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C R Wolf

Publications and source records attributed to C R Wolf.

At least 55 records · Page 3Linked to original sources
PubMed1999

Effects of human cytochrome b5 on CYP3A4 activity and stability in vivo.

Cytochrome P450s (P450) form a superfamily of membrane-bound proteins that play a key role in the primary metabolism of both xenobiotics and endogenous compounds such as drugs and hormones, respectively. To be enzymically active, they require the presence of a second membrane-bound protein, NADPH P450 reductase, which transfers electrons from NADPH to the P450. Because of the diversity of P450 enzymes, much of the work on individual forms has been carried out on purified proteins, in vitro, which requires the use of complex reconstitution mixtures to allow the P450 to associate correctly with the NADPH P450 reductase. There is strong evidence from such reconstitution experiments that, when cytochrome b5 is included, the turnover of some substrates with certain P450s is increased. Here we demonstrate that allowing human P450 reductase, CYP3A4, and cytochrome b5 to associate in an in vivo-like system, by coexpressing all three proteins together in Escherichia coli for the first time, the turnover of both nifedipine and testosterone by CYP3A4 is increased in the presence of cytochrome b5. The turnover of testosterone was increased by 166% in whole cells and by 167% in preparations of bacterial membranes. The coexpression of cytochrome b5 also resulted in the stabilization of the P450 during substrate turnover in whole E. coli, with 109% of spectrally active CYP3A4 remaining in cells after 30 min in the presence of cytochrome b5 compared with 43% of the original P450 remaining in cells in the absence of cytochrome b5.

Cloning, Molecular↗
PubMed1999

Nitric oxide synthases catalyze the activation of redox cycling and bioreductive anticancer agents.

Nitric oxide synthases (NOSs) play a crucial role in the control of blood flow, memory formation, and the immune response. These proteins can be structurally divided into oxygenase and reductase domains. The reductase domain shares a high degree of sequence homology with P450 reductase, which is thought to be the major enzyme responsible for the one-electron reduction of foreign compounds, including bioreductive antitumor agents currently undergoing clinical trials. In view of the structural similarities between NOS and P450 reductase, we investigated the capacity of NOS to reduce the hypoxic cytotoxin tirapazamine, the antitumor agent doxorubicin, and also the redox cycling compound menadione. All three isoforms exhibited high levels of activity toward these compounds. In the case of doxorubicin and menadione, the activity of NOS II was 5-10-fold higher than the other enzymes, whereas with tirapazamine, the activities were broadly similar. NOS-mediated metabolism of tirapazamine resulted in a large increase in plasmid DNA strand breaks, demonstrating that the reduction was a bioactivation process. In addition, tirapazamine inhibited NOS activity. Because nitric oxide is implicated in maintaining tumor vascular homeostasis, it is conceivable that tirapazamine could potentiate its own toxicity by increasing the degree of hypoxia. This study suggests that the NOSs could play a key role in the therapeutic effects of tirapazamine, particularly because NOS activity is markedly increased in several human tumors. In addition, the presence of NOS in the heart indicates that these enzymes may contribute to the cardiotoxicity of redox cycling drugs, such as doxorubicin.

Animals↗
PubMed1999

Determinants of specificity for aflatoxin B1-8,9-epoxide in alpha-class glutathione S-transferases.

We have used homology modelling, based on the crystal structure of the human glutathione S-transferase (GST) A1-1, to obtain the three-dimensional structures of rat GSTA3 and rat GSTA5 subunits bound to S-aflatoxinyl-glutathione. The resulting models highlight two residues, at positions 208 and 108, that could be important for determining, either directly or indirectly, substrate specificity for aflatoxin-exo-8,9-epoxide among the Alpha-class GSTs. Residues at these positions were mutated in human GSTA1-1 (Met-208, Leu-108), rat GSTA3-3 (Glu-208, His-108) and rat GSTA5-5 (Asp-208, Tyr-108): in the active rat GSTA5-5 to those in the inactive GSTA1-1; and in the inactive human GSTA1-1 and rat GSTA3-3 to those in the active rat GSTA5-5. These studies show clearly that, in all three GSTs, an aspartate residue at position 208 is a prerequisite for high activity in aflatoxin-exo-8,9-epoxide conjugation, although this alone is not sufficient; other residues in the vicinity, particularly residues 103-112, are important, perhaps for the optimal orientation of the aflatoxin-exo-8,9-epoxide in the active site for catalysis to occur.

