STUDIES ON CELL METABOLISM AND CELL DIVISION : V. CYTOCHROME OXIDASE ACTIVITY IN THE EGGS OF ARBACIA PUNCTULATA

1. An enzyme capable of oxidizing reduced cytochrome c (i.e. a cytochrome oxidase) has been obtained from Arbacia eggs. In 0.02 M hydroquinone, the cytochrome oxidase was half activated at a cytochrome c concentration of approximately 4 x 10^–6 M. The concentration of the cytochrome oxidase was found to be nearly the same in unfertilized and fertilized eggs, the amount of the enzyme—as measured by means of its activity toward cytochrome c as a representative substrate—being more than sufficient to account for the highest rate of oxygen utilization yet observed in the intact, living, fertilized eggs, and of the same order as that in certain rat tissues. 2. The Arbacia cytochrome oxidase was strongly inhibited by carbon monoxide in the dark, the inhibition being almost completely reversed by light. The inhibition constant was not greatly altered by variation in the concentration of cytochrome c or the concentration of hydroquinone used as reductant for the cytochrome c, having a value of 3 to 5 under the conditions used. The inhibition constant was about 2 with p-phenylenediamine as reductant for the cytochrome c, but apparently had the surprisingly low value of about 0.5 with 0.02 M cysteine as reductant. 3. The cytochrome oxidase was completely inhibited by sufficiently high concentrations of sodium cyanide, sodium azide, and sodium sulfide. It was also completely inhibited in 0.6 M sodium chloride. It was not inhibited by two inhibitors of copper containing enzymes, 8-hydroxyquinoline and sodium diethyldithiocarbamate. It was also not significantly inhibited by 2,4-dinitrothymol, 2,4-dinitro-o-cyclohexylphenol, phenylurethane, 5-isoamyl-5-ethylbarbituric acid, or iodoacetic acid. 4. Quantitative examination of the fertilized eggs showed that cytochrome c, if present at all, occurred in a concentration of less than 2 micrograms per gram of wet fertilized Arbacia eggs. On the basis of these data and those of Fig. 2, above, it seems safe to conclude that cytochrome c cannot carry a significant fraction of the oxygen consumption of fertilized Arbacia eggs. It was also found that, in contrast to similar preparations from certain other animal tissues, the Arbacia cytochrome oxidase preparation displayed no succinic dehydrogenase activity when tested manometrically in the presence of excess cytochrome c. 5. Extending previously reported (3) experiments with other inhibitors, the effects of sodium azide and sodium sulfide on the respiration and cell division of fertilized Arbacia eggs were determined, the eggs being initially exposed to the reagents 30 minutes after fertilization at 20°C. With either reagent cleavage was completely blocked by a concentration of reagent which reduced the respiration to approximately 50 per cent of the normal level. 6. On the basis of certain theoretical considerations regarding the possible mechanism of action of cyanide and other respiratory inhibitors it is suggested that a fraction of the respiration apparently concerned with supplying energy for division processes in the fertilized Arbacia egg may be keyed into the respiratory cycle through a carrier having a somewhat higher potential than those which carry the larger portion of the egg respiration. The theory is also employed in an effort to resolve a number of hitherto apparently paradoxical observations regarding the effects of cyanide, azide, and carbon monoxide on cell respiration.     

STUDIES ON CELL METABOLISM AND CELL DIVISION : II. STIMULATION OF CELLULAR OXIDATION AND REVERSIBLE INHIBITION OF CELL DIVISION BY DIHALO AND TRIHALOPHENOLS

