Andrea Sapone

CYP3A4 allelic variants with amino acid substitutions in exons 7 and 12 : Evidence for an allelic variant with altered catalytic activity

To determine the existence of mutant and variant CYP3A4 alleles in three racial groups and to assess functions of the variant alleles by complementary deoxyribonucleic acid (cDNA) expression.

The human peroxisome proliferator-activated receptor alpha gene: identification and functional characterization of two natural allelic variants.

Peroxisome proliferator-activated receptor (PPAR)alpha-null mice have a defect in fatty acid metabolism but reproduce normally. The lack of a detrimental effect of the null phenotype in development and reproduction opens up the possibility for null or variant PPARalpha gene (PPARA) alleles in humans. To search the coding region and splice junctions for mutant and variant PPARalpha alleles, the human PPARalpha gene was cloned and characterized, and sequencing by polymerase chain reaction was carried out. Two point mutations in the human gene were found in the DNA binding domain at codons for amino acids 131 and 162. The allele containing the mutation in codon 162 (CTT to GTT, L162V) designated PPARA*3, was found at a high frequency in a Northern Indian population. Transfection assays of this mutant showed that the non-ligand dependent transactivation activity was less than one-half as active as the wild-type receptor. PPARA*3 was also unresponsive to low concentrations of ligand as compared to the wild-type PPARA*1 receptor. However, the difference is ligand concentration-dependent; at concentrations of the peroxisome proliferator Wy-14 643 > 25 microM, induction activity was restored in this variants transactivation activity to a level five-fold greater as compared with wild-type PPARA*1 with no ligand. The mutation in codon 131 (CGA to CAA, R131Q), designated PPARA*2 is less frequent than PPARA*3, and the constitutive ligand independent activity was slightly higher than PPARA*1. Increasing concentrations of Wy-14 643 activated PPARA*2 similar to that observed with PPARA*1. The biological significance of these novel PPARalpha alleles remains to be established. It will be of great interest to determine whether these alleles are associated with differential response to fibrate therapy.

β-Carotene: a cancer chemopreventive agent or a co-carcinogen?

Evidence from both epidemiological and experimental observations have fueled the belief that the high consumption of fruits and vegetables rich in carotenoids may help prevent cancer and heart disease in humans. Because of its well-documented antioxidant and antigenotoxic properties, the carotenoid beta-carotene (betaCT) gained most of the attention in the early 1980s and became one of the most extensively studied cancer chemopreventive agents in population-based trials supported by the National Cancer Institute. However, the results of three randomized lung cancer chemoprevention trials on betaCT supplementation unexpectedly contradicted the large body of epidemiological evidence relating to the potential benefits of dietary carotenoids. Not only did betaCT show no benefit, it was associated with significant increases in lung cancer incidence, cardiovascular diseases, and total mortality. These findings aroused widespread scientific debate that is still ongoing. It also raised the suspicion that betaCT may even possess co-carcinogenic properties. In this review, we summarize the current data on the co-carcinogenic properties of betaCT that is attributed to its role in the induction of carcinogen metabolizing enzymes and the over-generation of oxidative stress. The data presented provide convincing evidence of the harmful properties of this compound if given alone to smokers, or to individuals exposed to environmental carcinogens, as a micronutrient supplement. This has now been directly verified in a medium-term cancer transformation bioassay. In the context of public health policies, while the benefits of a diet rich in a variety of fruits and vegetables should continue to be emphasized, the data presented here point to the need for consideration of the possible detrimental effects of certain isolated dietary supplements, before mass cancer chemoprevention clinical trials are conducted on human subjects. This is especially important for genetically predisposed individuals who are environmentally or occupationally exposed to mutagens and carcinogens, such as those found in tobacco smoke and in industrial settings.

Nomenclature for human DPYD alleles.

To standardize DPYD allele nomenclature and to conform with international human gene nomenclature guidelines, an alternative to the current arbitrary system is described. Based on recommendations for human genome nomenclature, we propose that each distinct allele be designed by DPYD followed by an asterisk and an Arabic numeral. The number specifies the key mutation and, where appropriate, a letter following the number indicates an additional mutation on the mutant allele. Criteria for classification as a distinct allele are also presented.