Aflatoxin B1↗
PubMed1999

Regulation of JNK signaling by GSTp.

Studies of low basal Jun N-terminal kinase (JNK) activity in non-stressed cells led us to identify a JNK inhibitor that was purified and identified as glutathione S-transferase Pi (GSTp) and was characterized as a JNK-associated protein. UV irradiation or H2O2 treatment caused GSTp oligomerization and dissociation of the GSTp-JNK complex, indicating that it is the monomeric form of GSTp that elicits JNK inhibition. Addition of purified GSTp to the Jun-JNK complex caused a dose-dependent inhibition of JNK activity. Conversely, immunodepleting GSTp from protein extracts attenuated JNK inhibition. Furthermore, JNK activity was increased in the presence of specific GSTp inhibitors and a GSTp-derived peptide. Forced expression of GSTp decreased MKK4 and JNK phosphorylation which coincided with decreased JNK activity, increased c-Jun ubiquitination and decreased c-Jun-mediated transcription. Co-transfection of MEKK1 and GSTp restored MKK4 phosphorylation but did not affect GSTp inhibition of JNK activity, suggesting that the effect of GSTp on JNK is independent of the MEKK1-MKK4 module. Mouse embryo fibroblasts from GSTp-null mice exhibited a high basal level of JNK activity that could be reduced by forced expression of GSTp cDNA. In demonstrating the relationships between GSTp expression and its association with JNK, our findings provide new insight into the regulation of stress kinases.

3T3 Cells↗
PubMed1999

Merits and limitations of recombinant models for the study of human P450-mediated drug metabolism and toxicity: an intralaboratory comparison.

A wide variety of pharmacological and toxicological properties of drugs are determined by cytochrome P450-mediated metabolism. Characterization of these pathways and of the P450 isoenzymes involved constitutes an essential part of drug development. Similarly, because P450s are catalyzing the toxication and detoxication of environmental pollutants, an understanding of these reactions facilitates risk assessment in environmental toxicology. Recently, a variety of recombinant expression systems has been employed to study the role of human P450s in these reactions. These include insect, bacterial, yeast, and mammalian models. As these were developed and characterized by different laboratories, evaluation of their merits and limitations is inherently difficult. To resolve this problem, we have established and characterized the latter three systems and present the key results here. In general, the catalytic properties of P450 isozymes in the various models were rather similar. However, taking technical considerations into account as well as the high level of functional expression of P450s achieved in bacteria make this system ideally suited for drug metabolism research, including the generation of milligram quantities of metabolites for structural determinations. For toxicological studies, however, expression of P450s in mammalian cells was most appropriate. This is exemplified here by studies into the role of human P450s in the activation and inactivation of chemotherapeutic drugs.

Amino Acid Sequence↗
PubMed1999

Characterization of cytochrome P450 expression in human oesophageal mucosa.