The dihalo and trihalophenols, and phenols containing both halo and nitro substituents in the same molecule, produce, in fertilized eggs of Arbacia punctulata, a rise in rate of oxygen consumption and a reversible block to cell division. To define the conditions which affect the degree of this activity, the following factors have been varied: the arrangement of substituents in the molecule, the concentration of reagent, and the time after fertilization at which the reagent is added. The stimulation of oxygen consumption and reversible block to cell division produced by the dihalophenols are qualitatively the same as those previously produced in fertilized Arbacia eggs by certain dinitrophenols. To yield optimum respiratory effect and maximum division block, it usually requires a higher concentration of dihalo than of the corresponding dinitrophenol. For example, with fertilized Arbacia eggs at 20°C. 2,4-dinitrophenol, in optimum concentration of 3 x 10^–5 molar, raises oxygen consumption to 292 per cent of normal (4). The corresponding values for two dihalo analogues are: 2,4-dichlorophenol, 10^–4 molar and 236 per cent; 2,4-dibromophenol, 6 x 10^–5 molar and 282 per cent. The halophenols differ from the nitrophenols in two interesting respects: (a) The monohalophenols produce little or no oxidative stimulation or division block in fertilized Arbacia eggs; p-nitrophenol is very active in both respects. (b) The symmetrical trihalophenols have an appreciable ability to stimulate oxygen consumption and block division; symmetrical trinitrophenol is inactive in both respects (4). The increases in oxygen consumption produced in fertilized Arbacia eggs by 2,4-dichloro and 2,4-dinitrophenol are larger than the percentage increases given by methylene blue and o-cresol indophenol under the same experimental conditions. The dihalo and dinitrophenols produce a reversible block to the cell division of fertilized marine eggs. The oxidation-reduction indicators, in contrast to the dihalo and dinitrophenols, block cell division irreversibly and fertilized eggs of Arbacia do not recover from optimum respiratory stimulating concentrations of these oxidation-reduction dyes. The present experiments with halophenols are in harmony with and lend considerable support to the hypothesis (4) that nitro and similarly substituted phenols derive their biological activity from the presence and properties of the phenolic OH group, as modified by proper substitution in the phenolic benzene ring.     

STUDIES ON CELL METABOLISM AND CELL DIVISION : III. OXYGEN CONSUMPTION AND CELL DIVISION OF FERTILIZED SEA URCHIN EGGS IN THE PRESENCE OF RESPIRATORY INHIBITORS

1. The effects of a number of respiratory inhibiting agents on the cell division of fertilized eggs of Arbacia punctulata have been determined. For eggs initially exposed to the reagents at 30 minutes after fertilization at 20°C., the levels of oxygen consumption prevailing in the minimum concentrations of reagents which produced complete cleavage block were (as percentages of the control): In 0.4 per cent O₂-99.6 per cent N₂, 32; in 0.7 per cent O₂-99.3 per cent CO, 32; in 1.6 x 10^–4 M potassium cyanide, 34; in 1 x 10^–3 M phenylurethane, 70; in 4 x 10^–3 M 5-isoamyl-5-ethyl barbituric acid, 20; in 3 x 10^–4 M iodoacetic acid, 53. 2. The carbon monoxide inhibition of oxygen consumption and cell division was reversed by light. The percentage inhibition of oxygen consumption by carbon monoxide in the dark is described by the usual mass action equation with K, the inhibition constant, equal to approximately 60, as compared to values of 5 to 10 for yeast and muscle. In 20 per cent O₂-80 per cent CO in the dark there was a slight stimulation of oxygen consumption, averaging 20 per cent. 3. Spectroscopic examination of fertilized and unfertilized Arbacia eggs reduced by hydrosulfite revealed no cytochrome bands. The thickness and density of the egg suspension was such as to indicate that, if cytochrome is present at all, the amount in Arbacia eggs is extremely small as compared to that in other tissues having a comparable rate of oxygen consumption. 4. Three reagents poisoning copper catalyses, potassium dithio-oxalate (10^–2 M), diphenylthiocarbazone (10^–4 M), and isonitrosoacetophenone (2 x 10^–3 M) produced no inhibition of division of fertilized Arbacia eggs. 5. These results indicate that the respiratory processes required to support division in the Arbacia egg may perhaps differ in certain essential steps from the principal respiratory processes in yeast and muscle.     

