholanthrene (MC), is not due to increase in the rate of breakdown but to decrease of de novo synthesis. Determinations of Vmax at time intervals in the transition from the high steady-state level induced by a carbohydrate-devoid casein diet, down to the low steady-state level of carbohydrate-containing basal diet, yielded two consecutive slopes; descent from the basal diet level to the lower steady-state level following pretreatment with MC yielded one slope. Plotting these slopes against the initial Vmax values gave a typical exponential curve (or straight line if the logs of slopes are used) indicating that the rate of enzyme decay in the MC-treated animals is not greater than that expected from normal enzyme catabolism. A multiplicity of effector sites appears to be involved in the repressor action of different structural types; for example, repression by MC (46.6%) and by phenobarbital (23.9%) in combination are approximately additive (62.0%), rather than competitive, indicating that the two agents act at different sites. A P-450 type cytochrome is involved in the demethylation of DMN. DMN-demethylase is inhibited by carbon monoxide, but the susceptibility to CO is far greater than that observed previously with 3,4-benzopyrene hydroxylation; inhibition of DMN-demethylase as a function of CO concentration follows typical enzyme kinetics. However, while both phenobarbital and MC powerfully repress the DMN-demethylase, we have confirmed that they are strong inducers of the synthesis of P-450 and P-448, respectively, as estimated from the difference spectra.

Dimethylnitrosamine‐demethylase: Molecular size‐dependence of repression by polynuclear hydrocarbons. Nonhydrocarbon repressors

Studies with 58 polynuclear aromatic hydrocarbons have shown that to repress demethylation of dimethylnitrosamine (DMN) in rat liver, the hydrocarbons must satisfy specific requirements of molecular geometry regarding size, shape, and coplanarity. Expressing the molecular size of these planar compounds by the two-dimensional area occupied, the size for maximal repressor activity ranges between about 85 and 150 A2. In addition to being within the correct molecular size range the hydrocarbons must have an elongated-rather than compact-molecular shape; circularly shaped and/or highly symmetrical hydrocarbons, such as coronene, triphenylene, ovalene, and tetrabenzonaphthalene, have very low activity or are inactive, in spite of being in the optimum size range. Coplanarity of the molecule is a critical requirement; thus, the potent carcinogen, 9,10-dimethyl-1,2-benzanthracene, is inactive as repressor of DMN-demethylase synthesis. Two exceptions, fluoranthene and benzol[ghi] fluoranthene, showed significant induction of DMN-demethylase. The molecular size distribution of hydrocarbons that repress the DMN-demethylase shows a mirror-image relationship with respect to the earlier reported molecular size requirement for indcution of azo dye N-demethylase. Compounds other than hydrocarbons also show the mirror-image relationship in the sense that pregnenolene-16alpha-carbonitrile, alpha- and beta-naphthoflavone, and Aroclor 1254 (known to be inducers of various mixed-function oxidases) are strong repressors of DMN-demethylase. Aminoacetonitrile, a strong inhibitor of carcinogenesis by DMN, is also a potent repressor of DMN-demethylase. The enzyme is inhibited by pretreatment of the animals with cobaltous chloride, an inhibitor of the synthesis of cytochrome P-450. Pregnenolone-16alpha-carbonitrile and 3-methylcholanthrene, despite their similarity of action on DMN-demethylase, have different effects on azo reductase, which is repressed by the former and induced by the latter compound.

Comparative effects of indole and aminoacetonitrile derivatives on dimethylnitrosamine-demethylase and aryl hydrocarbon hydroxylase activities