The clinical role of cytochrome p450 genotypes in Helicobacter pylori management

OBJECTIVE:The aim of this pharmacogenomics study was to investigate the influence of different cytochrome P450 (CYP) genotypes in Helicobacter pylori eradication therapy.METHODS:The study involved 143 consecutive Italian Caucasian patients with H. pylori infection diagnosed and treated with 1-wk triple therapy according to European Helicobacter Pylori Study Group guidelines. Using human genomic DNA, CYP2C19 (*2 and *3) and CYP3A4 alleles (*1B, *2, and *3) were evaluated by polymerase chain reaction–restriction fragment length polymorphism assays and confirmed by sequencing the amplicons.RESULTS:According to the endoscopy-based gold standard, 93 patients achieved H. pylori eradication. Regarding CYP2C19 genotype, the 50 patients who remained infected were all homozygous or heterozygous extensive metabolizers (homEM or hetEM). Carriers of homEM fared significantly less well than those of hetEM; homEM genotype was also predictive of failure at univariate/multivariate analysis. Carriers of CYP3A4 polymorphisms achieved favorable eradication rates similar to patients bearing CYP2C19. All four patients with single CYP3A4*2 polymorphism achieved eradication, and only 29% (5/17) of all CYP3A4*1B carriers did not achieve eradication. All nine patients carrying CYP3A4 polymorphisms in the CYP2C19 hetEM subgroup were cured, suggesting the possibility of a positive synergism between CYP3A4 and CYP2C19.CONCLUSIONS:This first pharmacogenomics study on the influence of different CYP genotypes on H. pylori therapy suggests that, as in Asian populations, CYP2C19 genotype patterns are probably also relevant in Caucasians receiving H. pylori eradication regimens that include omeprazole. The possibility of a favorable drug interaction mediated by CYP2C19 and CYP3A4 requires investigation.

Effect of licorice and glycyrrhizin on murine liver CYP-dependent monooxygenases

This study is aimed to investigate the effect of the prolonged intake of conspicuous amounts of licorice (LE), or its natural constituent glycyrrhizin (G) on murine liver CYP-catalyzed drug metabolism. For this purpose the modulation of the regio- and stereo-selective hydroxylation of testosterone, together with the use of highly specific substrates as probes for different CYP isoforms such as ethoxyresorufin (CYP1A1), methoxyresorufin (1A2), pentoxyresorufin (2B1), p-nitrophenol (2E1) and aminopyrine (3A), were investigated. Daily doses of licorice root extract (3,138 or 6,276 mg/kg b.w. per os), or G (240 or 480 mg/kg b.w. per os), were administered to different groups of Swiss Albino CD1 mice of both sexes for 1, 4 or 10 consecutive days. While a single LE or G dose was unable to affect the multienzymatic CYP-system, using both schedules of repeated treatment, either LE or G were able to significantly induce hepatic CYP3A- and, to a lesser extent, 2B1- and 1A2-dependent microsomal monooxygenase activities, as well as 6beta- (mainly associated to CYP3A), 2alpha-, 6alpha- (CYP2A1, 2B1), 7alpha-, 16alpha- (CYP2B9) and 16beta-testosterone hydroxylase (TH) activities in male and female mice. Data on CYP3A modulation, the major isoform present in human liver, was confirmed by using Western immunoblotting with anti-CYP3A1/2 rabbit polyclonal antibodies raised against purified rat CYP3A. Northern blotting analysis using CYP3A cDNA biotinylated probe showed that the expression of such isozyme is regulated at the mRNA level. These results suggest that the induction of cytochrome P450-dependent activities by the prolonged intake of high LE or G doses, may result in accelerated metabolism of coadministered drugs with important implications for their disposition. The adverse effects associated with CYP changes such as toxicity/cotoxicity and comutagenicity may also have clinical consequences.

Determination of lipophilicity by means of reversed-phase thin-layer chromatography: II. Influence of the organic modifier on the slope of the thin-layer chromatographic equation

The RM values measured for a series of steroids were very close to those determined more than 15 years ago using the same chemicals. This finding supports the reliability of RM values as a lipophilicity parameter. However, the point at issue in this work was the influence of the organic solvent in the mobile phase on the slopes of the TLC equations. In fact, the slopes of the TLC equations were shown to be related to the reciprocal of the solvent strength (1/E0). As a consequence, the ratio between the slopes of the TLC equations in different solvent systems are close to the ratio between the 1/E0 values for the corresponding solvent pairs. A further interesting aspect seems to arise from the analysis of the equations correlating slopes and intercepts of the TLC equations. In particular, the ratios between the b values of such equations in different solvent systems are close to the ratios between the corresponding E0 values.