Action of nitro- and halophenols upon oxygen consumption and phosphorylation by a cell-free particulate system from arbacia eggs

1. The ability of 4,6-dinitrocresol and eight other substituted phenols to stimulate oxygen uptake and inhibit phosphorylation by a cell-free particulate system from unfertilized Arbacia eggs has been determined. Five of those agents can produce both stimulation of oxygen consumption and inhibition of phosphorylation; one inhibits both oxygen consumption and phosphorylation; and two have no effect on either oxygen consumption or phosphorylation. In every case the effects of these substituted phenols upon the cell-free particulate systems parallel those upon oxygen consumption and cleavage in the intact fertilized Arbacia eggs. 2. The data suggest that energy for cleavage of the Arbacia egg is provided at least in part by oxidative phosphorylation and that substituted phenols may block cleavage by interfering with generation and transfer of high-energy phosphate groups.     

STUDIES ON CELL METABOLISM AND CELL DIVISION : VI. OBSERVATIONS ON THE GLYCOGEN CONTENT,CARBOHYDRATE CONSUMPTION,LACTIC ACID PRODUCTION,AND AMMONIA PRODUCTION OF EGGS OF ARBACIA PUNCTULATA

1. Under the present conditions of experiment, Arbacia eggs were found to contain an average of 110 mg. of acid-hydrolyzable carbohydrate (calculated as glucose) per gm. of egg protein. This carbohydrate was almost all in the egg proper, little or none being found in the jelly. To permit conversion of the data to other bases of reference the relation of nitrogen content to wet and dry weight and to egg number were determined. The eggs were found to contain 23.9 per cent solids, 0.10 mg. nitrogen per mg. dry weight, and 5.93 mg. nitrogen per 10⁶ cells. From these results, about 7 per cent of the egg dry weight is acid-hydrolyzable carbohydrate and about 65 per cent is protein. 2. Approximately one-half of the total acid-hydrolyzable carbohydrate was isolated in the form of an alkali-stable, alcohol-precipitable carbohydrate. This substance gave a typical glycogen color test with iodine, yielded glucose on acid hydrolysis, and had, within the limits of experimental error, the same optical rotation as glycogen from other animal sources. Since known amounts of glycogen were completely recovered when carried through the isolation process, the nature of one-half of the acid-hydrolyzable carbohydrate of Arbacia eggs remains undetermined. 3. In order to gain some estimate of the extent to which Arbacia eggs utilize their total carbohydrate for development, determinations of the oxygen consumption, respiratory quotient, carbohydrate consumption, lactic acid production, and ammonia production were made. While all samples of eggs were found to utilize carbohydrate from the 15th to the 24th hours of development at 20°C., certain samples of eggs consumed little or no carbohydrate from the 1st to the 6th hours, the period during which cell division proceeds most rapidly. In a number of instances where carbohydrate breakdown was lacking, a substantial proportion of the oxygen consumption could be accounted for on the basis of processes involving oxidation of protein or protein breakdown products.     

THE EFFECTS OF CAFFEINE ON OXYGEN CONSUMPTION AND CELL DIVISION IN THE FERTILIZED EGG OF THE SEA URCHIN,ARBACIA PUNCTULATA

