Metabolic gene polymorphism frequencies in control populations.

Using the International Project on Genetic Susceptibility to Environmental Carcinogens (GSEC) database containing information on over 15,000 control (noncancer) subjects, the allele and genotype frequencies for many of the more commonly studied metabolic genes (CYP1A1, CYP2E1, CYP2D6, GSTM1, GSTT1, NAT2, GSTP, and EPHX) in the human population were determined. Major and significant differences in these frequencies were observed between Caucasians (n = 12,525), Asians (n = 2,136), and Africans and African Americans (n = 996), and some, but much less, heterogeneity was observed within Caucasian populations from different countries. No differences in allele frequencies were seen by age, sex, or type of controls (hospital patients versus population controls). No examples of linkage disequilibrium between the different loci were detected based on comparison of observed and expected frequencies for combinations of specific alleles.

Adrenal 21-hydroxylase gene mutations in Slovenian hyperandrogenic women: evaluation of corticotrophin stimulation and HLA polymorphisms in screening for carrier status.

OBJECTIVE: To study the incidence of 21-hydroxylase deficiency in Slovenian hyperandrogenic women, at the gene level. Previous endocrine studies indicated large differences in the incidence of 21-hydroxylase deficiency in hyperandrogenic women. The predictive values of the 17-hydroxyprogesterone (17-OHP) response to ACTH stimulation and of HLA typing in screening for carrier status were re-evaluated. DESIGN: Molecular analysis of CYP21 gene, ACTH stimulation and human leucocyte antigen (HLA) typing were performed in 83 consecutive Slovenian hyperandrogenic women. MEASUREMENTS: Cortisol and 17-OHP concentrations were measured at baseline and 60 min after ACTH stimulation. Basal adrenal androgen concentrations were also measured. RESULTS: None of 83 hyperandrogenic patients was affected with non-classical 21-hydroxylase deficiency, but 12 of 81 patients (14.8%) had high concentrations of 17-OHP after stimulation, indicative of carrier status. The increase in 17-OHP concentrations could be explained by a carrier status for CYP21 gene mutations in only three of 12 patients (25%), whereas seven of 69 patients (10. 1%) with normal concentrations of 17-OHP after stimulation were found to be carriers of CYP21 gene mutations, indicating low positive predictive values of ACTH stimulation as a screening test for carriers of 21-hydroxylase deficiency. In total, 11 carriers were identified among 83 patients: seven CYP21 gene deletions/conversions, two Gln(318)Stop and one Val(281)Leu mutation and one gene conversion extending from exon 4 to exon 7 were found. The association between Val(281)Leu mutation and HLA-B14 antigen was confirmed in this Slovenian population. CONCLUSIONS: Basal or ACTH-stimulated 17-OHP concentrations are not a good indicator of the carrier status for 21-hydroxylase deficiency among Slovenian hyperandrogenic patients. Reliable screening for carriers of 21-hydroxylase deficiency is possible only by molecular analysis of the CYP21 gene.

Mutational spectrum of congenital adrenal hyperplasia in Slovenian patients: a novel Ala15Thr mutation and Pro30Leu within a larger gene conversion associated with a severe form of the disease.

OBJECTIVE: To analyse the mutational spectrum, the associated haplotypes and the genotype-phenotype correlation, and to design a reliable and rational approach for CYP21 mutation detection in Slovenian congenital adrenal hyperplasia (CAH) patients. DESIGN: Molecular analysis of the CYP21 gene was performed in 36 CAH patients and 79 family members. METHODS: Southern blotting, sequence-specific PCR amplification (PCR-SSP), sequence-specific oligonucleotide hybridisation (PCR-SSO) and sequencing were used to detect CYP21 gene deletions, conversions and point mutations. RESULTS: CYP21 gene deletion was the most frequent mutation (36.4%). Large gene conversions detectable only by Southern blotting represented 12.1%, and gene conversions involving the promoter region represented 7.6% of the mutated alleles. The most frequent point mutations were: intron 2 splice mutation 16.7%, Ile172Asn mutation 7.6%, Gln318Stop 7.5% and Pro30Leu 12.2% of alleles. A correlation between the genotype and the clinical phenotype similar to those described for large populations was observed. The finding of Pro30Leu mutation linked to a gene conversion could explain the simple virilising (SV) phenotype in compound heterozygotes for the Pro30Leu and a severe mutation. In two siblings with a salt wasting form of CAH (SW-CAH), a novel mutation Ala15Thr was found on the allele characterised by Pro30Leu mutation and gene conversion involving the promoter region. CONCLUSIONS: Our genotyping approach allowed reliable diagnosis of CAH in the Slovenian population. The high frequency of CYP21 gene aberrations on Pro30Leu positive alleles justified systematic searching for a gene conversion in the promoter region using the PCR-SSP reaction.