The expression of cytochrome (CYP) P450 enzymes in human oesophageal mucosa was investigated in a total of 25 histologically non-neoplastic surgical tissue specimens by using specific antibodies in immunoblots and by RT-PCR mRNA analysis. The presence of CYP1A, 2E1, 3A and 4A enzymes was demonstrated by both techniques; CYP2A reactive protein was also detected by immunoblot. The presence of CYP4B1 mRNA was established but no specific antibody was available for detection of the corresponding protein by immunoblot. CYP2B6/7 mRNA was not detected in any sample. The mRNA transcripts for CYP1A1, 2E1, 4A11 and 4B1 were consistently detected in the majority of samples (>84%), whereas CYP1A2 mRNA was only detected in 11 of 19 specimens examined. An RT-PCR method to differentiate CYP3A4 and 3A5 mRNA was developed. This demonstrated CYP3A5 mRNA expression in all samples tested, whereas CYP3A4 mRNA was not detectable, suggesting that CYP3A5 is the major CYP3A protein in human oesophagus. There were significant interindividual variations in the amount of proteins, ranging from 8-fold for CYP4A to 43-fold for CYP2E1. For each patient, data on exposure to risk factors for oesophageal cancer were available, including tobacco smoke, alcohol, gastro-oesophageal reflux and hot beverage consumption. None of these risk factors or other patient characteristics (age, sex, tumour location and tumour stage) were correlated with the protein level of the individual CYP enzymes as determined by quantitation of immunoblot staining. However, the small series of samples precludes any strong conclusion concerning the lack of such correlations. There were no differences between squamous cell carcinomas and adenocarcinomas in either the qualitative or quantitative expression of the CYP enzymes. These data demonstrate that a range of CYP enzymes are expressed in human oesophageal mucosa and indicate that this tissue has the capacity to activate chemical carcinogens to reactive DNA binding metabolites.

Adult↗
PubMed1999

Polymorphisms in NAT2, CYP2D6, CYP2C19 and GSTP1 and their association with prostate cancer.

The development of prostate cancer is dependent on heredity, androgenic influences, and exposure to environmental agents. A high intake of dietary fat is associated with an increased risk of prostate cancer, either through influence on steroid hormone profiles or through production of carcinogenic compounds that require biotransformation by enzymes. The polymorphic glutathione S-transferase (GST), N-acetyltransferase (NAT), and cytochrome P450 (CYP) enzymes are of particular interest in prostate cancer susceptibility because of their ability to metabolize both endogenous and exogenous compounds, including dietary constituents. Association between different NAT2, CYP2D6, CYP2C19 and GSTP1 genotypes and prostate cancer was studied in a Swedish and Danish case-control study comprising 850 individuals. The combined Swedish and Danish study population was analysed by polymerase chain reaction for the NAT2 alleles *4, *5A, *5B, *5C, *6 and *7, and for the CYP2D6 alleles *l, *3 and *4. The Swedish subjects were also analysed for the CYP2C19 alleles *1 and *2, and the GSTP1 alleles *A, *B and *C. No association was found between prostate cancer and polymorphisms in NAT2, CYP2D6, CYP2C19 or GSTP1. An association between CYP2D6 poor metabolism and prostate cancer was seen among smoking Danes; odds ratio 3.10 (95% confidence interval 1.07; 8.93), P = 0.03, but not among smoking Swedes; odds ratio 1.19 (95% confidence interval 0.41; 3.42), P = 0.75. Smoking is not a known risk factor for prostate cancer, and the association between CYP2D6 poor metabolism and prostate cancer in Danish smokers may have arisen by chance.

Acetylation↗
PubMed1999

Crystal structure of the FMN-binding domain of human cytochrome P450 reductase at 1.93 A resolution.

The crystal structure of the FMN-binding domain of human NADPH-cytochrome P450 reductase (P450R-FMN), a key component in the cytochrome P450 monooxygenase system, has been determined to 1.93 A resolution and shown to be very similar both to the global fold in solution (Barsukov I et al., 1997, J Biomol NMR 10:63-75) and to the corresponding domain in the 2.6 A crystal structure of intact rat P450R (Wang M et al., 1997, Proc Nat Acad Sci USA 94:8411-8416). The crystal structure of P450R-FMN reported here confirms the overall similarity of its alpha-beta-alpha architecture to that of the bacterial flavodoxins, but reveals differences in the position, number, and length of the helices relative to the central beta-sheet. The marked similarity between P450R-FMN and flavodoxins in the interactions between the FMN and the protein, indicate a striking evolutionary conservation of the FMN binding site. The P450R-FMN molecule has an unusual surface charge distribution, leading to a very strong dipole, which may be involved in docking cytochrome P450 into place for electron transfer near the FMN. Several acidic residues near the FMN are identified by mutagenesis experiments to be important for electron transfer to P4502D6 and to cytochrome c, a clear indication of the part of the molecular surface that is likely to be involved in substrate binding. Somewhat different parts are found to be involved in binding cytochrome P450 and cytochrome c.