1. By means of the Warburg-Barcroft microrespirometer apparatus and the Warburg direct method, the relative effect of caffeine upon the O₂ consumption of the fertilized egg of Arbacia punctulata was shown for the following concentrations in sea water: 0.002 per cent (M/10,000), 0.004 per cent (M/5,000), 0.02 per cent (M/1,000), 0.1 per cent (M/200), 0.2 per cent (M/100), 0.5 per cent (M/40), and 2 per cent (M/10). 2. In comparison with the normal eggs (uninhibited, non-caffeine-treated controls), caffeine in concentrations including and greater than 0.1 per cent (M/200) depressed the average uptake from approximately 25 to 61 per cent over the 3 hour period. In a number of instances, as typified by Experiment 10, the effective inhibitory concentration ranged from 0.02 per cent (M/1,000) upward and the degree of depression of the O₂ consumption ranged from 10.6 per cent to 60.6 per cent. 3. All caffeine concentrations including and above 0.02 per cent (M/1,000) in the series used, resulted in decreasing the normal rate of cleavage division in the fertilized Arbacia eggs. 4. The higher concentrations (0.5 and 2 per cent) produced a complete blockage of the cleavage process. 5. Complete cleavage inhibition was noted only when the O₂ uptake had been depressed to 50 per cent or more of the normal controls. 6. O₂ consumption-time relationship data indicate an average depression, in O₂ consumption over a 3 hour period, ranging from 25 per cent with a caffeine concentration of 0.1 per cent to a 61 per cent inhibition with a concentration of 2 per cent. 7. Concentrations of less than 0.1 per cent (certainly of less than 0.02 per cent) give variable results and indicate no significant effect. 8. It is inferred from the respiration data presented that it is probable that the inhibition of the O₂ consumption in fertilized Arbacia eggs is due to the influence of caffeine upon the main (activity or primary) pathway. It will be observed that there are certain similarities of the caffeine data to the degree of inhibition accomplished by sodium cyanide. Moreover, it has been demonstrated that the cyanide probably acts on the cytochrome oxidase step in the cytochrome oxidase-cytochrome chain of reactions constituting the O₂ uptake phase of respiratory metabolism. It is not improbable, therefore, that caffeine also may act upon the cytochrome oxidase enzyme. 9. From the viewpoint of environmental conditions influencing reproductive phenomena, it is of interest that caffeine can affect the normal metabolism of the zygote.     

THE PYRUVATE METABOLISM OF SEA URCHIN EGGS DURING THE PROCESS OF CELL DIVISION

The eggs of Arbacia and starfish contained about 70 and 25 micrograms of pyruvate per gm. of dry cells respectively. Arbacia eggs utilized added pyruvate, although the O₂ uptake did not increase. On fertilization the utilization of pyruvate increased sevenfold. This pyruvate seems to be metabolized, as in other cells, with diphosphothiamine as coenzyme. The diphosphothiamine content of fertilized and non-fertilized eggs was about 16 micrograms; that of sperm, 30 micrograms. Penetration of sperm into the egg and fertilization with cell division to the pluteus stage did not bring forth appearance of succino-dehydrogenase. The possible mechanism of fertilization and cell division is discussed.     

STUDIES ON CELL METABOLISM AND CELL DIVISION : VIII. THE DIPHOSPHOPYRIDINE NUCLEOTIDE (COZYMASE) CONTENT OF EGGS OF ARBACIA PUNCTULATA

1. The diphosphopyridine nucleotide content of Arbacia eggs has been measured manometrically and found to be approximately 250–500 micrograms per gm. wet weight of eggs, the value varying with individual egg samples and with the state of development of the eggs. Of the total diphosphopyridine nucleotide present, approximately 25–40 per cent is in an alkali-stable, presumably the dihydro, form. 2. Tests for triosephosphate and glutamic acid dehydrogenases carried out on Arbacia egg cytolysates were negative.     

THE EFFECTS OF URETHANE AND CHLORAL HYDRATE ON OXYGEN CONSUMPTION AND CELL DIVISION IN THE EGG OF THE SEA URCHIN,ARBACIA PUNCTULATA

