Regulation of p-nitroanisole O-Demethylation in Perfused Rat Liver

Transcription

1 Biochem. J. (1979) 184, Printed in Great Britain 675 Regulation of p-nitroanisole O-Demethylation in Perfused Rat Liver ADENINE NUCLEOTIDE INHIBITION OF NADP+-DEPENDENT DEHYDROGENASES AND NADPH-CYTOCHROME c REDUCTASE By Frederick C. KAUFFMAN,* Roxanne K. EVANS,* Lester A. REINKEt and Ronald G. THURMANt *Department ofpharmacology and Experimental Therapeutics, University of Maryland School of Medicine, 660 W. Redwood Street, Baltimore, MD 21201, U.S.A., and tdepartment of Pharmacology, University ofnorth Carolina School of Medicine, Chapel Hill, NC 27514, U.S.A. (Received 13 June 1979) Perfusion of rat livers with 10mM-fructose or pretreatment of the rat with 6-aminonicotinamide (70mg/kg) 6h before perfusion decreased intracellular ATP concentrations and increased the rate of p-nitroanisole O-demethylation. This increase was accompanied by a decrease in the free [NADP+]/[NADPH] ratio calculated from concentrations of substrates assumed to be in near-equilibrium with isocitrate dehydrogenase. After pretreatment with 6-aminonicotinamide the [NADP+]/[NADPH] ratio also declined. Reduction of NADP+ during mixed-function oxidation may be explained by inhibition of one or more NADPH-generating enzymes. Glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase and 'malic' enzyme, partially purified from livers of phenobarbital-treated rats, were inhibited by ATP and ADP. Inhibitor constants of ATP for the four dehydrogenases varied considerably, ranging from 9,UM for 'malic' enzyme to 1.85mm for glucose 6-phosphate dehydrogenase. NADPH-cytochrome c reductase was also inhibited by ATP (K, 2.8 mm) and by ADP (K; 0.9 mm), but not by AMP. Concentrations of ATP and ADP that inhibited glucose 6-phosphate dehydrogenase and the reductase were comparable with concentrations in the intact liver. Thus agents that lower intracellular ATP may accelerate rates of mixed-function oxidation by a concerted mechanism involving deinhibition of NADPH-cytochrome c reductase and one or more NADPH-generating enzymes. The mixed-function oxidation system in liver requires NADPH and molecular 02 to convert drugs and endogenous lipophiles into more polar products. During the process of mixed-function oxidation stoicheiometric amounts of NADPH and drug substrate are utilized. Thus changes in rates of NADPH formation could alter rates of mixedfunction oxidation, since NADPH supply may be rate-limiting for this system in intact cells (Thurman et al., 1977a). In hepatocytes the consumption of NADPH by the mixed-function oxidase system and generation of NADPH do not appear to be precisely balanced. Oxidation of NADPH has been detected in liver during the mixed-function oxidation of hexobarbital (Sies & Kandel, 1970) and aminopyrine (Thurman & Scholz, 1973), but not with p-nitroanisole (Kauffman et al., 1977). p-nitroanisole also differed from hexobarbital and aminopyrine in its effects on adenine nucleotides. It markedly diminished ATP and increased ADP and AMP in perfused livers, whereas hexobarbital and aminopyrine had no effect on concentrations of these nucleotides (Kauffman et al., 1977). Changes in nicotinamide and adenine nucleotides that accompany mixed-function oxidation have significant effects on intermediary metabolism, as indicated by changes in steady-state concentrations of intermediates associated with glycolysis, the pentose phosphate pathway and citric acid cycle (Sies & Kandel, 1970; Kauffman et al., 1977). A possible link between energy status and rates of NADPH formation may occur at the level of glucose- 6-phosphate dehydrogenase, since this enzyme is inhibited by ATP (Passonneau et al., 1966; Brand et al., 1970; Afolayan, 1972). A decrease in the concentration of ATP in the liver caused by a toxic chemical could theoretically activate metabolism of hexose phosphates via the oxidative enzymes of the pentose phosphate pathway, which in turn could lead to enhanced NADPH generation. In the present work rates of p-nitrophenol formation from p-nitroanisole were examined when intracellular ATP concentrations were depleted experimentally with fructose added to perfusion media or by pretreatment of animals with 6-aminonicotinamide. Altered rates of p-nitroanisole 0-