Animals↗
PubMed1999

Treatment of mammalian cells with the endoplasmic reticulum-proliferator compactin strongly induces recombinant and endogenous xenobiotic metabolizing enzymes and 3-hydroxy-3-methylglutaryl-CoA reductase in vitro.

Some xenobiotics induce membrane-bound drug metabolizing enzymes (Xme) and a profound proliferation of the endoplasmic reticulum (ER) in vivo. However these effects are much weaker in vitro, possibly due to absence of certain transcription factors. We tested the possibility that ER proliferation can affect the level of ER-resident enzymes even in the absence of transcriptional activation. For this purpose we analysed the effects of compactin, which has been shown to induce ER proliferation in vitro, on recombinant Xme, which were expressed from a constitutive viral promoter. High levels of recombinant UDP-glucuronosyltransferase UGT1A6 were achieved by amplification of the UGT1A6 cDNA using the dihydrofolate reductase cDNA as selectable marker in DHFR- CHO cells. Treatment of the resulting cell lines with lipoprotein-deficient serum in the absence and presence of compactin for 5 days resulted in a 1.3- and 2.3-fold, respectively, increase of the UGT enzyme activity towards 4-methylumbelliferone, paralleled by an induction of immunoreactive UGT1A6 protein. Similarly, treatment with this 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor increased the endogenous P450 reductase activity 2.6-fold, concomitant with an increase of immunodetectable protein. As expected compactin induced the level of 3-hydroxy-3-methylglutaryl-CoA reductase. Increased levels of this protein have been associated with a proliferation of the ER. Compactin treatment of a separate cell line that expressed recombinant human P450 reductase increased this enzyme activity fivefold. Pulse-chase experiments revealed that the induction of the recombinant Xme by compactin was most likely due to decreased protein degradation. Our results show that enzyme systems unrelated to those involved in cholesterol biosynthesis are affected by compounds known to affect membrane biogenesis. Since this effect extends to heterologously expressed enzymes, it also provides an efficient means by which to increase the levels of recombinant ER proteins.

Animals↗
PubMed1999

Pharmacogenetics.

Inter-individual variability in drug response is a major clinical problem. Adverse drug reactions (ADRs) are common, are responsible for a number of debilitating side effects following drug therapy and are a significant cause of death. It is now clear that much of the observed variability in drug response has a genetic basis, arising as a result of genetically-determined differences in drug absorption, disposition, metabolism or excretion. The best characterised pharmacogenetic polymorphisms are those within the phase I cytochrome P450 family of drug metabolising enzymes. One of these enzymes, CYP2D6 (debrisoquine hydroxylase), metabolizes one-quarter of all prescribed drugs and is inactive in 6% of the Caucasian population. Individuals at risk of developing ADRs as a result of genetically-determined variation in genes such as CYP2D6 can now be identified using DNA-based tests. A detailed knowledge of the genetic basis of individual drug response is potentially of major clinical and economic importance and could provide the basis for a rational approach to drug prescription. This would have significant benefits for human health.

Cytochrome P-450 Enzyme System↗
PubMed1999

Adaptive responses to environmental chemicals.

Adaption to chemical agents in the environment is a fundamental part of the evolutionary process and a large number of genes have evolved to specifically detoxify potentially harmful chemical agents. These genes can act at various levels within cells and determine circulating chemical or toxin concentrations, and uptake and efflux rates as well as intracellular detoxification enzymes, such as the cytochrome P-450-dependent mono-oxygenases and glutathione S-transferases. These multigene families of proteins play a central role in chemical and drug detoxification and their polymorphic expression may well be a factor in disease susceptibility.

Adaptation, Physiological↗
PubMed1999

Competition between cytochrome P-450 isozymes for NADPH-cytochrome P-450 oxidoreductase affects drug metabolism.