The effects of a series of concentrations of the narcotics, ethyl carbamate and chloral hydrate, have been determined on the consumption of oxygen by fertilized and unfertilized eggs of the sea urchin Arbacia punctulata. In the fertilized eggs the effects of the two inhibitors on cell division were also examined. The following observations were made: 1. Assuming that the narcotic acts upon a single catalyst in the unfertilized egg the degree to which the consumption of oxygen is inhibited in this resting cell can be related to the narcotic concentration by an expression derived from the law of mass action. 2. To account for the relation between the concentration of the narcotic and its effect on respiration in the fertilized eggs, it is necessary to conclude that in them the narcotic acts on two parallel respiratory systems. The experimental data can be quantitatively predicted (1) if the reaction of the narcotic on the two systems is governed by the law of mass action and (2) if 40 per cent of the oxygen consumption is mediated by one system, the "activity" system, and the remainder by the other, the "resting" or "basal" system. 3. The mass law constants applying to the resting system in the fertilized egg are similar to those for the single system functioning in the unfertilized egg so that these two respiratory systems are probably identical. 4. The concentrations of the narcotics just sufficient to abolish cell division affect primarily the activity system, the existence of which was inferred from the respiratory experiments. It is concluded that normal cell division requires specifically the normal function of the activity system, that in fact the energy for cell division is made available through that system.     

STUDIES ON CELL METABOLISM AND CELL DIVISION : VII. OBSERVATIONS ON THE AMOUNT AND POSSIBLE FUNCTION OF DIPHOSPHOTHIAMINE (COCARBOXYLASE) IN EGGS OF ARBACIA PUNCTULATA

1. Methods suitable for the determination of diphosphothiamine (cocarboxylase) in eggs of Arbacia punctulata have been developed. Quantitative extraction of the cocarboxylase was effected by combining the use of thiamine hydrochloride in the extraction fluid with critical adjustment of the pH of extraction to pH 6.3–6.7. 2. The unfertilized eggs were found to contain the equivalent of 2 to 3 micrograms of natural yeast cocarboxylase per gm. of wet eggs; the cocarboxylase content of the 30 minute and 10 hour fertilized eggs was somewhat less (Table III). 3. In preliminary experiments, Arbacia egg cytolysates were found to cause pyruvic acid to disappear. The rate of such disappearance was apparently greater under aerobic than under anaerobic conditions; it was also greater for cytolysates from fertilized eggs than for cytolysates from unfertilized eggs (Table IV).     

THE EFFECT OF URETHANE ON THE CONSUMPTION OF OXYGEN AND THE RATE OF CELL DIVISION IN THE CILIATE TETRAHYMENA GELEII

1. The inhibition of oxygen consumption produced by a series of concentrations of ethyl carbamate has been measured in the protozoan Tetrahymena geleii. 2. The relation found between the narcotic concentration and its effect on respiration leads to the conclusion that urethane has two distinct modes of action in this cell. The respiratory data can be accurately predicted by assuming that the inhibitor acts on two independent parallel respiratory systems. 3. Complete suppression of cell division in this organism is brought about by approximately 0.1 M urethane. 4. Urethane concentrations up to 0.1 M affect primarily only one of the two postulated respiratory systems. The mechanism of the narcosis of cell division in this organism by urethane thus appears to be inhibition of this "activity" system.     

ACTION OF PAPAIN AND FICIN ON TADPOLES AND ARBACIA EGGS

Living tadpoles and Arbacia eggs are not digested by ficin or papain although the dead organisms are. Arbacia eggs develop in papain solutions but the cells become separated. Development is normal in ficin and trypsin solutions.     

Studies in the Principles of Phytotoxicity: III. THE pH FACTOR AND THE TOXICITY OF 3:5-DINITRO-o-CRESOL, A WEAK ACID

It has previously been shown that the concentration of 3:5-dinitro-ocresolrequired in the external medium to halve (a) the growth rateof the mould Trichoderma viride or (b) the respiration rateof yeast increased same 75–100 times as the pH changedfrom 4 to 7. In the present investigation it has been foundthat when solutions or suspensions of 3:5-dinitro-o-cresol areapplied as sprays to seedlings of Brassica alba the concentrationneeded to kill half the plants changes by a factor of less than2 over the same pH range. On the other hand, when Lemma minor is grown in nutrient solutioncontaining the toxicant, the concentration required to halvethe rate of frond multiplication increases sharply as the pHis raised from 5.4 to 6.8. Moreover, when leaf disks of B. albaare vacuum infiltrated with buffered solutions of 3:5-dinitro-o-cresolthe concentration needed to halve the respiration rate rises50-fold between pH 4 and 8. It is considered that the essential condition which determinesthe similarity of the result for L. minor, the leaf disks, andmicro-organisms is that a relatively large volume of solutioncontaining the toxicant is in direct contact with the tissues.In contrast, under the conditions of spraying the volume ofspray droplets relative to that of plant tissue is small. Thusthe pH effect ie masked since the buffering capacity of thecells exceeds that of the droplets, with the result that 3:5-dinitro-o-cresollargely acts upon the cells at a pH determined by the tissues,a pH which may bear little relation to that of the originalsolution. Supporting evidence is produced that the pH factor operatesin the same way for other weak acids, such as the chlorophenoxyaceticacids.     

