Metabolism of cyclophosphamide by purified cytochrome P-450 from microsomes of phenobarbital-treated rats

Incubation of [³H]-sidechain-labeled and [¹⁴C]-C(4)-ring-labeled cyclophosphamide (CPA) with purified cytochrome P-450 from liver microsomes of rats treated with phenobarbital resulted in the production of a major metabolite that contained both labels, was unaffected by diazomethane, possessed high polarity, was identical in TLC and HPLC behavior to a synthetic standard, didechlorodihydroxy-CPA, and was converted to CPA and bis(2-chloroethyl)amine by thionyl choloride. These results indicate that phenobarbital-inducible cytochrome P-450 is able to dechlorinate CPA and may account, in part, for the inability of phenobarbital to enhance the therapeutic activity and toxicity of this important anticancer and immunosuppressive agent.     

Protection by N-acetylcysteine of cyclophosphamide metabolism - related invivo depression of mixed function oxygenase activity and invitro denaturation of cytochrome P-450

Cyclophosphamide (CP) at high or repeated doses results in the depression of mixed function oxygenase activities of the liver. Recent studies have attributed this to the interaction between acrolein, a metabolite of CP, and sulfhydryl groups in cytochrome P-450. The present report demonstrates the protection afforded by N-acetylcysteine against acrolein-induced denaturation of cytochrome P-450 $\text{in}\text{vitro}$ and CP-related depression of mixed function oxygenase $\text{in}\text{vivo}$. Co-administration of CP and innocuous chemicals that provide free sulfhydryl groups should, in the future, be useful in enhancing the therapeutic index of CP by either reducing some of the toxicity and/or by allowing the use of repeated treatment with lower but effective doses of CP.     

Denaturation of cytochrome P-450 by cyclophosphamide metabolites

Cyclophosphamide (CP) metabolites, acrolein and 4-hydroxy-CP, were found to denature rat liver microsomal cytochrome P-450, whereas another metabolite, phosphoramide mustard, CP $\text{per}\text{se}$ or its analog Ifosfamide had no effect. The denaturation produced by CP metabolites could be blocked by cysteine, suggesting an interaction between CP metabolite(s) and sulfhydryl groups in cysteine and probably in cytochrome P-450. These studies might explain the biochemical basis of the specific depression of various microsomal mixed function oxygenase activities produced by high doses of CP.     

Cytochrome P-450 and drug metabolism in intestinal villous and crypt cells of rats: effect of dietary iron.

The amino acid sequence of the $\text{Spirulina maxima}$ ferredoxin has been determined. $\text{Spirulina maxima}$ is a blue green algae and is a procaryote. The ferredoxins of the plant-algal type sequenced to date have all been isolated from eucaryotes. The $\text{S. maxima}$ ferredoxin was composed of 98 amino acids arranged in a single polypeptide chain.The sequences of the various procaryote-eucaryote ferredoxins are compared and the differences discussed.     

Effect of ACTH on rabbit adrenal microsomal 17 alpha-hydroxylase activity, cytochrome P-450 and protein electrophoretic patterns

To determine whether a change in microsomal proteins can be correlated with adrenocorticotropic hormone (ACTH)-stimulation of rabbit adrenal 17 alpha-hydroxylase activity, rabbit adrenal microsomes were subjected to electrophoresis on polyacrylamide gels in the presence of sodium dodecylsulfate. Microsomes were obtained from rabbits stimulated with ACTH for 0, 2, 4, and 6 days. A protein band with a molecular weight of 53,000 was found to increase 31.1, 27.2 and 61.0 percent in 2-, 4-, and 6-day ACTH-stimulated microsomes as compared to controls; but 17 alpha-hydroxylase activity showed no apparent correlation, increasing 5-6 fold in all experiments. No new protein bands were found after ACTH stimulation, and no other changes in microsomal protein electrophoretic patterns after ACTH stimulation were found to correlate with the increases in 17 alpha-hydroxylase activity. The specific activity (nmol/mg protein) of cytochrome P-450 remained nearly the same throughout the stimulation periods. Tetramethylbenzidine staining for heme prosthetic groups on the electrophoretic gels displayed bands with molecular weights of 61,000, 58,000 and 53,000.     

Spironolactone and cytochrome P-450: impairment of steroid hydroxylation in the adrenal cortex

The effect of spironolactone administration on the content of adrenal microsomal cytochrome P-450 and on the activity of adrenal 17α-hydroxylase was examined in male cortisol and corticosterone-producing animals. Decreases in the content of microsomal cytochrome P-450 and in the activity of the 17α-hydroxylase after spironolactone treatment occur only in those animals which predominantly produce cortisol rather than corticosterone and which have a high activity of adrenal steroid 17α-hydroxylase. The administration of spironolactone to cortisol-producing animals, namely the guinea pig and the dog, caused a 50 to 80% loss of microsomal cytochrome P-450 with a concomitant decrease in the activity of the microsomal 17α-hydroxylase. In contrast to its effect in cortisol-producing animals, the administration of spironolactone caused either an increase or slight alteration in the concentration of adrenal microsomal cytochrome P-450 in corticosterone-producing animals such as the rat and the rabbit.     

