Catalysis of the cysteine conjugation and protein binding of acetaminophen by microsomes from a human lymphoblast line transfected with the cDNAs of various forms of human cytochrome P450.

We have previously found that for acetaminophen kinetic differences exist between the hepatic microsomal catalyzed protein binding and cysteine conjugation. We have also observed that the protein binding of acetaminophen is only to intralumenal proteins. Together these data suggested that two pools of the reactive metabolite, N-acetyl-p-benzoquinone imine (NABQI), are formed during the oxidative metabolism of acetaminophen: one on the cytosolic surface and the other within the lumen of the microsomes. This would indicate that some of forms of cytochrome P450 (CYP) catalyzing NABQI formation have their active site on the cytosolic surface and others on the lumenal surface. We have examined this question by comparing the rates of cysteine conjugation and protein binding of acetaminophen by microsomes from lymphoblasts transfected with the cDNAs for human CYPs. We found that CYP2D6 catalyzed only cysteine conjugation; CYP1A2 and 3A4 catalyzed only protein binding; CYP2E1 catalyzed both; and CYP1A1, CYP2A6 and CYP2B6 catalyzed neither. These data suggest that CYP2D6 has its active site only on the cytosolic surface; CYP1A2 and CYP3A4 only on the lumenal surface; and CYP2E1 has catalytic sites on both the lumenal and cytosolic surfaces of the membrane. In mouse studies we have found that ethanol administration increased acetaminophen protein binding by 265% but cysteine conjugation by only 61%. CYP2E1 and CYP2B increased, whereas CYP3A decreased and the others did not change. These data suggest that in control mice CYP2E1 catalyzes the bulk of protein binding, whereas CYP2D catalyzes slightly more cysteine conjugation than does CYP2E1.

Studies comparing the kinetics of cysteine conjugation and protein binding of acetaminophen by hepatic microsomes from male mice.

A large body of evidence indicates that acetaminophen toxicity is mediated through the formation of the reactive metabolite, N-acetyl-p-benzoquinone imine (NABQI). Two assays have been employed to monitor NABQI formation by hepatic microsomes: the conjugation with a thiol trap, such as cysteine or glutathione, and the binding of NABQI to microsomal proteins. Studies from our laboratory with rat hepatic microsomes have suggested that the two assays may not be equivalent. We now find with mouse hepatic microsomes that there are also marked differences between these two assays. Among these the rate of cysteine conjugation was almost three orders of greater than that of protein binding. Furthermore, ethanol feeding increased protein binding by 97%, but cysteine conjugation by only 33%. Protein binding was linear for 20 min while cysteine conjugation was linear for only 5 min. CO, imidazole and metyrapone inhibited cysteine conjugation much more than protein binding while SKF-525A and KCN had similar effects on both reactions. Both reactions increased linearly with increasing [NADPH] up to 0.32 mM. At higher concentrations, the rate of cysteine conjugation markedly decreased while the rate of protein binding plateaued. The addition of equimolar concentrations of NADH decreased protein binding, but had no effect on cysteine conjugation. NADPH reduced the protein binding of added NABQI while NADH had little effect. The reduction of NABQI back to acetaminophen was equal for NADPH and NADH. These data indicate that the formation of the cysteine conjugate of NABQI has markedly different kinetic characteristics than the microsomal protein binding. These data might suggest that the two reactions are catalyzed in part by different isoforms of cytochrome P-450.

The covalent binding of [14C]acetaminophen to mouse hepatic microsomal proteins: the specific binding to calreticulin and the two forms of the thiol:protein disulfide oxidoreductases.

Numerous in vitro studies have indicated that acetaminophen is activated by mouse hepatic microsomal cytochrome P450 to form N-acetylbenzoquinone imine. This in turn covalently binds through a Michael addition to protein sulfhydryl and amino groups. Although acetaminophen adducts of several cytosolic proteins have been purified after its administration in vivo, no adducts of specific microsomal proteins have been reported. We find that, after the in vitro incubation of mouse hepatic microsomes with [ring-14C] acetaminophen in the presence of an NADPH generating system, 95% of the bound radioactivity was associated with adducts to three intraluminal microsomal proteins: calreticulin and the two forms of thiol:protein disulfide oxidoreductase, Q2 and Q5. The acetaminophen bound to 0.35, 1.32, and 0.25 mol/mol of the three proteins, respectively. Sequencing of the 14C-labeled tryptic peptides indicated that the acetaminophen bound to lysine 103 of Q2, lysines 202, 209 or 210 and 354 of Q5 and lysines 233 or 239 of calreticulin. No adducts of cysteine residues were observed. Our data might suggest that acetaminophen hepatotoxicity results from the formation of the reactive metabolite within the endoplasmic reticulum. This then binds to these essential proteins and blocks the posttranslational modification of secretory and membrane proteins. This inhibition could then lead to cellular injury and death.