A NOTE ON THE RESPIRATION OF ARBACIA EGGS

Methods used in preparing Arbacia eggs for respiration studies, in carrying through the manometric determinations, and in estimating egg quantities have been reexamined. Discrepancies in previous results are almost entirely due to a steady error in measuring egg volume by centrifuging. Volumes so obtained averaged 80 per cent too high. The respiration of unfertilized eggs of Arbacia punctulata at 21°C. is 0.9 c.mm. O₂ per hour per 10 c.mm. of eggs.     

Dinitrocresol, Cyanide, and the Pasteur Effect in Yeast

At pH 5·0 the respiration of yeast is stimulated by lowconcentrations of 3:5-dinitro-o-cresol, reaching a peak levelof 170 per cent, at 10⁵ M. Concentrations above this inhibitoxygen uptake and cause aerobic fermentation to appear, whichin turn reaches a peak value and is then inhibited. The rateof carbohydrate breakdown, or glycolysis, calculated from therates of respiration and aerobic fermentation, increases steadilyup to 3 x 10^–5 M., at which concentration it is 5 timesfaster than the control: higher concentrations depress the rateof glycolysis. The rate of fermentation under nitrogen is abouttwice that of respiration, and it is inhibited over the sameconcentration range as aerobic fermentation. It was found earlier that oxidative assimilation of glucoseby yeast is progressively inhibited by increasing concentrationsof dinitrocresol, and it is now shown that this parallels theincrease in the rate of aerobic glycolysis. It is argued thatdinitrocresol is here acting as an uncoupling agent and thatboth oxidative assimilation and the rate of glycolysis are controlledby the level of energy-rich phosphate. With cyanide there is no stimulation of oxygen uptake, aerobicfermentation only appears when respiration becomes inhibited,and after an initial slight decrease the rate of glycolysisrises to 575 per cent. of the control value at 5 x 10^–4M. It is suggested that the rate of glycolysis only increaseswhen respiration has been inhibited sufficiently to reduce therate of formation of energy-rich phosphate.     

THE RELATION BETWEEN THE ELECTRICAL CONDUCTIVITY OF THE EXTERNAL MEDIUM AND THE RATE OF CELL DIVISION IN SEA URCHIN EGGS

Dilution of sea water with isotonic sugar solution leaves the rate of cleavage of Arbacia eggs almost unchanged until the proportion of sea water is decreased to 20 or 25 volumes per cent. From this point cleavage becomes progressively slower with further dilution. Many eggs fail to cleave at dilutions of 5 to 6 volumes per cent. No cleavage occurs in 2 volumes per cent sea water or in pure sugar solution. Eggs returned from these media to sea water resume cleavage and development. There is thus no relation between the rate of cleavage and the electrical conductivity of the medium, except possibly within the range of dilutions from 20 to 5 volumes per cent sea water. In this range cleavage rate decreases as conductivity decreases, but the relation is not a linear one.     