The role of cytochrome P450(11 alpha) in detoxification of steroids in the filamentous fungus Rhizopus nigricans.

The evidence was presented that steroid hydroxylating enzyme complex induced by substrate in the filamentous fungus Rhizopus nigricans (R. nigricans) alleviated toxic effect(s) of the steroid on fungal growth. The growth inhibition of fungal mycelium observed in steroid-containing culture(s) became much more obvious when fungal mycelia were grown in the simultaneous presence of inducing steroid and the P450(11 alpha) inhibitor metyrapone. On the other hand, in experiments where we followed the fate of radioactively labelled progesterone added to the mycelial suspension, we noticed that steroid, after being initially accumulated in the microorganism, was, after some time, released from it; the latter phenomenon was not observed if induction of 11 alpha-hydroxylase was prevented by cycloheximide. Results of experiments presented in this communication can be regarded as the first strong indication that the biological role of P450(11 alpha) induction in R. nigricans is in removal of steroids which are toxic for the mycelium.

Estradiol binding components in intestinal mucosa of female rats.

The presence of estrogen binding components (EBC) in intestinal mucosa of female rats was investigated by competitive-binding assay using radiolabelled and nonlabelled estradiol 17 beta (E2). EBC were found exclusively in the nuclear fraction and were absent from the cytosolic and from the microsomal fractions. Two types of nuclear EBC with different binding characteristics and capacities were found: Kd1 = 4.8 +/- 0.8 nM, n1 = 18.4 +/- 4.2 fmol/mg protein (n1 = 83.4 +/- 12.5 fmol/mg DNA) and Kd2 = 31.1 +/- 6.8 nM, n2 = 91.1 +/- 18.5 fmol/mg protein (412.7 +/- 80.0 fmol/mg DNA). Type 1 component showed slightly greater affinity for estrogens as compared to progesterone and dexamethasone whereas type 2 component bound other competitors with even greater affinity than E2.

The properties of cytochrome P-450 induced in the rat small intestine by estradiol.

The properties of cytochrome P-450 induced in the rat small intestine by estradiol were investigated. The interaction of substrates with intestinal microsomal cytochrome P-450 was compared to that of the enzyme induced in the rat liver by phenobarbital. The results obtained indicate that in the rat small intestine estradiol increases the concentration of the enzyme which differs from the liver type cytochrome P-450 but resembles the liver type cytochrome P-448. The difference spectroscopy data were supported by a parallel study on the action of specific inhibitors of the hydroxylation reaction.

Effect of estrogen pretreatment on the intestinal cytochrome P-450 in female rats.

The effect of estrogen hormones on the cytochrome P-450-linked hydroxylation system of rat small intestine was investigated. Estrogens were found to increase the activity of the intestinal arylhydrocarbon hydroxylase as well as the level of cytochrome P-450 in female rats. There was essentially no difference in the increase of the concentration of cytochrome P-450 due to the duration of pretreatment.

Genetic susceptibility to environmental carcinogenesis in Slovenian melanoma patients.

The skin acts as the first defence barrier against external environmental pollutants, including chemicals and UV radiation. Cytochrome P450 CYP1A1 and glutathione S-transferases (GSTs) found in melanocytes and skin basal layers were shown to participate both in the metabolism of xenobiotics and in detoxification of reactive oxygen species (ROS). In our study we analysed the distribution of single and combined CYP1A1, GSTM1, GSTT1 and GSTP1 genotypes contributing to inter-individual differences in metabolism of xenobiotics and ROS in 125 Slovenian healthy individuals and in 140 patients with sporadic malignant melanoma. Our results showed no statistically significant differences between melanoma patients and healthy controls in the frequency of polymorphic CYP1A1 and GST genotypes. The risk of developing melanoma was not significantly increased in individuals homo- or heterozygous for the CYP1A1*2A allele combined with GSTM1*0 genotype (OR: 1.86; 95% CI: 0.36-7.71), but increased slightly in carriers of CYP1A1*2A combined with both GSTM1*0 and GSTT1*0 genotypes (OR: 3.42; 95% CI: 0.36-29.6). Our results indicate that factors other than the polymorphic genes coding xenobiotic metabolising enzymes play a major role in protection against environmental carcinogenesis in human skin.