The effect of in vivo administration of indole and five 3-indolyl derivatives including L-tryptophan, as well as of aminoacetonitrile and 3 of its derivatives, were studied on the carcinogen-metabolizing hepatic mixed-function oxidases dimethylnitrosamine (DMN)-demethylase I and II and aryl hydrocarbon hydroxylase (AHH). Indole, 3-indolylmethanol, 3-indolyl-acetonitrile, 3-indolylacetone and L-tryptophan induce AHH activity from 3- to 6-fold of the control level, whereas beta-3-indolylethanol has no effect; the latter compound produces a 21% decrease of the endoplasmic reticulum content in the tissue. Only L-tryptophan induces DMN-demethylase I and only L-tryptophan and 3-indolylmethanol induce DMN-demethylase II, representing a doubling of enzyme activity in all 3 instances. Aminoacetonitrile is a potent repressor of DMN-demethylase I. Substitutions on the amino group bring about strong decrease or abolishment of mixed-function oxidase repressor activity; thus, iminodiacetonitrile has only about 1/5th the repressor activity of the parent compound, whereas nitrilotriacetonitrile and dimethylaminoacetonitrile appear to be inactive. Aminoacetonitrile and its derivatives studied have no effect on DMN-demethylase II and AHH activities. The mixed-function oxidase-modifying effects of the indole compounds and of aminoacetonitrile and its derivatives illustrate the potential complexity of effects of dietary constituents on the carcinogenic responses.

Structural limits of specificity of methylcholanthrene-repressible nitrosamine N-dealkylases. inhibition by analog substrates

The dealkylation of dsimethyl-, diethyl- and dipropylnitrosamine by hepatic microsomes of Sprague-Dawley rats is repressed by pretreatment of the animals with 3-methylcholanthrene (MC), and this repression progressively decreases with the increase of alkyl chain length. In contrast to its effect on the demethylation of dimethylnitrosamine (DMN), in vivo phenobarbital induces rather than represses the deethylation of diethylnitrosamine. The rates of demethylation of the DMN analog substrates (dimethylformamide, dimethylacetamide, dimethylpropionamide, and dimethylbutyramide), although low as compared to DMN, increase with the acyl chain length. These analogs are potent in vitro inhibitors of DMN demethylation when used in combination with DMN as substrates, and the inhibition decreases with the length of the acyl chain. Dimethylaminoacetone, which corresponds to the insertion of a CH2 group between the N atom and the carbonyl group in dimethylacetamide, is not an in vitro inhibitor of DMN demethylation; the demethylation rates are additive when this compound is used as substrate in combination with DMN. The rate of demethylation of dimethylaminoacetone is substantially higher than the rates of the dimethylacylamides, and is significantly repressed by MC-pretreatment. The rate of demethylation of methylphenylnitrosamine is not influenced by MC-pretreatment; the compound is, however, a potent inhibitor of demethylation when used as substrate in combination with DMN. The demethylation rates of 1,1-dimethylhydrazine (the reduction product of DMN) and dimethylaniline are not influenced by MC-pretreatment; neither do they affect the overall rate of demethylation when used as substrate in combination with DMN.

Mitochondrial ultrastructure and energy transduction in rat heart during progressive thiamine deficiency

SummaryChanges in ultrastructure, mitochondrial respiration and enzyme activities of rat myocardium were studied during progressive dietary thiamine depletion. Pronounced changes in mitochondrial fine structure and enlargement in size and increase in number were seen as early as 1 week. By the 5th week on thiamine-devoid diet the mitochondria were further enlarged, and the cristae were reduced in number and irregularly oriented.During feeding thiamine-deficient (0.7 mg/kg) diet for 10 weeks, the mitochondrial respiratory control index with pyruvate, α-ketoglutarate and glutamate (in the oxidation of which lipothiamide pyrophosphate is involved) decreased throughout the experiment, while respiratory control with β-hydroxybutyrate and succinate decreased only in the late stage. All changes in respiratory control resulted from decrease of State 3 respiration while the State 4 respiration remained at the control level. The ADP:O ratios with all five substrates were unchanged during the whole course of thiamine depletion.There was a pronounced decrease in activity of pyruvate kinase from heart muscle after 2 weeks of thiamine deficiency with a large minimum at 3–5 weeks and a return to normal by 8–10 weeks. The activity of cardiac α-glycerophosphate-cytochrome c reductase-in a mirror-image-like manner to the above—increased in activity with a maximum at 3–6 weeks and gradually decreased thereafter.Using NADH2 as substrate, the respiratory rate of normal rat heart mitochondria was enhanced by addition of dihydroxyacetone phosphate. This difference disappeared when EDTA, a competitive inhibitor of mitochondrial α-glycerophosphate dehydrogenase (glycero-phosphate oxidase), was present.