2 676 F. C. KAUFFMAN, R. K. EVANS, L. A. REINKE AND R. G. THURMAN demethylation were compared with changes in the oxidation-reduction state of NADP+ calculated from substrates assumed to be in near-equilibrium with 'malic' enzyme and NADP+-dependent isocitrate dehydrogenase. In addition, the effects of adenine nucleotides on the activities of four major NADPHgenerating enzymes, glucose 6-phosphate dehydrogenase (EC ), 6-phosphogluconate dehydrogenase (EC ), NADP+-dependent isocitrate dehydrogenase (EC ) and 'malic' enzyme (EC ), as well as NADPH-cytochrome c reductase (EC ), were studied. The results indicate that rates of mixed-function oxidation are enhanced when intracellular concentrations of ATP are lowered possibly by deinhibition of enzymes that generate NADPH and deinhibition of NADPHcytochrome c reductase. A preliminary account of these results has been presented (Evans et al., 1978). Methods Perfusion ofrat liver Female Sprague-Dawley rats ( g) were treated with phenobarbital (1 mg/ml of drinking water) for 2 weeks before perfusion experiments or isolation of hepatic enzymes. Some animals received 6-aminonicotinamide (70mg/kg intraperitoneally) 6h before perfusion. Livers were perfused with Krebs-Henseleit bicarbonate buffer, ph 7.4, as described previously (Thurman et al., 1977a). p-nitrophenol formation from p-nitroanisole was monitored continuously in the haemoglobin-free perfusion medium at 436nm (Thurman et al., 1977a). Glucuronide plus sulphate conjugates of p-nitrophenol were measured in perfusate by incubating portions (1.Oml) at room temperature for 90min with a mixture of 8-glucuronidase and sulphatase from Helix pomatia (Sigma, Louis, MO, U.S.A.). Samples were incubated with 250 Fishman units of f6-glucuronidase, which was sufficient to hydrolyse more than 95 % standards ofp-nitrophenyl sulphate (50.uM) and p-nitrophenylglucuronide (50AuM) added to the perfusate. The p-nitrophenol liberated under these conditions was measured at 436nm. Metabolite measurements Metabolites were determined in HCI04 extracts of livers that had been freeze-clamped with liquid-n2- chilled aluminium tongs. Extracts were prepared and metabolites, except for isocitrate, were determined by direct enzymic procedures (Kauffman et al., 1969; Lowry & Passonneau, 1972). Isocitrate was measured in HCl04 extracts of freeze-clamped livers by an enzymic cycling procedure. Neutralized HC104 extracts equivalent to 1 mg of wet tissue were incubated at room temperature for 30min in 20,pl of reagent containing 0.025M-Tris/ HCI, ph8.0, 0.1mM-MnCI2, 0.005% bovine serum albumin, 0.1 mm-nadp+ and 1 g of isocitrate dehydrogenase/ml. The reaction was stopped and excess NADP+ was destroyed by adding an equal volume of 0.2M-NaOH and heating at 80 C for 30min. NADPH formed from isocitrate was measured in a 25,ul portion of the reaction mixture by enzymic cycling in looul of cycling reagent for NADPH as described by Lowry & Passonneau (1972). Tissue and reagent blanks as well as isocitrate standards were carried through the entire procedure. Isocitrate concentrations ranging between 0.2 and 5,umol/kg of wet tissue could be reliably measured with this technique. Assay ofenzyme activities All analyses of NADP+-dependent dehydrogenases were performed on enzymes partially purified from livers of phenobarbital-treated rats. Livers were homogenized in lovol. of 0.02M-sodium phosphate buffer, ph 7.0. Homogenates were centrifuged at 17000g for 0min to obtain a high-speed supernatant fraction containing the four NADP+-dependent dehydrogenases. Protein fractions precipitating between 0.5M- and 3.0M-(NH4)2SO4 were collected, dissolved in a small volume of 0.02M-phosphate buffer, ph7.0, and dialysed against the same buffer for 4h in a hollow-fibre device (Bio-Rad, Richmond, CA, U.S.A.). A portion of the dialysate containing approx. 