Cytochrome System Research Articles

Severe hepatotoxicity associated with the combination of enfuvirtide and tipranavir/ritonavir: case report

Tipranavir (TPV), a new non-peptidic protease inhibitor (PI) was approved in summer 2005 after demonstration of its efficacy in patients with multiple-PI resistant virus [1]. In particular TPV worked much better in reducing HIV viral load in highly treatment experienced patients when combined with the fusion inhibitor enfuvirtide (T-20) [2]. Recently it was shown that the co-administration of TPV/ritonavir (RTV) with T-20 potentially affected volume of distribution and elimination half-life of TPV and RTV resulting in higher nadir levels of TPV and RTV [3]. This, together with the results of the RESIST 1 and 2 trials in which 10% of participants taking TPV developed new grade 3 or 4 alanine aminotrasferase (ALT) or aspartate aminotransferase (AST) elevations (compared to 3% taking a comparison boosted PI) suggests that potential hepatotoxic effects could result because of the higher TPV levels under co-administration with T-20 [4]. Case report We here report a 52-year-old white male (Centers for Disease Control and Prevention stage C3) with a 20-year history of multidrug-resistant HIV. In February 2005 he was started on an antiretroviral regimen with TPV and RTV (500/200 mg twice daily), continuing the existing regimen of zidovudine/lamivudine (300/150 mg twice daily) and T-20 (90 mg twice daily), which was started 12 months before without hepatic disorders. Initial laboratory values were CD4 count 312 cells/μl, viral load 86 × 106 copies/ml, with normal liver function tests (LFT). The patient was known to have chronic hepatitis B virus (HBV) infection with no evidence of delta virus infection. Two weeks after the initiation of the TPV containing regimen, the patient developed elevated liver enzymes with AST, ALT and gamma-glutamyltransferases (G-GT) up to 261, 538 and 238 U/l respectively. Abdominal ultrasonography showed normal bile ducts; HBV DNA viral load was low with 20 000 copies/ml, autoimmune markers for hepatitis and cirrhosis were negative, excessive alcohol consumption was denied. Percutaneous liver biopsy showed a mild, non-active hepatitis with moderate lymphocyte infiltration in the portal areas and no intrahepatic steatosis. Following the patient's wish to interrupt the T-20 treatment because of severe local skin reactions, and under the assumption of a hepatotoxic effect of the antiretroviral regimen T-20 was discontinued for 6 weeks. Shortly after, liver function improved with a 50% decrease in LFT values. Due to a weight loss of 10 kg and an elevation of the viral load treatment with T-20 was recommenced and 2 weeks later after an increase of the G-GT TPV/RTV was discontinued. The liver function tests returned to normal within 4 weeks. The patient's CD4 cell count is currently stable but the viral load has increased to 17 000 copies/ml. No further deterioration in the liver function tests has been observed. Discussion Hepatotoxicity as a result of TPV containing therapy is well documented, although the mechanism for this toxicity remains unknown. In contrast to TPV, a strong inducer of CYP3A4, and RTV, a strong inhibitor of CYP3A4, T-20 does not affect the P450 cytochrome system. Our patient developed grade 4 levels of LFT under the co-administration of TPV/RTV and T-20 and recovered clearly after T-20 was discontinued. It is hypothesized that the hepatotoxicity was induced by TPV/TPV aggravated by the usage of T-20 causing higher TPV/RTV serum levels. Unfortunately no drug level tests were performed in our patient. Further studies are needed concerning the metabolic pathways and interactions of this potentially fatal drug combination. Physicians who treat patients with HIV need to be aware of the potential additive effect regarding hepatotoxicity when administering TPV/RTV and T-20. Close monitoring of TPV serum levels and dosage adaption could help to avoid hepatotoxicity. Furthermore, a reduction or even discontinuation of RTV should be considered when TPV is combined with T-20 due to its potential boosting effect.