Induction of cytochrome CYPIA1 and formation of toxic metabolites of benzo[a]pyrene by rat aorta: a possible role in atherogenesis.

Cigarette smoking is a leading risk factor for atherosclerosis. Endothelial injury may be the initial event in this process. The carcinogenic metabolites of the polycyclic aromatic hydrocarbons found in cigarette smoke tars could cause this injury. We tested this model by examining the effect of 3-methylcholanthrene administration on aortic polycyclic aromatic hydrocarbon metabolism. Immunoblotting with a monoclonal antibody (mAb 1-7-1) specific for cytochromes CYPIA1 and CYPIA2 showed that aortic microsomes from treated, but not from control, animals contained CYPIA1; the CYPIA1 was primarily in the endothelium. Aortic microsomes from induced animals metabolized benzo[a]pyrene (BaP) to the 7R,8S,9,10-tetrahydrotetrol-, 7,8-dihydrodiol-, 1,6 quinone-, 3,6 quinone-, 6,12 quinone-, 3-hydroxy-, and 9-hydroxy-BaP. mAb 1-7-1 inhibited the formation of the tetrahydrotetrol, the dihydrodiol-BaP, and the 3-hydroxy-BaP but did not inhibit the quinones or the 9-hydroxy-BaP. Arachidonic acid did not affect metabolism. These data suggest that the aortas of induced animals metabolize the BaP in cigarette smoke to carcinogenic and toxic products and that this metabolism may initiate vessel injury and lead to the accelerated atherosclerosis seen in cigarette smokers.

The role of NADPH- and reduced glutathione-dependent enzymes in the norepinephrine modulation of the ATP-dependent, hepatic microsomal calcium pump: a new pathway for the noradrenergic regulation of cytosolic calcium in the hepatocyte.

The authors have recently found that the hepatic, microsomal ATP-dependent Ca++ pump activity is decreased through oxidation by cytochrome P-450-generated reactive oxygen species. This inhibition is reversed by reduced glutathione and only partially reversed by reactive oxygen scavengers. In view of these observations, the authors have sought to determine whether norepinephrine could regulate the Ca++ pump by differential modulation of the oxidative and reductive pathways. They find that, in the presence of superoxide dismutase (15 micrograms/ml), catalase (65 micrograms/ml), reduced glutathione (5 mM) and NADP+ (0.39 mM), the pump activity was maximal 142% of no norepinephrine at 10(-11) to 10(-10) M norepinephrine and decreased with increasing concentrations of norepinephrine. NADPH had no effect on uptake at 10(-11) M norepinephrine, but, between 10(-10) and 10(-8) M norepinephrine, it significantly decreased uptake compared with NADP+. At 10(-7) to 10(-6) M norepinephrine, with either NADP+ or NADPH, the uptake was significantly lower than at other norepinephrine concentrations. This decrease in the uptake seen at 10(-7) to 10(-6) M norepinephrine disappeared on the addition of 0.25 microM l-(S)-propranolol. The NADPH inhibition of the pump was blocked by imidazole-histidine buffer but not by inhibitors of mitochondrial metabolism. ATP and norepinephrine had little effect on mitochondrial uptake. These studies suggest that norepinephrine may modulate the hepatic, microsomal ATP-dependent Ca++ pump through alterations in the balance between oxidative and reductive pathways.

The effect of cytochrome P-450 and reduced glutathione on the ATP-dependent calcium pump of hepatic microsomes from male rats.

In the presence of ATP hepatic microsomes sequester calcium. This sequestration is thought to be important in the modulation of free cytosolic calcium concentration. We find that on the addition of NADPH the uptake of calcium by the hepatic microsomes is inhibited 27-85%. This inhibition is reversed by the addition of 1 mM reduced glutathione (85-91% of control), incubation under a nitrogen atmosphere (112% of control), or incubation in a 80% carbon monoxide/20% oxygen atmosphere (75% of control). Superoxide dismutase had no effect on the inhibition, while catalase reversed the inhibition by 35%. The addition of 1 mM reduced glutathione at 2 and 5 min after the addition of NADPH led to uptakes of calcium which paralleled the uptake seen when the reduced glutathione was added at the beginning of the incubation. The effect of reduced glutathione showed saturation kinetics with a Km of 10 microM. Together these data suggest that cytochrome P-450 reduces the activity of the microsomal ATP-dependent calcium pump both by the production of hydrogen peroxide and by the direct oxidation of the protein thiols. The reversal of this effect by reduced glutathione appears to be enzymatically catalyzed.