10mg of protein was applied to a column (2cmx20cm) of DEAE-cellulose equilibrated with 0.02M-sodium phosphate buffer, ph7.0. The four NADP+-dependent dehydrogenases were eluted with a linear gradient of NaCl ( M) in 0.02M-sodium phosphate buffer, ph 7.0. All four activities were purified approx. 15-fold and were recovered in yields of about 50%. The activities of the four NADP+-dependent dehydrogenases were assayed fluorimetrically (Harkonen & Kauffman, 1974). Substrates and cofactor concentrations were varied as indicated in Figures and legends. NADPH-cytochrome c reductase was determined in microsomes isolated from phenobarbital-treated rats as described previously (Strobel & Dignam, 1978). Results Effects offructose on hepatic ATP concentrations and on rates ofp-nitrophenolformationfromp-nitroanisole Previous workers (Maenpaa et al., 1968; Burch et al., 1970; Woods et al., 1970) have shown that fructose lowers hepatic ATP by rapid formation of fructose 1-phosphate via ketohexokinase. We have therefore used fructose to investigate the relationship 1979

3 p-nitroanisole O-DEMETHYLATION IN PERFUSED RAT LIVER 677 Table 1. Effect offructose on adenine nucleotides and [NA DP+]/[NA DPH] ratios in perfused liversfrom starvedphenobarbitaltreated rats Values are means + S.E.M. for the numbers of livers indicated in parentheses. Livers were perfused with either Krebs- Henseleit buffer alone or buffer containing 10mM-fructose for 8min before being clamped with aluminium tongs chilled to liquid-n2 temperature. Livers were perfused with Krebs-Henseleit buffer containing 0.2mM-p-nitroanisole before infusion of fructose. Before infusion ofp-nitroanisole, ATP concentrations were #mol/kg wet wt., and [ATPJ/(ADPJ ratios were [NADP+]/[NADPH] ratios were calculated from concentrations of pyruvate, *Results significantly different from controls, P<0.05; **results significantly malate, a-oxoglutarate and isocitrate. different from controls, P< Control (3) Fructose (5) 102 x [NADP+]/[NADPH] Concentration (,umol/kg wet wt.) 'Malic' Isocitrate ATP ADP AMP [ATP]/[ADP] enzyme dehydrogenase ** ± ** * 0 6 *;: : noo 0 E 0. 1 z 1 L p-nitroanisole Fructose tl_ s X S'~~~~~~~~~i I Time of perfusion (min) Fig. 1. Effect offructose on 0-demethylation of p-nitroanisole in isolated perfused livers from phenobarbitaltreated starved rats Livers were perfused with Krebs-Henseleit bicarbonate buffer in a non-recirculating system. The broken line indicates total p-nitrophenol production (free plus conjugated) determined after incubations of perfusate with p-glucuronidase plus sulphatase as described in the Methods section. Each point represents p-nitrophenol recovered in a 1 ml sample of the perfusate obtained at various time intervals. The solid line is a continuous trace of free p-nitrophenol formed during the course of the experiment. Addition of p-nitroanisole (0.2mM) or fructose (10mM) is indicated by horizontal bars and arrows. This is a typical experiment. between intracellular ATP concentrations and rates of mixed-function oxidation. The effect of fructose on p-nitrophenol production from p-nitroanisole and on concentrations of ATP was examined in perfused livers from starved phenobarbital-treated rats. Starved rats were chosen because conjugation of p-nitrophenol is markedly diminished by food deprivation (Reinke et al., 1978) and depletion of carbohydrate substrates minimizes the effects of altered ATP concentrations on conjugation reactions. Infusion of fructose (10mM) for 10min produced a dramatic 3-fold decrease in hepatic ATP concentrations from 1756 to 572,umol/kg wet wt (Table 1). Concentrations of ADP were unaltered; however, the concentration of AMP was increased 3-fold. Total adenine nucleotides were not altered significantly as a consequence of fructose treatment. During infusion of p-nitroanisole, a steady-state rate of total p-nitrophenol production was reached in 5 min (Fig. 1). Subsequent infusion offructose (10mM) increased the rate of p-nitrophenol production by approx. 