Agmatine is transported into liver mitochondria by a specific electrophoretic mechanism

Agmatine, a divalent diamine with two positive charges at physiological pH, is transported into the matrix of liver mitochondria by an energy-dependent mechanism the driving force of which is DeltaPsi (electrical membrane potential). Although this process showed strict electrophoretic behaviour, qualitatively similar to that of polyamines, agmatine is most probably transported by a specific uniporter. Shared transport with polyamines by means of their transporter is excluded, as divalent putrescine and cadaverine are ineffective in inhibiting agmatine uptake. Indeed, the use of the electroneutral transporter of basic amino acids can also be discarded as ornithine, arginine and lysine are completely ineffective at inducing the inhibition of agmatine uptake. The involvement of the monoamine transporter or the existence of a leak pathway are also unlikely. Flux-voltage analysis and the determination of activation enthalpy, which is dependent upon the valence of agmatine, are consistent with the hypothesis that the mitochondrial agmatine transporter is a channel or a single-binding centre-gated pore. The transport of agmatine was non-competitively inhibited by propargylamines, in particular clorgilyne, that are known to be inhibitors of MAO (monoamine oxidase). However, agmatine is normally transported in mitoplasts, thus excluding the involvement of MAO in this process. The I2 imidazoline receptor, which binds agmatine to the mitochondrial membrane, can also be excluded as a possible transporter since its inhibitor, idazoxan, was ineffective at inducing the inhibition of agmatine uptake. Scatchard analysis of membrane binding revealed two types of binding site, S1 and S2, both with mono-co-ordination, and exhibiting high-capacity and low-affinity binding for agmatine compared with polyamines. Agmatine transport in liver mitochondria may be of physiological importance as an indirect regulatory system of cytochrome c oxidase activity and as an inducer mechanism of mitochondrial-mediated apoptosis.

A history of research on yeasts 9: regulation of sugar metabolism1

A history of research on yeasts 9: regulation of sugar metabolism1

Relationship between hepatic phenotype and changes in gene expression in cytochrome P450 reductase (POR) null mice

Cytochrome P450 reductase is the unique electron donor for microsomal cytochrome P450s; these enzymes play a major role in the metabolism of endogenous and xenobiotic compounds. In mice with a liver-specific deletion of cytochrome P450 reductase, hepatic cytochrome P450 activity is ablated, with consequent changes in bile acid and lipid homoeostasis. In order to gain insights into the metabolic changes resulting from this phenotype, we have analysed changes in hepatic mRNA expression using microarray analysis and real-time PCR. In parallel with the perturbations in bile acid levels, changes in the expression of key enzymes involved in cholesterol and lipid homoeostasis were observed in hepatic cytochrome P450 reductase null mice. This was characterized by a reduced expression of Cyp7b1, and elevation of Cyp7a1 and Cyp8b1 expression. The levels of mRNAs for other cytochrome P450 genes, including Cyp2b10, Cyp2c29, Cyp3a11 and Cyp3a16, were increased, demonstrating that endogenous factors play a role in regulating the expression of these proteins and that the increases are due, at least in part, to altered levels of transcripts. In addition, levels of mRNAs encoding genes involved in glycolysis and lipid transport were also increased; the latter may provide an explanation for the increased hepatic lipid content observed in the hepatic null mice. Serum testosterone and oestradiol levels were lowered, accompanied by significantly decreased expression of Hsd3b2 (3beta-hydroxy-Delta5-steroid dehydrogenase-2), Hsd3b5 (3beta-hydroxy-Delta5-steroid dehydrogenase-5) and Hsd11b1 (11beta-hydroxysteroid dehydrogenase type 1), key enzymes in steroid hormone metabolism. These microarray data provide important insights into the control of metabolic pathways by the cytochrome system.

Effects of D-003 on Hepatic Drug-Metabolizing Enzyme Activities in Rats

D-003 is a mixture of very-long-chain aliphatic acids with cholesterol-lowering and concomitant anti-platelet effects. The microsomal cytochrome P-450 system comprises a superfamily of proteins present in hepatic and extrahepatic tissues that is responsible for the metabolism of many drugs. The present study was undertaken to investigate the effects of D-003 on in vivo drug-metabolizing hepatic enzymes. Two experimental series (n = 6 animals/group) were performed. In the first series rats were randomly distributed in one control and two groups treated with D-003 at 1,000 and 2,000 mg/kg for 14 days. In the second one they were distributed in one control and three groups treated with D-003 (250, 500, and 1,000 mg/kg) for 6 months. All treatments were orally administered by gastric gavage. Control rats were orally treated only with acacia gum/water vehicle. The content of microsomal P-450, b (5) cytochromes, total sulfhydryl groups, nonprotein sulfhydryl groups, and protein-bound sulfhydryl groups as well as the activities of NADPH cytochrome c reductase, aminopyrine demethylase, dimethylnitrosamine N-demethylase, 7-ethoxyresorufin O-deethylation, 7-pentoxyresorufin O-depentylation, and cytosolic glutathione S-transferase were assessed. D-003 administered up to 2,000 mg/kg or 1,000 mg/kg during 14 days or 6 months did not affect the activities of the hepatic drug-metabolizing enzymes investigated. It is concluded that D-003 is not metabolized by the liver cytochrome system and that potential risk derived from drug-to-drug interactions between D-003 and concomitant drugs appears to be low.