Activation of aflatoxin B1 by primary cultures of adult rat hepatocytes: effects of hepatocyte density

Primary monolayer cultures of adult rat hepatocytes readily activate aflatoxin B1 as determined by bacterial mutagenesis (Ames test) and the extent of apparent covalent binding of aflatoxin B1 residues to hepatocyte macromolecules. For intact cultures inoculated with 3 X 10(5)-3 X 10(6) cells/dish, the efficiency of activation decreases with increasing cell density whereas permeabilized hepatocytes prepared from similarly-handled monolayer cultures show with increasing protein proportional increases in the capacity to activate aflatoxin B1. The density effect observed with intact cultured hepatocytes appears not to be due to substrate (aflatoxin B1) or oxygen depletion. These findings have apparent relevance to studies of carcinogen metabolism and in the design of carcinogen/mutagen testing protocols which utilize cultured hepatocytes.     

Inhibition of NADPH-cytochrome P450 reductase by cyclophosphamide and its metabolites

Cyclophosphamide (CP) administration to rats produced a dose-dependent loss of hepatic NADPH-cytochrome-P450 reductase and microsomal mixed function oxidase (MFO) activities. $\text{In vitro}$ CP, its metabolites (acrolein, phosphoramide mustard, 4-keto CP and nor-nitrogen mustard) and Ifosfamide, which is an analog of CP, were tested for their effects on the reductase activity. Only acrolein produced a significant loss of the reductase (66%). This loss of activity could be prevented by the presence of cysteine in the incubation mixture. Acrolein also produced a dose dependent loss of the activity when incubated with the purified reductase. These data suggest that CP-induced loss of the reductase results from interaction between CP metabolite acrolein and critical sulfhydryl groups in the reductase.     

Influence of flavin addition and removal on the formation of superoxide by NADPH-Cytochrome P-450 reductase: a spin-trap study

Several structural derivatives of the dipeptide, l (and d)-cysteinyl-l-proline were synthesized and shown to be very potent competitive and noncompetitive inhibitors of human serum angiotensin I-converting enzyme. Only if the sulfhydryl group of the cysteine was blocked with benzyl, trityl, or benzyloxycarbonyl protecting groups, was the dipeptide a noncompetitive inhibitor. Compounds with free sulfhydryl groups were competitive inhibitors with K_i values in the 10^?8m range. d-Cys-l-Pro, our most potent inhibitor (k₁ = 0.0055 μM), was an order of magnitude more potent than l-Cys-l-Pro consistent with findings of Cushman et al. (1977, Biochemistry16, 5484) that -CH₃ group substitution improves binding if the configuration is d but diminishes binding if the configuration is l. Zinc and calcium ions released inhibition by some of the noncompetitive, but only one, of the competitive inhibitors. The noncompetitive inhibitor, l-cysteinyl(benzyl)-l-proline, and the competitive inhibitor, l-cysteinyl-l-proline, were used as affinity ligands to obtain near homogenous (25 units/mg) enzyme from human plasma. The observation that compounds with a free sulfhydryl group are competitive inhibitors and those in which the sulfhydryl groups are blocked are noncompetitive inhibitors can be rationalized if the active site of the converting enzyme is an extended linear trench.     

The final step of aldosterone biosynthesis is catalyzed by an NADPH-dependent and molecular oxygen-requiring enzyme

Using sonicated mitochondria fraction prepared from bovine adrenal glomerulosa cells, aldosterone biosynthesis from 18-hydroxycorticosterone was examined as its final step, as production of [³H]-aldosterone from [³H]-corticosterone was strongly reduced by addition of non-radioactive 18-hydroxycorticosterone during the incubation. Significant conversion of 18-hydroxycorticosterone to aldosterone by the mitochondria sonicate was observed in the presence of NADPH, but not NADP⁺. This reaction was almost completely inhibited in the atmosphere of 100% carbon monoxide in the presence of either NADP⁺, or NAD⁺, and significantly reduced in the mixture of carbon monoxide and oxygen (90:10) in the presence of NADPH. Several drugs, such as SU compounds, spironolactone, amphenone B and SKF 525A which affect cytochrome P-450 blocked production of aldosterone from 18-hydroxycorticosterone. From these results, we conclude that a mixed function oxidase involving a cytochrome P-450 is engaged in the final course of aldosterone biosynthesis.     