70% (Fig. 1); however, when fructose infusion was terminated, rates of p-nitrophenol production did not change appreciably. Fructose (1OmM) had noeffect uponp-nitroanisole O-demethylation in isolated microsomes from phenobarbitaltreated rats (results not shown). Effect offructose on the oxidation-reduction state of NADP+ in the perfused liver The oxidation-reduction state of the free NADP+ system was calculated from substrates assumed to be in near-equilibrium with 'malic' enzyme and isocitrate dehydrogenase (Table 1). Substrates assumed to be in near-equilibrium with isocitrate dehydrogenase indicated a reduction of NADP+ after infusion of fructose. However, substrates assumed to be in nearequilibrium with 'malic' enzyme did not indicate an alteration in the [NADP+]/[NADPH] ratio. Effect of6-aminonicotinamide on rates ofp-nitrophenol production and metabolite concentrations in the perfused liver Pretreatment of rats with 6-aminonicotinamide, a substance converted in vivo to an analogue of NADP that is a potent inhibitor of 6-phosphogluconate dehydrogenase (Kohler et al., 1970), produced an unexpected increase in free p-nitrophenol production

4 678 F. C. KAUFFMAN, R. K. EVANS, L. A. REINKE AND R. G. THURMAN from p-nitroanisole in perfused livers (Thurman et al., 1977b). Consequently, we examined the effect of 6-aminonicotinamide pretreatment on total p-nitrophenol formation as well as on adenine nucleotides and substrates for 'malic' enzyme and isocitrate dehydrogenase. Treatment of rats with 6-aminonicotinamide 6h before perfusion increased rates of total p-nitrophenol production by about 27 % (Table 2). This increase was accompanied by a decrease in ATP from 1.98 ± 0.56 to 0.64 ± 0.18 mmol/ kg, which was reflected by a more than 2-fold decrease in [ATP]/[ADP] ratios (Table 2). With the decline in [ATP]/[ADP] ratios, the [NADP+]/[NADPH] ratios calculated from substrates for 'malic' enzyme and isocitrate dehydrogenase decreased 2-3-fold (Table 2). Before infusion of p-nitroanisole, [ATP]/[ADP] ratios in perfused livers were 4.0 ± 0.3. Inhibition of NADP+-dependent dehydrogenases by ATP Each of the four major NADPH-generating enzymes from rat liver was inhibited by ATP; however, the inhibitor constants for these enzymes varied considerably (Table 3). The inhibition constant for glucose 6-phosphate dehydrogenase as determined by a simple Dixon plot is about 1.8mm. This value is comparable with that reported earlier for this enzyme isolated from ascites-tumour cells (Brand et al., 1970) and from brain (Passonneau et al., 1966). Analysis of the data by Dixon and Cornish- Bowden plots (Cornish-Bowden, 1974) (results not shown) rules out uncompetitive and non-competitive inhibition of the enzyme by ATP. A similar pattern of inhibition ofatp was observed for 6-phosphogluconate dehydrogenase and 'malic' enzyme; however, the inhibitor constants were considerably lower than for glucose 6-phosphate dehydrogenase (Table 3). The K1 of ATP for 'malic' enzyme was only 9AM, and for 6-phosphogluconate dehydrogenase the value was 114AM (Table 3). NADP+-dependent isocitrate dehydrogenase was also inhibited by ATP; however, the inhibition was non-linear and was diminished by Mg2+. Addition of equimolar amounts of ATP and Mg2+ reduced the Table 2. Effect of 6-aminonicotinamide pretreatment on p-nitrophenol production from p-nitroanisole, adenine nucleotides and [NA DP+]/[NA DPH] ratio Rates of p-nitrophenol production and metabolites were measured in perfused livers from fed rats pretreated with phenobarbital. Metabolites were measured in livers that were freeze-clamped 6min after perfusion with 0.2mM-pnitroanisole; this is the same period at which steady-state rates ofp-nitrophenol were measured. Values are means ± S.E.M. for the numbers of livers in parentheses. Total 102 x [NADP+]/[NADPH] p-nitrophenol production 'Malic' Isocitrate (,amol/h per g) [ATP]/[ADP] enzyme dehydrogenase Control (1 1) 8.6± Aminonicotinamide (4) ± Table 3. Summary ofinhibitor constants (K,) and kinetic constants (Ki)for NADP+-dependent enzymes from livers ofphenobarbital-treated rats Inhibitor constants were determined by Dixon plots by using about 8,ug of protein/ml for each of the partially purified NADP+-dependent dehydrogenases and 0.02pg of microsomal protein/ml for NADPH-cytochrome c reductase. Dixon plots were constructed from rates determined in the presence of ATP concentrations ranging from 0.05 to 2 mm for the NADP+-dependent dehydrogenases and from 0.5 to 4mM for NADPH-cytochrome c reductase. All assays were performed in duplicate at 22 C with at least four separate concentrations of ATP or ADP. KA(AM) Km Km NADP+ [Substrate] substrate [NADP+] ATP ADP (pm) (pm) CUM) (PM) Glucose 6-phosphate dehydrogenase 6-Phosphogluconate dehydrogenase 'Malic' enzyme Isocitrate dehydrogenase NADPH-cytochrome c * * Inhibitor constant for ATP determined with MgATP. t NADPH t

5 p-nitroanisole O-DEMETHYLATION IN PERFUSED RAT LIVER 679 inhibition of this enzyme by ATP and changed the kinetics from non-linear to linear. The K, of ATP for isocitrate dehydrogenase in the presence of Mg2+ was estimated to be 181 um (Table 3). Inhibitor constants of ADP for 'malic' enzyme and 6-phosphogluconate dehydrogenase were considerably higher than that of ATP for these enzymes; however, inhibition of glucose 6-phosphate dehydrogenase and isocitrate dehydrogenase by ADP was comparable with that observed for ATP (Table 3). AMP appeared to be a very weak inhibitor of the four dehydrogenases (K, > 4mM). For comparison, the Km values of several of the enzymes for carbohydrate substrates were also determined and are summarized in Table 3. Effect ofadenine nucleotides on NADPH-cytochrome c reductase The influence of ATP and ADP on the activity of NADPH-cytochrome c reductase in isolated microsomal preparations was determined in the presence of 7,UM- and 67#M-NADPH. Both nucleotides inhibited the enzyme in a non-competitive manner. The inhibitor constant for ATP was 2770#M and that of ADP was 900,UM (Table 3). AMP had no effect on this enzyme when added in concentrations as high as 5 mm. Discussion ATP inhibition of NADP+-dependent dehydrogenases Four major NADPH-generating enzymes in liver are inhibited by ATP and ADP (Table 3). Since both nucleotides are competitive with the cofactor and share structural similarities with NADP, it is likely that they inhibit these dehydrogenases by binding at the cofactor-binding site. Dixon plots of the data for 'malic' enzyme, 6-phosphogluconate dehydrogenase and glucose 6-phosphate dehydrogenase are linear with respect to ATP concentration. However, non-linearity is observed for isocitrate dehydrogenase. Linearity of the isocitrate dehydrogenase reaction is restored by the addition of Mg2+ to the reaction mixture. Since the degree of inhibition is greater in the absence than in the presence of Mg2+, the free adenine nucleotide appears to be a more potent inhibitor of the enzyme than the Mg2+ salt. Glucose 6-phosphate dehydrogenase and 'malic' enzyme may also be more sensitive to free nucleotide, since addition of equimolar amounts of Mg2+ with ATP decreased the inhibition. Addition of equimolar amounts of Mg2+ with ATP lowers the inhibition of glucose 6-phosphate dehydrogenase by about 30% and that of 'malic' enzyme by about 65 %. Although the four NADPH-generating enzymes in liver are inhibited by ATP, the sensitivity of each enzyme varies considerably. For example, the K, of ATP for 'malic' enzyme is at least 200-fold lower than that for glucose 6-phosphate dehydrogenase. Inhibitor constants of ATP for isocitrate dehydrogenase and 6-phosphogluconate dehydrogenase as well as 'malic' enzyme are considerably below intracellular concentrations of ATP; therefore these enzymes may be markedly inhibited under physiological conditions. Intracellular ATP concentrations in liver are 2-3 mm (Kauffman et al., 1977; Elbers et al., 1974), and amounts of ATP are known to be higher in cytosol than in