The effect of over-expression of the alternative oxidase in the procyclic forms of Trypanosoma brucei

Trypanosome alternative oxidase (TAO) is the cyanide-resistant but SHAM-sensitive terminal oxidase of the mitochondrial electron transport chain in African trypanosomes. The bloodstream forms of Trypanosoma brucei lack cytochromes and respire exclusively via TAO. On the other hand, the insect, or procyclic form possesses a fully developed cytochrome system, and down regulates TAO several folds by reducing the stability of the TAO transcript. We expressed an ectopic copy of TAO in the procyclic form from a tetracycline regulated stable expression vector, in which the TAO 3′-UTR was replaced by T. brucei aldolase 3′-UTR. The TAO transcript produced from the ectopic copy was stably accumulated in the procyclic form. Upon induction with doxycycline, TAO protein level was gradually increased about five-fold within 72 h. TAO over-expression did not show any effect on the growth of the parasite. The rate of respiration and the SHAM-sensitive respiratory pathway capacity was increased about two- and five-fold, respectively, and the cytochrome-mediated respiratory pathway capacity was reduced two- to three-folds within 5 days after induction of TAO. Doxycycline induced TAO + cells preferentially utilized CN-resistant, SHAM-sensitive pathway of respiration, whereas, in the control cells 70–80% of total respiration was via the CN-sensitive pathway. Moreover, we have found that increased expression of TAO caused about two-fold down regulation of cytochrome oxidase subunit IV, and cytochrome c 1 protein level and also caused a four-fold up-regulation of the expression of the surface coat protein, GPEET procyclin in the procyclic form. This suggests that the expression of two terminal oxidases and the coat protein is linked in T. brucei.

Binding and release of cytochrome c in brain mitochondria is influenced by membrane potential and hydrophobic interactions with cardiolipin.

Factors influencing the release and anchorage of cytochrome c to the inner membrane of brain mitochondria have been investigated. Metabolic activity of mitochondria caused a decrease in the membrane potential delta psi(m), accompanied by detachment of the protein from the inner membrane. In a model system of cytochrome c reconstituted in cardiolipin (CL) liposomes, phosphate was used to breach the hydrophilic lipid-protein interactions. About 44% cytochrome c was removable when heart CL (80% 18:2n-6) was employed, whereas the remaining protein accounted for the tightly bound conformation characterized by hydrophobic lipid-protein interactions. Cytochrome c release from brain CL liposomes was higher compared to heart CL, consistent with lower polyunsaturated fatty acid content. The release was even higher with CL extracted from metabolically stressed mitochondria, exhibiting more saturated fatty acid profile compared to control (30% vs. 17%). Therefore, weakening of the hydrophobic interactions due to saturation of CL may account for the observed cytochrome c release from mitochondria following metabolic stress. Moreover, mitochondria enriched with polyunsaturated CL exhibited higher delta psi(m), compared to less unsaturated species, suggesting that CL fatty acid composition influences delta psi(m). Mitochondria incorporated exogenous cytochrome c without protease-sensitive factors or delta psi(m). The internalized protein anchored to the inner membrane without producing swelling, as monitored by forward and side light scattering, but produced delta psi(m) consumption, suggesting recovery of respiratory activity. The delta psi(m) decrease is ascribed to a selected mitochondrial population containing the incorporated cytochrome c.

Clinical pharmacokinetics of everolimus.