Studies on the effect of chronic consumption of moderate amounts of ethanol on male rat hepatic microsomal drug-metabolizing activity.

Weanling, male Sprague-Dawley rats given 10% ethanol in the drinking water and food ad lib. for up to 8 weeks consumed 17% of their calories as ethanol. The alanine aminotransferase (ALT), aspartate aminotransferase (AST), and liver histology by light microscopy were unaffected by this treatment. Similarly, hepatic microsomal NADPH-cytochrome c reductase, ethylmorphine N-demethylase and benzphetamine N-demethylase activities were also not affected by ethanol consumption. On the other hand, cytochrome P-450 content, aniline hydroxylase activity and acetaminophen metabolism as measured by both the cysteine conjugate and the [3H]acetaminophen covalently-bound to microsomal protein were increased significantly by ethanol consumption. The maximal effect was seen by 6 weeks. The 2- to 3-fold increase in aniline and acetaminophen metabolism, the absence of liver damage, and the similarity in weight gains and caloric intakes for controls and treated animals suggest that the rat on 10% ethanol in the drinking water is a reasonable model for studies of the effect of moderate alcohol consumption on specific biochemical pathways.

Role of substrate lipophilicity on the N-demethylation and type I binding of 3-O-alkylmorphine analogues.

A series of 3-O-alkylmorphine analogues was synthesized to determine if there was a good correlation between the rate of metabolism, type I binding affinity, and lipid solubility. The data indicate that the Km for the N-demethylation declines with increasing chain length from C1 to C9, while for increasing chain length the Vmax for the N-demethylation increases to a maximum of 15.20 nmol min-1 (mg of protein)-1 for the butyl analogue (C4) and then slowly declines with analogues with chain lengths greater than butyl (C4). The decyl (C10) and dodecyl (C12) analogues showed no activity. There was a good correlation between the lipophilicity and Km values, except for codeine and the C10 and C12 analogues. The type I binding dissociation constants (Ks) also decreased with increasing alkyl chain length with an excellent correlation between the Ks and log P. The ODmax did not change with increasing the chain length of the analogues. Our data suggest that in male rat hepatic microsomes the catalytic site for N-demethylation and the site for type I binding in this series of compounds are similar but distinct.

The effect of NADH and low oxygen pressure on the hepatic microsomal ethylmorphine N-demethylase activity of the male rat.

Plots of reciprocal initial rate of formaldehyde formation as a function of reciprocal ethylmorphine concentration provide parallel lines for oxygen concentrations ranging from 5.41 to 211 microM, and the apparent Km values for ethylmorphine at these extremes are 167 and 417 microM, respectively. Similarly, the apparent Km for O2 decreases from 7.69 microM at 2 mM ethylmorphine to 3.64 microM at 0.2 mM ethylmorphine. Reciprocal plots of 1/Km app and 1/Vm for ethylmorphine against 1/[O2] give Km values for O2 of 9.35 microM and 9.09 microM, respectively. Similar plots give Km values for ethylmorphine of 0.28 mM and 0.36 mM. Similarly, NADH causes an uncompetitive stimulation. These data suggest that hepatic microsomal ethylmorphine N-demethylase follows parallel plot sequential kinetics. These results, along with studies of other workers, suggest that cytochrome P-450 forms an active complex with O2 which is stable for at least many milliseconds.

Evaluation of a parallel plate membrane plasma exchange system.

A new parallel plate membrane plasma exchange system (Centry TPE System) has been developed and clinically evaluated. A series of 27 consecutive plasma exchanges were performed on 14 patients with autoimmune diseases. Blood access was obtained by routine means identical to centrifugal procedures. The blood flow rate was 94 +/- 20 ml/min and plasma filtration rate was 37 +/- 9 ml/min, with an average patient hematocrit of 36%. The sieving coefficients for a wide range of plasma solutes including immunoglobulins, immune complexes, and lipids, clustered about 1.00, indicating nonselective plasma filtration. The average reduction in patient plasma solutes of 60 to 70% was in close agreement with the predicted value for unhindered plasma filtration. There was no appreciable loss of blood cells, as compared to a study with a centrifugal system showing a 56% reduction in circulating platelets. Complement activation measured by C'3 conversion was undetectable in patient samples and either undetectable or minimal in plasma filtrate samples. The system was convenient to operate and problems were rare. No untoward patient effects attributable to the membrane system were observed. These results show that the parallel plate membrane plasma exchange system described in this report is capable of delivering safe, efficient plasma exchange.