mitochondria (Elbers et al., 1974; Soboll et al., 1978). Since the inhibitor constant of glucose 6-phosphate dehydrogenase for ATP is comparable with cellular ATP concentrations, ATP could serve a major function in regulating the activity of this enzyme. All four NADP+-dependent dehydrogenases are inhibited by ATP in media containing 5mM-Mg2+, 0.1 mm-ca2+, 160mM-K+ and 5mM-PO42- at ph 7.0 (R. K. Evans & F. C. Kauffman, unpublished work). This medium has been designed to resemble intercellular ph and ionic conditions (Lowry & Passonneau, 1964). In general, inhibition constants tend to be somewhat higher in this medium. For example, the K, of ATP for 6-phosphogluconate dehydrogenase increases from 1 14#UM to about 500pM. Enzymes, such as 6-phosphogluconate dehydrogenase and 'malic' enzyme, which have relatively low activities in liver (Kauffman et al., 1 977) and may be strongly inhibited in intact hepatocytes, might not contribute to maintaining the NADP+ pool in the reduced form. Previous suggestions that 'malic' enzyme is part of a substrate shuttle mechanism that provides reducing equivalents for drug metabolism (Thurman & Scholz, 1969) are difficult to reconcile with the marked inhibition of this enzyme by ATP (Table 3). If the NADP+-dependent dehydrogenases are strongly inhibited in the cell, as suggested above, calculation of [NADP+]/(NADPH] ratios on the basis of the assumption of near-equilibrium (Krebs & Veech, 1969) may not be justified. If enzyme activity is diminished by intracellular concentrations of adenine nucleotides, the assumption that nearequilibrium conditions exist may not be valid. Alternatively, ATP and ADP may be largely bound in cells, and intracellular adenine nucleotide concentrations available to the NADP+-dependent dehydrogenases may be much lower than that measured in tissue. This possibility is supported by the observation that Mg2+ diminishes the inhibition of isocitrate dehydrogenase, 'malic' enzyme and glucose 6-phosphate dehydrogenase by ATP. NADPH-cytochrome c reductase has been shown to be the rate-limiting component of mixed-function oxidation by reconstituted microsomal electrontransport systems from phenobarbital-induced livers (Vermilion & Coon, 1978). Therefore effectors that change the activity of this enzyme would be expected

6 680 F. C. KAUFFMAN, R. K. EVANS, L. A. REINKE AND R. G. THURMAN to alter rates of mixed-function oxidation. The possibility that adenine nucleotides participate in the regulation of this flavoprotein in vivo is strengthened by the observation that the K1 for ATP and ADP is in the physiological range (Table 3). The enzyme is also inhibited by concentrations of ADP (K, 0.9 mm) that are similar to those observed in liver, whereas AMP has no effect. Thus the total profile of adenine nucleotides must be taken into account to explain their effect on the flavoprotein. Interrelationship between cellular energetics and rates of mixed-function oxidation The observation that ATP strongly inhibits NADP+-dependent dehydrogenases and NADPHcytochrome c reductase leads to the possibility that altered cellular energetics influence rates of mixedfunction oxidation in the whole cell. For example, a decrease in intracellular ATP concentration could enhance NADPH production by deinhibition of NADP+-dependent dehydrogenases and at the same time promote cofactor utilization by a similar effect on NADPH-cytochrome c reductase. These concerted actions on cofactor supply and utilization would be expected to increase rates of mixed-function oxidation. To test this hypothesis the effect of fructose, an agent known to lower intracellular ATP concentrations (Woods et al., 1970; Maenpaa et al., 1968), on the O-demethylation ofp-nitroanisole in the perfused rat liver was examined. Infusion of fructose (10mM) markedly diminishes intracellular ATP concentrations, has no effect on ADP, but increases AMP 3-fold (Table 1). The increase in AMP in the absence of a significant decline in total adenine nucleotides differs from decreases