Everolimus is an immunosuppressive macrolide bearing a stable 2-hydroxyethyl chain substitution at position 40 on the sirolimus (rapamycin) structure. Everolimus, which has greater polarity than sirolimus, was developed in an attempt to improve the pharmacokinetic characteristics of sirolimus, particularly to increase its oral bioavailability. Everolimus has a mechanism of action similar to that of sirolimus. It blocks growth-driven transduction signals in the T-cell response to alloantigen and thus acts at a later stage than the calcineurin inhibitors ciclosporin and tacrolimus. Everolimus and ciclosporin show synergism in immunosuppression both in vitro and in vivo and therefore the drugs are intended to be given in combination after solid organ transplantation. The synergistic effect allows a dosage reduction that decreases adverse effects. For the quantification of the pharmacokinetics of everolimus, nine different assays using high performance liquid chromatography coupled to an electrospray mass spectrometer, and one enzyme-linked immunosorbent assay, have been developed. Oral everolimus is absorbed rapidly, and reaches peak concentration after 1.3-1.8 hours. Steady state is reached within 7 days, and steady-state peak and trough concentrations, and area under the concentration-time curve (AUC), are proportional to dosage. In adults, everolimus pharmacokinetic characteristics do not differ according to age, weight or sex, but bodyweight-adjusted dosages are necessary in children. The interindividual pharmacokinetic variability of everolimus can be explained by different activities of the drug efflux pump P-glycoprotein and of metabolism by cytochrome P450 (CYP) 3A4, 3A5 and 2C8. The critical role of the CYP3A4 system for everolimus biotransformation leads to drug-drug interactions with other drugs metabolised by this cytochrome system. In patients with hepatic impairment, the apparent clearance of everolimus is significantly lower than in healthy volunteers, and therefore the dosage of everolimus should be reduced by half in these patients. The advantage of everolimus seems to be its lower nephrotoxicity in comparison with the standard immunosuppressants ciclosporin and tacrolimus. Observed adverse effects with everolimus include hypertriglyceridaemia, hypercholesterolaemia, opportunistic infections, thrombocytopenia and leucocytopenia. Because of the variable oral bioavailability and narrow therapeutic index of everolimus, blood concentration monitoring seems to be important. The excellent correlation between steady-state trough concentration and AUC makes the former a simple and reliable index for monitoring everolimus exposure. The target trough concentration of everolimus should range between 3 and 15 microg/L in combination therapy with ciclosporin (trough concentration 100-300 microg/L) and prednisone.

Mitochondrial NADH Dehydrogenase gene (mtND2) Phylogeny of Egyptian Tilapia species

NADH dehydrogenase is a very important protein and is expressed by the mitochondrial NADH dehydrogenase gene (mtND2). Dehydrogenase enzyme is used to remove hydrogen from its substrate, which is used in the cytochrome (hydrogen carrier) system in respiration to produce a net gain of ATP. Also, it reversibly catalyses the oxidation of NADH to NAD and reduced acceptor. The size of mtDN2 of Tilapia species and their hybrids is ~1050 base pairs and was detected by using the polymerase chain reaction technique. To identify the molecular phylogeny and the physical characteristics of mtND2 gene of Tilapia species were done by using the restriction fragment length polymorphisms (RFLPs) with some restriction endonucleases(AccI, AvaII, AvaI, StyI, Bg1I and EaeI). The PCR-RFLPs of NADH dehydrogenase gene of Tilapia species and their hybrids may prove that the gene is quite evolution phylogenetic difference from one species to another. At the same time, This study investigated the feasibility of mitochondrial DNA (mtDNA) based approaches in addressing problems of identification of Tilapia species and their hybrids, isolated from the River Nile by using the PCR-RFLPs analysis of mtND2 gene.