Clinical evaluation of a flat-plate membrane plasma exchange system.

A new flat-plate membrane plasma separation system specifically designed for therapeutic plasma exchange (TPE) was clinically evaluated in both research and routine clinical settings. The study included a comparison to a currently available centrifugal cell separation system employed for TPE. A total of 267 membrane procedures were performed on 39 patients over a 14-month period. Both qualitative and quantitative studies showed that membrane plasma exchange procedures were equivalent to centrifugal procedures in the removal of plasma constituents from patients. A notable difference between the two types of procedure was the effect on the peripheral blood platelet count: the plasma filtrate from the membrane system was essentially cell-free and platelet counts fell only 11% during the procedure, compared to a 53% decrease during the centrifugation runs. Patient responses to both types of procedure were similar and the frequency of side-effects was low. A sampling of patient opinion revealed a preference for the membrane system for a variety of reasons. Procedure times were shorter with the membrane system because of higher achievable blood flow rates, and thus higher plasma exchange rates, while the overall nursing time requirement was lower. The results show that this flat-plate membrane TPE system enables rapid and effective plasma exchange therapy, and offered a number of monitoring and control functions that provided a safer, more efficient therapeutic procedure in the majority of patient treatments performed in this study.

The metabolism of phenytoin by isolated hepatocytes and hepatic microsomes from male rats.

Previous in vivo and in vitro studies have indicated that the Km for phenytoin hydroxylation is about 30 microM. Yet, the drug shows dose-dependent kinetics suggesting a Km of about 5 microM. The present studies indicate the discrepancy is not due to active transport of the drug in the hepatocyte or a decrease in the Km due to the low pO2 of the portal vein leading to uncompetitive inhibition. Studies in both hepatocytes and microsomes indicate the presence of a high affinity hydroxylase with a Km of 2 to 5 microM. These data suggest that this enzyme is the one primarily involved in the metabolism of phenytoin.

The kinetics of ethylmorphine N-demethylation in isolated hepatocytes: the effect of drug transport and oxygen concentration.

In previous studies we have observed that the Km for ethylmorphine N-demethylase was only 50 to 100 microM in the isolated hepatocyte as opposed to 250 to 340 microM in isolated microsomes. These data suggested that either the drug is actively transported into the cell or that the enzymes in the cell had a reduced turnover number. Our current study indicates that ethylmorphine is transported into the hepatocyte with a Km of 250 microM and Tm of 20 nmol/min 10(6) cells. On the basis of temperature and inhibitor studies, this would appear to be active transport. When the turnover number of the ethylmorphine N-demethylase was decreased by reducing the oxygen from 20% of the gas phase to 5%, the activity was partially uncompetitively inhibited with the Km decreasing from 182 to 105 microM. The Vm decreased from 1.85 nmol/min 10(6) to 0.71 nmol/min 10(6) cells. These data suggest that the low Km we initially observed was probably due to active transport of the substrate into the hepatocyte. Further, our observations indicate that the effects of oxygen pressure on the kinetic parameters should be considered in predicting in vivo kinetics from in vitro studies.

The effect of exogenous intrauterine progesterone on the amount and prostaglandin F2alpha content of menstrual blood in dysmenorrheic women.

Women with complaints of moderate or severe dysmenorrhea received intrauterine progesterone contraceptive system (16 patients) or placebo systems releasing no hormone (8 patients). Tampons were collected during the period prior to insertion and from 11 and 6 women, respectively, in the two groups at the second and fourth postinsertion periods. Prostaglandins in menstrual blood were extracted, and the amount and concentration of PGF2alpha analyzed for each patient. The menstrual blood loss (MBL) was determined by the method of Hallberg and Nilsson. The total PGF2alpha content was significantly lower in the group using progesterone systems than in the placebo group at collections 2 and 4 and was well below the preinsertion level; in placebo users the content tended to be slightly higher than it had been before insertion. The MBL increased approximately 60% above preinsertion levels in five of the six women using placebo units and decreased approximately 40% in 10 of 11 women with progesterone systems. Of the eight women in the progesterone group who had reported severe dysmenorrhea prior to insertion, seven reported an improvement; three of six in the placebo group reported a lower degree of improvement. These findings suggest that the decreased biosynthesis of PGF2alpha is a concomitant of intrauterine progesterone administration and may be a basis for the ability of the Progestasert system to diminish menstrual pain.