in AMP and total adenine nucleotides noted in an earlier study of the effect of fructose on metabolism in perfused livers (Woods et al., 1970). The reason for this disparity is not known, but could be related to the different perfusion media used in the two studies. In the present study fructose produced a 50% increase in the rate of total p-nitrophenol production from p-nitroanisole in liver of starved rats. Since amounts of p-nitrophenol conjugated with glucuronide and sulphate are relatively low in this condition (Fig. 1), stimulation by fructose appears to occur mainly by enhanced oxidation of p-nitroanisole. Fructose did not affect p-nitroanisole O-demethylation in isolated microsomes. Thus the following sequence of events is suggested to explain the action of fructose on mixed-function oxidation in the intact liver. First, fructose is actively converted into fructose 1-phosphate via the action of fructokinase. The phosphorylation of fructose has been reported to be about 3 times faster than that of glucose in liver (Mendeloff & Weichselbaum, 1953). Further, metabolism of fructose 1-phosphate may be decreased as a result of inhibition of aldolase by IMP, which may be increased after fructose loading (Woods et al., 1970). Formation of fructose 1-phosphate leads to a marked depletion of intracellular ATP in the absence of a significant change in ADP, which results in deinhibition of NADP+-dependent dehydrogenases and NADPH-cytochrome c reductase. Finally, enhanced NADPH generation and increased NADPH utilization at the NADPH-cytochrome c reductase step leads to increased rates of mixedfunction oxidation ofp-nitroanisole. Other agents that deplete intracellular ATP may act via similar mechanisms. For example, pretreatment of rats with the anti-metabolite 6-aminonicotinamide produces a decrease in [ATP]/[ADP] ratios and a stimulation of p-nitrophenol production from p-nitroanisole. The finding that the free [NADP+]/ [NADPH] ratio decreases when either fructose is infused or the rat is pretreated with 6-aminonicotinamide suggests that NADPH generation exceeds its utilization via p-nitroanisole O-demethylation under these conditions (Table 3). Thus toxic agents that compromise the energetics of the cell could theoretically enhance their own metabolism through deinhibition of NADP+-dependent dehydrogenases and NADPH-cytochrome c reductase long before any induction of cytochrome P-450 occurs. This work was supported in part by U.S. Public Health Service grants CA and CA L. A. R. is a postdoctoral fellow, supported, in part, by grants CA and CA R. G. T. is the recipient of Career Scientist Development Award AA References Afolayan, A. (1972) Biochemistry 22, Brand, K., Deckner, K. & Musil, J. (1970) Hoppe-Seyler's Z. Physiol. Chem. 351, Burch, H. B., Lowry, 0. H.. Mernhardt, L. & Max, P., Jr. (1970) J. Biol. Chem. 245, Cornish-Bowden, A. (1974) Biochem. 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7 p-nitroanisole O-DEMETHYLATION IN PERFUSED RAT LIVER 681 Maenpaa, P. H., Raivio, K. 0. & Kekomaki, M. P. (1968) Science 161, Mendeloff, A. I. & Weichselbaum, T. E. (1953) Metab. Clin. Exp. 2, 450 Passonneau, J. V., Schulz, D. W. & Lowry, 0. H. (1966) Fed. Proc. Fed. Am. Soc. Exp. Biol. 25, 219 Reinke, L. A., Thurman, R. G. & Kauffman, F. C. (1978) Pharmacologist 37, 522 Sies, H. & Kandel, M. (1970) FEBS Lett. 9, Soboll, S., Scholz, R. & Heldt, H. W. (1978) Eur. J. Biochem. 87, Strobel, H. W. & Dignam, J. D. (1978) Methods Enzymol. 52, Thurman, R. G., Marazzo, D. P., Jones, L. S. & Kauffman, F. C. (1977a) J. Pharmacol. Exp. Ther. 201, Thurman, R. G., Lurquin, M., Evans, R. & Kauffman, F. C. (1977b) in Microsomes and Drug Oxidations (Ullrich, V., Roots, I., Hildebrant, A., Estabrook, R. & Conney, A., eds.), pp , Pergamon Press, Oxford Thurman, R. G. & Scholz, R. (1969) Eur. J. Biochem. 10, Thurman, R. G. & Scholz, R. (1973) Eur. J. Biochem. 38, Vermilion, J. L. & Coon, M. J. (1978) J. Biol. Chem. 253, Woods, H. F., Eggleston, L. V. & Krebs, H. A. (1970) Biochem. J. 119,