Possible Interaction of Clozapine and Lisinopril

Back to table of contents Previous article Next article Letter to the EditorFull AccessPossible Interaction of Clozapine and LisinoprilGEORGE ABRAHAM, M.D., BEATRICE GRUNBERG, M.D., and SYLVIA GRATZ, M.D., GEORGE ABRAHAMSearch for more papers by this author, M.D., BEATRICE GRUNBERGSearch for more papers by this author, M.D., and SYLVIA GRATZSearch for more papers by this author, M.D., Philadelphia, Pa.Published Online:1 Jun 2001https://doi.org/10.1176/appi.ajp.158.6.969AboutSectionsView EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InEmail To the Editor: Clozapine levels should be at 350–450 ng/ml for clinical efficacy and minimal side effects (1). Factors affecting its metabolism or excretion can influence clinical efficacy. We report on a patient who possibly experienced an interaction effect of clozapine and lisinopril.Mr. A, a 39-year-old Asian man with treatment-resistant schizophrenia, was receiving 300 mg/day of clozapine; his blood level of the drug was 490 ng/ml. In addition, he had diabetes that was controlled with glipizide, 10 mg/day. One year later, he developed hypertension, so lisinopril treatment, 5 mg/day, was initiated. Shortly afterward, his blood levels of clozapine and one its metabolites, norclozapine, were 966 ng/ml and 512 ng/ml, respectively. Six months later, his dose of lisinopril was increased to 10 mg/day. His blood levels of clozapine and norclozapine, as measured on two consecutive occasions 1 month apart, were 1092 and 380 ng/ml and 1245 and 392 ng/ml, respectively.Mr. A became more disorganized and had frequent episodes of irritability and angry outbursts. He experienced severe sleep disturbances and frequent nightmares and awakenings. He also salivated excessively. Other known side effects of clozapine, such as anticholinergic toxicity or seizures, were not observed. Laboratory values, including measures of renal functions, were found to be within normal limits.Mr. A’s clozapine dose was decreased to 200 mg/day. His blood levels, measured after 6 weeks, remained high: clozapine, 1335 ng/ml, and norclozapine, 428 ng/ml. When we suspected a drug interaction, his antihypertensive was changed from lisinopril to diltiazem, 240 mg/day. Repetition of laboratory tests after 6 weeks resulted in clozapine and norclozapine levels that had decreased to 693 and 254 ng/ml, respectively. Although Mr. A continued to experience psychotic symptoms and sialorrhea, his sleep disturbances and irritability had improved.An interaction in which lisinopril raises the level of clozapine or its metabolites has not been reported, to our knowledge. Angiotensin-converting enzyme inhibitors, by dilating efferent arterioles, decrease overall renal vascular resistance. This in turn decreases intraglomerular pressure, which reduces effective filtration pressure and slows the glomerular filtration rate, causing reversible renal impairment (2). This impairment may not be reflected by routine tests of renal function. Increased levels of norclozapine suggest impaired excretion.Lisinopril does not influence the cytochrome systems, and it is excreted unchanged in urine. Clozapine is metabolized by cytochrome 1A2 and 3A4 isoenzymes. It is a well-known phenomenon that excess levels of substrates cause abnormal enzymatic feedback control (3). In our patient, levels of both clozapine and norclozapine were elevated. It is not known whether accumulation of clozapine metabolites can cause negative feedback inhibition in the cytochrome P450 system. If so, it can cause an accumulation of the parent compound. The toxic effects of clozapine are known to cause sleep disturbances, with intensification of dream activity and immediate onset of REM sleep.

Pharmacokinetic interactions of statins.

Statins have shown high efficacy in managing hypercholesterolemia in patients requiring chronic drug treatment, particularly those who show comorbidity and thus receive concomitant medication for other pathologies. According to the reported data extensively reviewed in this work, absorption and elimination are the kinetic processes mainly affected by this type of interaction, while distribution and protein binding is only slightly modified. Products (drugs or food) with the ability to affect the activity of protein-mediated transport and/or P450 cytochrome systems, particularly the P-glycoprotein and/or CYP3A4, respectively, are expected to cause pharmacokinetic interactions with statins. The intensity of the interaction is dependent on the statin kinetic profile and the capacity of the coadministered product to alter the systems mentioned above. Modification of the total HMG-CoA inhibitors instead of just the parent drug profile is to be considered when evaluating the clinical relevance of the interaction. Interindividual variability must also be taken into account when extrapolating results from studies performed in small groups of relatively healthy individuals. Patients treated with other drugs that have the potential ability to interact with statins should be monitored.

Gender difference: Hype or fact?

Summary The number of trials looking at the gender effecton anesthetic agents is very limited. What evidence exists is not clear, but the general pattern suggests a difference in pharmacokinetics and pharmacodynamics between men and women for certain drugs. These differences may not be predictable within the same group of drugs, such as benzodiazepines; the gender effect may vary and sometimes may be completely opposite. Important gender factors, such as menopause, pregnancy, and use of oral contraceptives, may affect the pharmacokinetics and dynamics of drugs significantly. Difference in body weight and composition alone result in significant pharmacokinetic differences in volume of distribution, initial plasma concentration, organ blood flow, rate of redistribution, and rate of filtration by the kidney. The intrinsic activity of the cytochrome system appears to be affected by gender: The activity of CYP3A4 is 1.4 times greater in women than men, with the resultant increased metabolism of certain drugs in women. One possible explanation for this increased intrinsic activity in women may be the interaction of the sex steroids with the cytochrome systems. Increased sensitivity to drugs may result from interaction of the sex steroids with receptors such as the μ-opioid receptors. The sex hormones may affect other intrinsic substances such as beta-endorphins. For example, beta-endorphin levels are increase in pregnancy and along with progesterone are thought to result in the decreased MAC associated with pregnancy. Clearly, further studies are needed to identify which drugs are affected by gender as well as the causes for these differences. Last, how big these differences are and to what degree they are clinically significant must be determined.