Influence of CYP2C9 polymorphisms, demographic factors and concomitant drug therapy on warfarin metabolism and maintenance dose.

Warfarin is an anticoagulant drug with narrow therapeutic index and high interindividual variability in dose requirement. S-warfarin is metabolized mainly by polymorphic cytochrome P450 (CYP) 2C9. We systematically quantified the influence of CYP2C9 genotype, demographic factors and concomitant drug treatment on warfarin metabolism and maintenance dose. The mean warfarin doses were lower in carriers of one (2.71 mg/day, 59 patients) and two polymorphic alleles (1.64 mg/day, 11 patients) than in carriers of two wild-type alleles (4.88 mg/day, 118 patients). Multiple regression analysis demonstrated that CYP2C9 genotype, age, concomitant treatment with warfarin metabolism inducers and lean body weight contributed significantly to interindividual variability in warfarin dose requirement (adjusted R(2)=0.37). The same factors, except for age, significantly influenced S-warfarin clearance (adjusted R(2)=0.42). These results can serve as a starting point for designing prospective studies in patients in the initiation phase of genotype-based warfarin therapy.

Purification of cytochrome P450 from filamentous fungus Rhyzopus nigricans.

It has been reported in our previous studies that steroid hydroxylation system of Rhizopus nigricans involves cytochrome P450 and NADPH-cytochrome P450 reductase in the electron transport system. Both enzymes are membrane bound and are located in the microsomal preparations of progesterone induced fungal mycelia. In order to identify and characterize the cytochrome P450 component of fungal monoxygenase system, microsomal proteins from induced mycelia were subjected to HIGH Q anion exchange and MONO P (FPLC) anion exchange chromatography. Four fractions containing cytochrome P450 have been resolved on MONO P column. They exhibit CO difference spectra and type II difference spectra with ketoconazole.

Purification and characterization of NADPH-cytochrome P450 reductase from filamentous fungus Rhizopus nigricans.

We report here the isolation and partial characterization of a flavoprotein, NADPH-cytochrome P450 (cytochrome c) reductase. The enzyme is a part of steroid 11 alpha-hydroxylating system and is associated with the microsomal fraction of the fungus Rhizopus nigricans. Fungal reductase was solubilized from microsomal membranes with Triton X-100 and purified to apparent homogeneity by affinity and high-performance ion-exchange chromatography. A 350-fold purification of the enzyme with specific activity of 37 mumol cytochrome c reduced/min/mg protein was achieved. A single protein band was obtained on SDS-PAGE analysis with an apparent molecular weight of 79 kDa. Purified reductase contained approximately equimolar quantities of flavin adenine dinucleotide and flavin mononucleotide per mole of the enzyme. Upon induction of the steroid hydroxylating system with progesterone the activity of microsomal NADPH-cytochrome c (P450) reductase increased 10-fold. This is in good correlation with the increase in content of fungal cytochrome P450. Purified fungal flavoprotein was active in a reconstituted system with cytochrome P450 C21 from adrenal gland but could not replace adrenodoxin reductase in the mitochondrial steroid 11 beta-hydroxylating system. We were able to confirm the role of the enzyme by reconstituting steroid 11 alpha-hydroxylating activity from the separated components NADPH-cytochrome P450 reductase and cytochrome P450, partly purified from fungal microsomes.

Human CYP2D6 gene polymorphism in Slovene cancer patients and healthy controls.