THE EFFECT OF 5-BROMODEOXYURIDINE ON DNA REPLICATION AND CELL DIVISION IN TETRAHYMENA PYRIFORMIS

Populations of Tetrahymena pyriformis were grown in a chemically defined medium containing the thymidine analogue 5-bromodeoxyuridine (BUdR). About 65% of the thymidine sites in DNA were substituted by BUdR. During the first generation in the presence of BUdR, all DNA became hybrid. After the following cell division, in about 80% of the cells the second DNA replication round was initiated but no further cell division took place. The cells could be rescued by removing BUdR and adding thymidine. New replication took place before the first cell division. However, although the cells contained double heavy as well as hybrid DNA, only the hybrid DNA was replicated. After a full replication of the hybrid DNA, normal growth was restored. Melting profiles of normal, hybrid, and double heavy DNA indicated a structural change of the double heavy DNA.     

OBSERVATIONS ON CELL GROWTH, MITOSIS, AND DIVISION IN THE FUNGUS BASIDIOBOLUS RANARUM

The paper describes the forward streaming, growth, and division of the vegetative cell of Basidiobolus ranarum. The cell is several hundred microns long and has a single large nucleus. Mitosis is invariably followed by cell division. Both processes have been studied in the living cell by ordinary and phase contrast microscopy. Mitosis is accompanied by a temporary coarsening of the organisation of the cytoplasm and a considerable slowing down of the rate of growth of the cell wall tube. Fixed and stained preparations have shown that there is a large number of small chromosomes and that the mitotic spindle is formed from the nucleolus. Basidiobolus appears suitable for observations on the cell duplication cycle and the physiology of mitosis.     

The action of 4,6-dinitro-o-cresol (DNOC) in soil

Summary The addition of initially poisonous amounts of the herbicide 4,6-dinitro-o-cresol (DNOC) to non-sterilized soil enhances the ultimate emergence percentage from cress seeds, if these are sown some time after the DNOC treatment of the soil.DNOC is removed from soil mainly by microbial activities.The increase in the emergence percentage cannot be ascribed to a stimulation, exerted either by DNOC in any concentration or by some breakdown product of DNOC.The ultimate emergence percentage of cress seedlings in sterilized soil surpasses that in non-sterilized soil.It is suggested that the more complete emergence after DNOC addition to non-sterilized soil is due to a partial sterilization of the soil by DNOC, advantages of which remain preserved after removal of the DNOC by the remainder of the microflora.The consequences of this view for an explanation of the yield-increasing effect of DNOC spraying in agriculture are discussed.     

The deleterious metabolic and genotoxic effects of the bacterial metabolite p-cresol on colonic epithelial cells

p-Cresol that is produced by the intestinal microbiota from the amino acid tyrosine is found at millimolar concentrations in the human feces. The effects of this metabolite on colonic epithelial cells were tested in this study. Using the human colonic epithelial HT-29 Glc^–/+ cell line, we found that 0.8 mM p-cresol inhibits cell proliferation, an effect concomitant with an accumulation of the cells in the S phase and with a slight increase of cell detachment without necrotic effect. At this concentration, p-cresol inhibited oxygen consumption in HT-29 Glc^–/+ cells. In rat normal colonocytes, p-cresol also inhibited respiration. Pretreatment of HT-29 Glc^–/+ cells with 0.8 mM p-cresol for 1 day resulted in an increase of the state 3 oxygen consumption and of the cell maximal respiratory capacity with concomitant increased anion superoxide production. At higher concentrations (1.6 and 3.2 mM), p-cresol showed similar effects but additionally increased after 1 day the proton leak through the inner mitochondrial membrane, decreasing the mitochondrial bioenergetic activity. At these concentrations, p-cresol was found to be genotoxic toward HT-29 Glc^–/+ and also LS-174T intestinal cells. Lastly, a decreased ATP intracellular content was observed after 3 days treatment. p-Cresol at 0.8 mM concentration inhibits colonocyte respiration and proliferation. In response, cells can mobilize their “respiratory reserve.” At higher concentrations, p-cresol pretreatment uncouples cell respiration and ATP synthesis, increases DNA damage, and finally decreases the ATP cell content. Thus, we have identified p-cresol as a metabolic troublemaker and as a genotoxic agent toward colonocytes.