NADPH-cytochrome P-450 oxidoreductase (CPR) is essential for the catalytic activity of cytochrome P-450 (P-450). On a molar basis, the amount of P-450 exceeds that of CPR in human liver. In this study, we investigated whether drug-drug interactions can occur as a result of competition between P-450 isozymes for this ancillary protein. For this purpose, combinations of P-450 isozymes were coexpressed together with P-450 reductase in Escherichia coli. We show that testosterone inhibited the CYP2D6-mediated bufuralol 1'-hydroxylase activity in bacterial membranes containing both CYP2D6 and CYP3A4 but not in membranes containing CYP2D6 alone. Conversely, bufuralol inhibited the CYP3A4-mediated testosterone 6beta-hydroxylase activity in bacterial membranes containing both CYP3A4 and CYP2D6 but not in membranes containing only CYP3A4. In each case, inhibition was seen even at a P-450 to P-450 reductase ratio of 1.9:1, which is more favorable than the ratio of 4 reported for human liver. The physiological significance of this mechanism was demonstrated by the observation that testosterone inhibited several prototypical P-450 enzyme activities, such as bufuralol 1'-hydroxylase, coumarin 7-hydroxylase, and 7-ethoxyresorufin O-dealkylase, in human liver microsomes, but not if tested against a panel of bacterial membranes containing the human P-450 isozymes that mainly catalyze these reactions.

Aryl Hydrocarbon Hydroxylases↗
PubMed1999

Cytochrome P450 CYP2D6.

Altered expression of CYP2D6 (debrisoquine hydroxylase), resulting from genetic polymorphism at the CYP2D6 gene locus, is responsible for pronounced interindividual variation in the metabolism of many clinically important drugs. Although CYP2D6 substrates are structurally diverse, most are small molecules that interact with the protein via an electrostatic interaction between a basic nitrogen which is common to the majority of CYP2D6 substrates and an aspartic acid residue in the active site of the protein. As CYP2D6 substrates have a wide range of pharmacological functions, any variation in CYP2D6 expression can have profound clinical consequences. CYP2D6 activity can be determined both by phenotyping methods with a variety of probe drugs and by genotyping methods where PCR-based techniques are used to investigate the inheritance of individual CYP2D6 alleles. Allele frequencies have been shown to vary widely between populations of different racial origin. For example, the PM genotype is particularly rare in Orientals. The inheritance of certain CYP2D6 allelic variants has been associated with altered susceptibility to Parkinson's disease and several types of cancer.

Alleles↗
PubMed1999

Chapter 21. Additional polymorphisms and cancer.

The purpose of this chapter is to investigate as many of the less-studied genes as possible in relation to their importance in cancer susceptibility. For example, within the cytochrome P450 superfamily there are genes whose protein product is solely responsible for the detoxification of specific carcinogens. For a number of these genes, although genetic polymorphisms have not been identified, defined phenotypic polymorphisms have been demonstrated in specific populations. Other examples are given in which functional metabolic polymorphisms have been elucidated yet pertinent experiments for the determination of cancer risk have not been performed. The status of work relating to the genes in question is summarized and areas demanding future study are highlighted.

Alleles↗
PubMed1998

cis-parinaric acid is a ligand for the human peroxisome proliferator activated receptor gamma: development of a novel spectrophotometric assay for the discovery of PPARgamma ligands.

Peroxisome proliferator activated receptor gamma (PPARgamma) is the subject of intense investigation as a target for drugs against diabetes, atherosclerosis and cancer. For this reason there is considerable interest in the spectrum of compounds that bind this receptor. In this paper we have identified cis-parinaric acid (CPA) as a novel hPPARgamma ligand. The binding of this fatty acid to the receptor increases its fluorescence and causes a shift in the UV spectrum. This spectral shift is reversible by competition with other known ligands for PPARgamma. This report represents the first direct demonstration of a fatty acid binding to PPARgamma.

Base Sequence↗
PubMed1998

The repressor protein, Bm3R1, mediates an adaptive response to toxic fatty acids in Bacillus megaterium.