The polymorphic CYP2D6 gene encoding debrisoquine hydroxylase has attracted much interest for its possible role in human pulmonary carcinogenesis. The purpose of this work was to determine the frequency of poor metabolizers (PM) and extensive metabolizers (EM) of debrisoquine in Slovene population of healthy individuals (n = 107), lung cancer patients (200) and melanoma patients (121). Polymorphism of CYP2D6 gene was studied by genotyping based on PCR analysis of the intron 3 exon 4 junction containing G to A mutation and one base pair deletion in exon 5, which are responsible for approximately 95% of poor metabolizer phenotype in Caucasians. In the healthy Slovene population 62.5% of individuals were identified as extensive metabolizers, 31% as extensive-heterozygous metabolizers and 6.5% as poor metabolizers of debrisoquine. The frequency of EM individuals was 70.5% in lung cancer patients and 64% in melanoma patients, the frequency of extensive-heterozygous subjects was 27% in lung cancer patients and 31% in melanoma patients. The frequency of PM individuals in the lung cancer patients was 2.5% and in melanoma patients 5%. The decrease in PM genotype in the group of Slovene lung cancer patients is similar to the decrease published for some other ethnic groups. Our results support the hypothesis that polymorphic CYP2D6 gene probably plays some, though not a prevalent role in chemical carcinogenesis. Poor metabolizer individuals appear to be less susceptible to lung cancer than EM individuals.

Induction of hsp70 in the fungus Rhizopus nigricans.

We characterised two positive clones, RnH20/2 and RnH2/3, isolated from the cDNA library prepared from the fungus Rhizopus nigricans previously exposed to heat shock. Nucleotide sequences of both cDNA clones contained open reading frames for polypeptides with a significant amino acid identity to each other and to cytoplasmic members of Hsp70s from different eukaryotic organisms. Northern blot analysis, using a fragment of RnH2/3 as a probe, revealed that in the fungus R. nigricans the Hsp70 transcripts were inducible with deoxycorticosterone and testosterone as well as with heat stress, ethanol, CuSO(4), and H(2)O(2). This is the first report on isolation of cDNAs encoding Hsp70s from the fungal phylum Zygomycota.

Ion binding to lysine-modified derivatives of cytochrome c.

The effect of Cl- and K+ ions on the apparent equilibrium constant of the reaction between horse ferricytochrome c and potassium ferrocyanide was studied. Unmodified cytochrome was compared with two lysine-modified derivatives. One, guanidinated, had all lysyl groups converted to homoarginine (but retained the same positive charge); the other was trinitrophenylated at one lysine (measured spectrophotometrically). Both modified derivatives had a somewhat larger equilibrium constant in the reaction of the reduced protein with ferricyanide, but, unlike trifluoroacetylated cytochrome c (which has a negative charge), the redox properties were not dramatically different. The native protein and the lysine-modified cytochromes showed differential K+ binding in Tris-cacodylate buffer at constant ionic strength (0.003-0.005 M). More K+ was bound to ferrocytochrome c. This redox-linked binding, however, was unaffected by modification of lysine. All three derivatives also showed redox-linked differential Cl- ion binding (more Cl- ion was bount to ferricytochrome); however, in this case, the binding was reduced in the lysine-modified molecules. This was interpreted as loss of a single anion site. This anion site critically depends on one or a few lysines which are more reactive with trinitrobenzene sulfonate.

Genetic polymorphism of xenobiotic metabolising enzymes in Slovenian lung cancer patients.

Most carcinogenic substances require metabolic activation in order to become ultimate carcinogens. Genetic polymorphism of xenobiotic metabolising enzymes cytochromes P450 may therefore influence human cancer susceptibility. The aim of our study was to investigate if CYP1A1 gene polymorphism contributes to lung cancer susceptibility in Slovenian patients. Two polymorphic sites in CYP1A1 gene were analysed in DNA samples from 100 healthy controls and 199 lung cancer patients using genotyping approach. Our results indicate that CYP1A1 may be one of the factors determining susceptibility to squamous cell carcinoma of lung in Slovenian population. However the frequency of CYP1A1 polymorphisms is too low to be a potentially useful marker of increased lung cancer risk.

Isolation, partial length sequence and expression of steroid inducible hps 70 gene from Rhizopus nigricans.

Previous studies have shown that filamentous fungus Rhizopus nigricans responds to addition of different steroids into growth medium with induction of hydroxylation system and that some steroids provoke stress response. The purpose of this study was to investigate whether those steroids provoke induction of Hsp70 gene(s), well studied markers of stress response in different cells and organisms. The expression studies of fungal Hsp70 gene(s) using Northern blot analysis showed that fungal hsp70 mRNA was upregulated after treatment of mycelia with deoxycorticosterone and testosterone, but not after exposure to progesterone. In addition, expression of fungal Hsp70 mRNA was elevated after exposure of mycelia to heat shock (32 degrees C), ethanol, heavy metal (CuSO4), and oxidative stressor (H2O2), whereas treatment of mycelia with osmotic stressor (KCl) didn't have any influence on stress protein expression. The partial nucleotide sequence and deduced amino acid sequence homology search revealed that the cDNA clone (lambda hs20/2), isolated from cDNA library prepared from heat shock treated fungal mycelia, contained Hsp70 gene of DnaK subfamily.