Quantitation of vitellogenin messenger RNA in the liver of male xenopus toads during primary and secondary stimulation by estrogen

From livers of estrogen-stimulated female Xenopus toads, large quantities of estrogen-induced, poly(A)-containing RNA could be isolated, showing the same characteristics as vitellogenin mRNA obtained from hormone-treated males.Using cDNA hybridization, vitellogenin mRNA was monitored in the cytoplasmic poly(A)-containing RNA of the liver of male toads during 13 days of primary and the initial phase of secondary stimulation with estrogen.During primary stimulation, low amounts of vitellogenin mRNA, not exceeding 0.18% of the cytoplasmic poly(A)-containing RNA, were first detected after 12 hr of hormone treatment, and vitellogenin mRNA was found to increase on the average to 34% of the cytoplasmic poly(A)-containing RNA on the seventh day of hormone treatment. After 3 days of primary stimulation, accumulation of vitellogenin mRNA leveled off, showing no significant increase in the cytoplasm up to 13 days of hormone treatment. As judged from incorporation of ³²PO₄ into blood plasma proteins of males during primary stimulation, vitellogenin was first detected after 1 day, and its synthesis was found to increase dramatically until the thirteenth day of hormone treatment. This implies that there is a coincidence between appearance and extent of synthesis of vitellogenin and the abundance of vitellogenin mRNA in the cytoplasm, but there is evidence that during later phase of primary stimulation (day 3–13), the increase in synthesis of vitellogenin cannot be attributed anymore to a significant accumulation of vitellogenin mRNA.In male Xenopus, estrogen-induced synthesis of vitellogenin is no more detectable 41 days after hormone injection, and the concentration of vitellogenin mRNA was found to be <0.03% of the cytoplasmic poly(A)-containing RNA. Secondary stimulation by estrogen of these animals results in an at least 30 fold faster accumulation of vitellogenin mRNA in the cytoplasm within the initial 12 hr of hormone treatment. This may explain the faster appearance of vitellogenin in the blood plasma.     

Studies on cytochrome P-450-dependent lipid hydroperoxide reduction

A reconstituted mixed-function oxidase system containing cytochrome P-450, cytochrome P-450 reductase, phosphatidylcholine, and NADPH catalyzed the reduction of 13-hydroperoxy-9,11-octadecadienoic acid to 13-hydroxy-9,ll-octadecadienoic acid. Activity was stimulated by the addition of type I substrates, while carbon monoxide and oxygen inhibited the reaction. Perfluoro-n-hexane stimulated the reduction of lipid hydroperoxide to lipid alcohol in the reconstituted system but not by cytochrome P-450 alone. Incubation of cytochrome P-450 with only lipid hydroperoxide resulted in destruction of the hemoprotein. Addition of substrates such as aminopyrine decreased cytochrome P-450 destruction. Addition of reducing equivalents from a reconstituted electron transport system also decreased cytochrome P-450 destruction.     

Oxidation-reduction potential of cytochrome C oxidase in mitochondria of yeast grown under various copper concentrations

The pore complex-lamina fraction obtained from nuclear envelope contains a protein phosphokinase activity capable of phosphorylating endogenous and exogenous protein substrates. Its specific activity in the presence of MgCl₂ is approximately twice that of intact nuclear envelope. However, when MgCl₂ is replaced by CoCl₂ in the reaction mixture, a 7 to 12-fold increase in incorporation of ³²P from γ-³²P-ATP into protein substrate occurs. This appears not to be due to an effect of the divalent cation on the substrate, or to inhibition of a phosphoprotein phosphatase activity. Substitution of CuCl₂, MnCl₂, CaCl₂, and ZnCl₂ for MgCl₂ results in a 20 to 30% decrease in incorporation of ³²P. Cyclic AMP and cyclic GMP at 1 μM were without apparent effect. Approximately 40% of the total protein phosphokinase activity of the nuclear envelope is associated with the pore complex-lamina fraction.     

Pepsins--a chromatographic and kinetic comparison

A simplified procedure for the preparation of pepsin from porcine pepsinogen has been developed. Chromatographic and kinetic data indicate that this procedure yields a pepsin product identical with that obtained by the conventional method.The K_m and k_cat values for the hydrolysis of Ac-Phe-Tyr-OMe by commercial and freshly prepared pepsins were determined. All pepsins studied were found to have the same value for k_cat. The parameter which kinetically distinguished each of the pepsins was K_m.     

The determination of dissociation constants by affinity electrophoresis on Cibacron Blue F3G A-agarose-polyacrylamide gels

The application of copolymerized agarose-polyacrylamide gels as the support for immobilized Cibacron Blue F3G A is demonstrated for the analytical electrophoresis of proteins possessing an affinity for this dye. Bovine serum albumin was used as a model protein to develop this technique. The optimal conditions for preparing matrices are described. These conditions produce gels with suitable mechanical strength and which allow rapid electrophoresis of proteins. The dye-agarose-polyacrylamide gels permit the determination of dissociation constants. The ease of preparation of these matrices recommends them for a variety of quantitative analytical investigations.     

A catalytic method for determination of trace amounts of iron in biological material

A rapid, simple, and reproducible method for determination of iron in biological material is suggested using the oxidation of p-phenetidine hydrochloride with hydrogen peroxide in a reaction catalyzed by Fe(III) and activated by 1,10-phenanthroline. The high sensitivity of the reaction allows a single determination to be carried out with as much as 1–5 mg fresh tissue.