Cloning and Characterization of the Gene for Phosphatidylcholine Synthase

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and can be synthesized by either of two pathways, the CDP-choline pathway or the methylation pathway. In prokaryotes only the methylation pathway was thought to occur. Recently, however, we could demonstrate (de Rudder, K. E. E., Sohlenkamp, C., and Geiger, O. (1999) J. Biol. Chem. 274, 20011-20016) that a second pathway for phosphatidylcholine biosynthesis exists in Sinorhizobium (Rhizobium) meliloti involving a novel enzymatic activity, phosphatidylcholine synthase, that condenses choline and CDP-diacylglyceride in one step to form PC and CMP. Using a colony autoradiography method we have isolated mutants of S. meliloti deficient in phosphatidylcholine synthase and which are no longer able to incorporate radiolabeled choline into PC. Complementation of such mutants with a sinorhizobial cosmid gene bank, subcloning of the complementing fragment, and sequencing of the subclone led to the identification of a gene coding for a presumptive CDP-alcohol phosphatidyltransferase. Amplification of this gene and its expression in Escherichia coli demonstrates that it codes for phosphatidylcholine synthase. Genomes of some pathogens (Pseudomonas aeruginosa and Borrelia burgdorferi) contain genes similar to the sinorhizobial gene (pcs) for phosphatidylcholine synthase. Although pcs-deficient S. meliloti knock-out mutants show wild type-like growth and lipid composition, they are unable to perform rapid PC biosynthesis that normally is achieved via the phosphatidylcholine synthase pathway in S. meliloti wild type.

Use of steroids in renal transplantation

LUCOCORTICOIDS (GC) have been the cornerstone of immunosuppressive therapy in organ transplantation for many years and are still used by most transplant centers for basic immunosuppression. Unfortunately, the administration of high-dose GC therapy has also represented a major cause of morbidity and mortality in transplant recipients. After the advent of cyclosporine (CyA), the doses of GC have been consistently reduced. However, steroid-related complications still account for significant morbidity in CyA-treated patients. 1 This iatrogenic morbidity in spite of the low-dose GC administration may be due to the fact that GC and CyA are metabolized by the same P-450 cytochrome system with possible retention of active metabolites of both drugs. On the other hand, the two agents may share side effects such as hypertension, diabetes, hypercholesterolemia, osteopenia, and cataracts. 2 Thus, only a steroid-free immunosuppression can actually prevent the steroid-related toxicity. Two types of steroidfree immunosuppression have been attempted in patients treated with calcineurin inhibitors: withdrawal of GC several months after transplantation in patients with stable allograft function or avoidance of GC immediately or early after transplantation.

CYP 3A proteins are expressed in human neutrophils and lymphocytes but are not induced by rifampicin

Cytochrome P-450 3A (CYP 3A) enzymes, the prominent subfamily in the cytochrome system, are expressed in various extrahepatic tissues. Until now, their expression has been demonstrated in human polymorphic neutrophils (PMNs) but not in lymphocytes using immunohistochemistry and immunoblot analysis. Moreover, their potential modulation has not been determined yet. To study such an expression in different peripheral blood cell populations, rifampicin (600 mg/day during 6 days) was given to 8 healthy subjects. PMNs and lymphocytes were isolated by centrifugation of whole white blood cell fractions using Ficoll gradients before drug administration, immediately after, and 3 days after drug withdrawal. PMN and lymphocyte smears and homogenates were subjected to immunostaining and immunoblotting, respectively, with a mouse monoclonal antibody recognizing all CYP 3A proteins. These proteins were quantified by densitometric analysis. Before and after rifampicin administration, a positive cytoplasmic staining was observed in all PMNs and in about 50% of lymphocytes. CYP 3A expression in lymphocytes was further confirmed by positive immunoblots for lymphocyte homogenates. Neither in PMNs nor in lymphocytes, induction of CYP 3A protein expression was observed after rifampicin treatment despite overall induction of CYP 3A activity assessed by the urinary excretion of 6β-hydroxycortisol. These results demonstrate that CYP 3A proteins are constitutively expressed not only in PMNs but also in lymphocytes. However, in both cell lineages CYP 3A protein expression was not induced by rifampicin.