Comparison of two monooxygenase systems with cytochrome P450 in filamentous fungus Rhizopus nigricans.

Besides the progesterone inducible steroid hydroxylase which catalyses the transformation of progesterone to 11alpha-hydroxyprogesterone the presence of another monooxygenase system in filamentous fungus Rhizopus nigricans (R. nigricans)--lanosterol demethylase was confirmed. Lanosterol demethylase was not inducible with progesterone. After subcellular fractionation both monooxygenase systems were found in microsomal fraction of fungus R. nigricans. To find out how to differentiate between the hydroxylase and demethylase systems the influence of inhibitors ketoconazole and metyrapone on both monooxygenase systems was studied. Both substances efficiently inhibited fungal steroid hydroxylase. Spectral studies used to characterize the interaction of inhibitors with cytochromes P450 showed that both inhibitors bind to induced fungal preparations whereas in noninduced fungal preparations interaction with P450 was found only with ketoconazole. This indicated the difference in interaction of the two inhibitors on both monooxygenase systems present in fungus R. nigricans.

Localization of the gene encoding steroid hydroxylase cytochrome P-450 from Rhizopus nigricans inside a HindIII fragment of genomic DNA.

The gene encoding steroid inducible cytochrome P450 of Rhizopus nigricans ATCC 6227b has been found inside a HindIII fragment of the genomic DNA by hybridization with a partial length cDNA probe. The latter was isolated by immunoscreening a cDNA library prepared in the lambda gt11 expression system and identified on the basis of inducibility and sequence analysis. The nucleotide sequence of the cDNA probe revealed a coding sequence for the heme binding segment characteristic of the P450 gene family.

Neural regulation of muscle acetylcholinesterase is exerted on the level of its mRNA.

In rat muscles, AChE activity drops rapidly after denervation, and the patterns of AChE molecular forms in slow and fast muscles differ considerably. Both observations imply that muscle AChE is regulated by the motor nerve. In order to obtain a better insight into the underlying mechanism, AChE regulation in rat muscles was examined on the level of its catalytic subunit mRNA using northern blot analysis. The level of two AChE transcripts (2.4 and 3.2 kb) was much higher in the fast sternomastoid (STM) than in the slow soleus muscle, which explains the difference in AChE activity between the two types of muscles. Expression of AChE mRNA in the extrajunctional region of STM muscle is fairly high so that little difference in the level of AChE mRNAs was observed in comparison to the region rich in the neuromuscular junctions. This indicates that very high AChE activity in the neuromuscular junctions is achieved by unique posttranslational modifications and cellular processing of AChE enhancing stability of the junctional in comparison to the extrajunctional AChE. Denervation as well as botulinum toxin evoked paralysis of STM muscle caused rapid decline of AChE transcripts to almost undetectable levels both in the junctional and extrajunctional regions. The low level of AChE mRNA is therefore largely responsible for low AChE activity in denervated rat muscles. It seems that either muscle activity and/or quantal ACh release enhance the level of AChE mRNA in the junctional as well as extrajunctional regions. In rat muscles, extrajunctional mRNA level of the catalytic subunit of AChE is neurally regulated in exact opposite fashion from that of acetylcholine receptor subunits.

Resolution and reconstitution of the NADPH-cytochrome c (P-450) reductase induced by progesterone in Rhizopus nigricans.

The NADPH-cytochrome c (P-450) reductase induced in the filamentous fungus Rhizopus nigricans as a component of 11 alpha-hydroxylase of progesterone was resolved by DEAE-cellulose chromatography into two components. One of the components is an iron-sulfur protein (rhizoporedoxin), whereas the other component is a protein with reductase activity dependent on NADPH (rhizoporedoxin reductase). As shown in the reconstitution assay, the NADPH-cytochrome c (P-450) reductase activity was restored upon combination of these two proteins.