Bm3R1 is a helix-turn-helix transcriptional repressor from Bacillus megaterium whose binding to DNA is inhibited by fatty acids and a wide range of compounds that modulate lipid metabolism. The inactivation of Bm3R1/DNA binding activity results in the activation of transcription of the operon encoding a fatty acid hydroxylase, cytochrome P450 102. The metabolic role of this operon is unknown. It is possible that it is involved in the synthesis of modified fatty acids as part of normal cellular metabolism or may represent a protective mechanism by which B. megaterium detoxifies harmful foreign lipids. In this report we demonstrate that polyunsaturated fatty acids (PUFA) activate the transcription of the CYP102 operon. These PUFA are the most potent activators of the CYP102 operon observed to date, and we show that their effects are due to binding directly to Bm3R1. In addition, cultures that have been treated with the CYP102 inducer, nafenopin, are protected against PUFA toxicity. Resistance to PUFA toxicity is also seen in a Bm3R1-deficient strain that constitutively expresses CYP102. The resistant phenotype of this Bm3R1 mutant strain is reversed by specific chemical inactivation of CYP102. These data demonstrate that Bm3R1 can act as a direct sensor of toxic fatty acids and, in addition, provide the first direct evidence of fatty acids binding to a prokaryotic transcription factor.

Adaptation, Physiological↗
PubMed1998

Differential modulation of CYP2E1 activity by cAMP-dependent protein kinase upon Ser129 replacement.

Many toxic compounds are activated by cytochrome P450 (CYP) 2E1 to reactive metabolites, which represents a potential hazard for cellular homeostasis. Therefore knowledge about CYP2E1 regulation could be of great biological importance. It has been shown that CYP2E1 is controlled transcriptionally and post-translationally by phosphorylation. In the present study we investigated the role of serine-129 (Ser129) in the protein kinase A (PKA) recognition sequence motif Arg-Arg-Phe-Ser129. To gain further insights into the possible relevance of Ser129 for CYP2E1 function, Ser129 was replaced by alanine (Ala) or glycine (Gly) by site-directed mutations of the cDNA coding for CYP2E1. The mutant cDNAs were transfected into Chinese hamster lung fibroblast V79 cells. Despite the mutation in the PKA phosphorylation motif, all strains produced catalytically active CYP2E1. However, there was a marked change in the substrate preference: The Gly129-containing strains hydroxylated p-nitrophenol (PNP) to a markedly higher extent than the wild-type cDNA-containing cells, while they demethylated N-nitrosodimethylamine (NDMA) to a markedly lower extent than the wild-type cells. All the strains activated NDMA to mutagenic products. Treatment with the membrane-permeating cAMP derivative db-cAMP reduced markedly both the PNP hydroxylase and the NDMA demethylase activities as well as the mutation frequency induced by NDMA in the Ser129-containing strain. This decrease in activity was not accompanied by a decrease in CYP2E1 content. In addition, the catalytic activities of CYP2E1 were decreased in microsomes from rat hepatocytes treated with db-cAMP. Also in this case, the decrease in activities was not accompanied by a decrease in enzyme protein. These findings argue that involvement of Ser129 and its phosphorylation is not in determining CYP2E1 protein level, but rather in controlling its catalytic activity. In contrast, in the strains containing Ala129 or Gly129, treatment with db-cAMP caused a marked increase in both PNP hydroxylase and NDMA demethylase. In these strains a similar db-cAMP-mediated increase was also observed in the mutation frequency, resulting from the treatment with the promutagen NDMA, which is activated by CYP2E1. Our results show that CYP2E1 in V79 cells responds in two separate ways to db-cAMP exposure depending on the amino acid residue present in the PKA recognition sequence. The enzyme is committed to a negative regulation by db-cAMP if Ser129 is the target amino acid for PKA, leading to a decrease in the metabolic activation to mutagenic and carcinogenic species. On the other hand, Ala129 or Gly129 substitution directed CYP2E1 toward a positive regulation by increasing its catalytic activities and metabolic activation to mutagenic intermediates in the presence of db-cAMP. We also obtained evidence that cAMP-mediated downregulation of wild-type (Ser129) CYP2E1 was not accompanied by its destruction but instead by its stabilization, which shows that Ser129 is not involved in CYP2E1 degradation but dictates requirements for its specific activities.

Amino Acid Substitution↗