Induction and suppression of cytochrome P450 isoenzymes and generation of oxygen radicals by procymidone in liver, kidney and lung of CD1 mice

Although chronic administration of procymidone (a widely used dicarboximide fungicide) leads to an increased incidence of liver tumors in mice, short-term genotoxicity studies proved negative. As cytochrome P450 (CYP) induction has been linked to non-genotoxic carcinogenesis, we investigated whether procymidone administration causes induction of CYP-dependent monooxygenases in liver, kidney and lung microsomes of male Swiss Albino CD1 mice after single or repeated (daily for three consecutive days) i.p. treatment with either 400 or 800 (1/10 or 1/20 of the DL(50)) mgkg(-1) b.w. procymidone. CYP content and CYP3A1/2, 1A1, 1A2, 2B1/2, 2E1, 2A, 2D9 and 2C11 supported oxidations were studied using either the regio- and stereo-selective hydroxylation of testosterone as multibiomarker or highly specific substrates as probes of various CYPs. While a single dose was uneffective, multiple procymidone administration lead to marked inductions of various monooxygenases: CYP3A1/2 in liver and lung (as measured by N-demethylation of aminopyrine and testosterone 6 beta-hydroxylase); CYP2E1 in liver (p-nitrophenol hydroxylation); CYP1A1 in liver and kidney (deethylation of ethoxyresorufin). Several hydroxylations were induced in the liver, including the CYP2A-linked 7 alpha (14-fold) as well as 6 alpha (22-fold), 6 beta, 16 beta and 2 beta hydroxylases. The pattern of inductions/suppressions recorded in the three different tissues suggests that procymidone exerts complex effects on the CYP profile. Tissue-specific trends included a large number of inductions in the liver and suppressions in the lung. The main inductions were corroborated by immunoblotting analyses and Northern blotting showed that inductions of CYP3A1/2, CYP2E1 and CYP1A1/2 were paralleled by increased mRNA levels. It was also found that CYP over-expression generates large amounts of reactive oxygen species (ROS), especially in liver. These data may explain why in vitro short-term genotoxicity studies on procymidone were negative, whereas in vivo long-term carcinogenesis studies turned out positive: long-term CYP induction (e.g. oxygen centered free radicals over-production) can have a co-carcinogenic and/or promoting potential.

Molecular non-genetic biomarkers related to Fenarimol cocarcinogenesis : organ- and sex-specific CYP induction in rat

Selective biochemical markers of effect have been used to evaluate some non-genotoxic cocarcinogenic properties of Fenarimol. Several CYP-dependent reactions have been monitored in liver, kidney and lung microsomes of male and female Sprague-Dawely rats treated (i.p.) with 200 or 400 mg/kg body wt dose of this pesticide. Highly specific substrates were used as probes of various isoforms, such as CYP1A1, 1A2, 2B1, 2E1 and 3A. A complex pattern of CYP induction, including organ- and sex-related differences in the inductive response by Fenarimol, has been recorded in this investigation, the kidney (mainly male) being more responsive when compared to other tissues. A 6.6-fold increase in the 2B1-like activity, probed by dealkylation of pentoxyresorufin was observed in the liver at a higher dose. On the contrary, a marked induction of CYP1A1 mediated ethoxyresorufin O-deethylase activity, ranging from 20- to 35-fold in female and male, respectively, was observed in the kidney at a lower dose tested. In the lung, at a higher dose, the p-nitrophenol hydroxylase activity (2E1) was enhanced up to 3.5-fold in male animals, whereas the 3A-like activity, probed by the N-demethylation of aminopyrine, was induced up to 2.6-fold in females. A weak, although significant reduction of CYP2B1 isoforms in lung was also recorded. Taken together, these data corroborated by means of Western immunoblotting analysis (using rabbit polyclonal antibodies anti-CYP 2B1/2, 1A1, 2E1, and 3A1/2) indicate a possible cotoxic, comutagenic cocancerogenic and promoting potential of this fungicide.

Induction of CYP2B1 mediated pentoxyresorufin O-dealkylase activity in different species, sex and tissue by prototype 2B1-inducers

The induction of CYP2B1 mediated pentoxyresorufin O-dealkylase (PROD) activity by various xenobiotics was explored in liver, kidney and lung from a variety of animal species of both sexes, in order to gain insights into the substrate specificity of induced CYPs. Marked species- and sex-related differences in the inducibility of PROD activity by tested chemicals were observed, the mouse being always more responsive when compared to hamster or rat. Induction by sodium phenobarbital (NaPB) led to a conspicuous increase in all situations, up to approximately 38-fold in female rat and mouse liver, with the exception of hamster kidney where PROD activity was only slightly affected. Unexpectedly, both sodium barbital (NaB) and phorone (PHR) moderately induce CYP2B1 isoforms in rat, the extent being highest in female kidney (PHR, 14-fold increase) and male lung (NaB, 4.5-fold). The degree of induction was maximal in the liver with some exceptions occurring in male mice where NaB induced up to 46- and 115-fold increases in lung and kidney and PHR up to 115-fold in kidney. Minimal, although significant induction of PROD activity following treatment with trans-1,2-dichloroethylene (1,2-DCE) occurred in all situations with the exception of hamster kidney and lung. Therefore, caution should be exercised when using PROD activity as specific enzymatic assay to probe CYP2B1-like induction.