Presence and Formation of Heme and Occurrence of Certain Heme Proteins in the Filarial ParasiteSetaria digitata

There is currently renewed interest in the biological significance of heme proteins. The most common heme proteins include hemoglobin, myoglobin, cytochromes, and redox enzymes such as catalase and peroxidase.Setaria digitatais a cattle filarial parasite, which is devoid of typical cytochrome systems. However, studies showed activities of δAminolevulinate synthase (ALAS), δAminolevulinate dehydratase (ALAD), and heme oxygenase in appreciable amounts, suggesting the presence of necessary equipment for the biosynthesis of heme. This is further confirmed by the end product inhibition of ALAS by heme and the observation of the death of the parasite by succinyl acetone, an inhibitor of the biosynthesis of heme. Though typical cytochrome systems are absent, microsomal cytochrome P 450 and elevated levels of heme containing enzymes such as catalase and peroxidase are present in the parasite. A unique hemoglobin is also detected which shows a difference in biological functions from the host system and that of the much-studied nematode parasite Ascaris sum.

Cooperative coupling and role of heme a in the proton pump of heme-copper oxidases

In the last few years, evidence has accumulated supporting the applicability of the cooperative model of proton pumps in cytochrome systems, vectorial Bohr mechanism, to heme-copper oxidases. The vectorial Bohr mechanism is based on short- and long-range protonmotive cooperative effects linked to redox transitions of the metal centers. The crystal structure of oxidized and reduced bovine-heart cytochrome c oxidase reveals, upon reduction, the occurrence of long-range conformational changes in subunit 1 of the oxidase. Analysis of the crystal structure of cytochrome c oxidase shows the existence of hydrogen-bonded networks of amino acid residues which could undergo redox-linked pK shifts resulting in transmembrane proton translocation. Our group has identified four proteolytic groups undergoing reversible redox-linked pK shifts. Two groups result in being linked to redox transitions of heme a 3. One group is apparently linked to Cu B. The fourth group is linked to oxido-reduction of heme a. We have shown that the proton transfer resulting from the redox Bohr effects linked to heme a and Cu B in the bovine oxidase displays membrane vectorial asymmetry, i.e., protons are taken up from the inner aqueous space (N), upon reduction, and released in the external space (P), upon oxidation of the metals. This direction of proton uptake and release is just what is expected from the vectorial Bohr mechanism. The group linked to heme a, which can transfer up to 0.9 H +/e at pHs around neutrality, can provide the major contribution to the proton pump. It is proposed that translocation of pumped protons, linked to electron flow through heme a, utilizes a channel (channel D) which extends from a conserved aspartate at the N entrance to a conserved glutamate located between heme a and the binuclear center. The carboxylic group of this glutamic acid, after having delivered, upon electron flow through heme a, pumped protons towards the P phase, once reprotonated from the N phase, moves to deliver, subsequently, to the binuclear center chemical protons consumed in the conservation of the peroxy to ferryl and of the latter to the oxy intermediate in the redox cycle. Site-directed mutagenesis of protolytic residues in subunit 1 of the aa 3-600 quinol oxidase of Bacillus subtilis to non-polar residues revealed that the conserved Lys 304 is critical for the proton pumping activity of the oxidase. Crystal structures of cytochrome c oxidase show that this lysine is at the N entrance of a channel which translocates the protons consumed for the production of the peroxy intermediate. Inhibition of this pathway, by replacement of the lysine, short-circuits protons from channel D to the binuclear center, where they are utilized in the chemistry of oxygen reduction.

Nefazodone in Major Depression

One hundred sixty-six patients suffering from major depressive disorders were treated for 8 weeks with nefazodone in an open study in dosage ranges from 200 to 600 mg. This report focuses primarily on the first week of therapy and on the concomitant use of several benzodiazepines, one of which is not metabolized by the cytochrome system (temazepam). Triazolam response was further evaluated as a function of two nefazodone dosage regimens provided during the first week of therapy, one group receiving nefazodone 200 mg/day for 7 days, and another group receiving nefazodone 200 mg/day for 3 days, followed by 4 days with 400 mg/day. Finally, a comparison of three different nefazodone dosages, the third being 400 mg from day 1 on, was also carried out. Outcome measures included Hamilton Rating Scale for Depression total and the total of the three Hamilton Rating Scale for Depression insomnia items, as well as global improvement, a daily completed sleep questionnaire, and adverse event assessment. A combination of nefazodone with a benzodiazepine (BZ) caused more sedation than nefazodone alone; triazolam, the BZ with the shortest half-life and the highest dependence on the cytochrome 450 system for its metabolism, caused the least amount of sedation, and alprazolam and diazepam, the two daytime benzodiazepines, caused the most sedation. Triazolam caused significant and identical reduction of insomnia in both nefazodone groups. Compared with nefazodone 200 mg given as monotherapy, insomnia was significantly improved--not only by triazolam, but also alprazolam and diazepam, but not temazepam. The addition of nefazodone raised triazolam plasma levels to almost 500%, the plasma level of desmethyl-diazepam 87%, and that of alprazolam 34%. Temazepam plasma levels remained unchanged. When prescribing nefazodone with a benzodiazepine, one should expect an improved sleep pattern initially, but at the cost of clinically relevant daytime sedation. The prediction that temazepam, the only BZ not dependent on the cytochrome mechanism for metabolism, should be the least sedating, and triazolam, because of its cytochromic metabolism interference with nefazodone should be the most sedating, could not be confirmed. In fact, triazolam 0.25 mg capsules seem to be the safest treatment of choice when one has to combine a benzodiazepine with nefazodone in initial stages of therapy, at least of the four benzodiazepines tested in this study.

Investigation of the electrode reaction of cytochrome c through mixed self-assembled monolayers of alkanethiols on gold(111) surfaces

Horse heart cytochrome c was immobilized on mixed self-assembled monolayers (SAMs) of carboxyl- and methyl-terminated alkanethiolates (HOOC(CH 2 ) 10 S + CH 3 (CH 2 ) 9 S) on Au(111) electrodes. The apparent standard rate constant k obs of electron transfer for the electrode system of cytochrome c ⧹mixed SAM⧹gold electrode was measured by a.c. impedance and a.c. modulated UV-visible electro reflectance techniques. A strong dependence of k obs on the composition of the mixed alkanethiol solution, from which the monolayers had been assembled, was observed. A maximum of k obs ≈ 200s −1 was found for SAMs formed from solutions containing a mole fraction χ ≈ 0.8 of carboxyl-terminated alkanethiols. This rate constant is about six times faster than that of a system with a single-component monolayer of HOOC(CH 2 ) 10 S ( ξ = 1). Reductive desorption suggested that the two alkane thiolates mix homogeneously in the monolayer and there is no sizable phase separation on the Au(111) electrode. Double-layer capacitance measurements determined the degree of protonation of the mixed SAMs in the pH range 6–9. The enhancement of electron-transfer rate is attributed to a favorable distribution of protein binding sites in the mixed monolayers.

The aquatic vertebrate embryo as a sentinel for toxins: zebrafish embryo dechorionation and perivitelline space microinjection.

Pollution of aquatic ecosystems poses a serious threat to aquatic organisms and ultimately the entire ecosystem. Understanding how a toxin affects embryonic development is key to determining the risk a pollutant represents to the environment. Extraembryonic membranes, such as the chorion of fish eggs, provide a protective barrier between the embryo and the environment. Although the fish chorion excludes many chemical pollutants, some noxious agents can still gain access to the aquatic embryo. Therefore a monitoring system that tests the effects directly upon the embryo must be established. Although exposure to a single toxin in the laboratory can determine the concentration at which a pollutant becomes a health or environmental hazard, embryos and adults in nature are not merely affected by a single chemical, but are exposed to mixtures of different pollutants. Zebrafish (Danio rerio) and medaka (Oryzias latipes) embryos were employed for the rapid observation of the effects of single chemicals and chemical mixtures on development. Using dechorionation and a perivitelline space microinjection system, the embryos were effective sentinels for low concentrations of aquatic pollutants. The developmental effects of small quantities of toxins were observed. Embryos treated during the late gastrula stage of development with hexachlorobenzene (HCB); 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); toluene; benzene; or mixtures of these chemicals developed cardiovascular abnormalities. The zebrafish dechorionation exposure technique, Micro Intrachorionic Zebrafish Embryo Live Laboratory test, was especially effective in testing the pollutant mixtures. Combinations of both TCDD and benzene (as well as the toluene and benzene combinations) were tested and the mixtures acted synergistically; the combinations were more toxic than either chemical by itself. Hexachlorobenzene- and TCDD-treated embryos tested positively for expression of cytochrome P450 1A indicating that the cytochrome metabolic pathways were already functional in these early embryos, and suggested that a product of the cytochrome system may be involved in HCB and TCDD pollution associated cardiovascular defects.