Gene expression profiling of systems involved in the metabolism and the disposition of xenobiotics: comparison between human intestinal biopsy samples and colon cell lines.

Intestinal cell lines are used as in vitro models for pharmacological and toxicological studies. However, a general report of the gene expression spectrum of proteins that are involved in the metabolism and the disposition of xenobiotics in these in vitro systems is not currently available. To fill this information gap, we systematically characterized the expression profile of 377 genes encoding xenobiotic-metabolizing enzymes, transporters, and nuclear receptors and transcription factors in intestinal mucosa (ileum, ascending colon, transverse colon, descending colon, and rectum) from five healthy subjects and in five commonly used intestinal cell lines (Caco-2, C2BBe1, HT29, T84, and FHC). For this, we performed a quantitative real-time reverse transcription-polymerase chain reaction analysis using TaqMan low-density arrays and analyzed the results by different statistical approaches: Spearman correlation coefficients, hierarchical clustering, and principal component analysis (PCA). A large variation in gene expression spectra was observed between intestinal cell lines and intestinal tissues. Both hierarchical clustering and PCA showed that two distinct clusters are visible, of which one corresponds to all cultured cell lines and the other to all intestinal biopsies. The best agreement between human tissue and the representative cell line was observed for human colonic tissues and HT29 and T84 cell lines. Altogether, these data demonstrated that gene expression profiling represents a new valuable tool for investigating in vitro and in vivo expression level correlation. This study has pointed out interesting expression profiles for various colon cell lines, which will be useful for choosing the appropriate in vitro model for pharmacological and toxicological studies.

Evidence for a functional genetic polymorphism of the Rho-GTPase Rac1. Implication in azathioprine response?

BACKGROUND: Adverse effects of thiopurine drugs occur in 15-28% of patients and the majority is not explained by thiopurine-S-methyltransferase deficiency. Furthermore, approximately 9% of patients with inflammatory bowel disease are resistant to azathioprine therapy. Recently, the small guanosine triphosphatase, Rac1, was identified as an important molecular target of 6-thioguanine triphosphate, one of the active metabolite of thiopurines such as azathioprine. To date, no functional genetic polymorphism of the human Rac1 gene had been reported. OBJECTIVES: Evidence for functional genetic polymorphisms of the human Rac1 gene and to investigate their relative contribution to the development of toxicity induced by azathioprine treatment in patients with inflammatory bowel disease. METHODS: We first screened for polymorphisms in the Rac1 gene in genomic DNA samples from 92 unrelated Caucasian individuals. The functional consequences of identified polymorphisms were assessed in vitro using transient transfection assays in Jurkat and A549 cell lines. The relationship between polymorphisms of Rac1 and thiopurine response or hematotoxicity was studied in 128 patients under thiopurine treatment. RESULTS: Three single nucleotide polymorphism and one variable number tandem repeat were identified in the promoter region of Rac1 gene. Interestingly, in Jurkat T cells, the c.-289G>C substitution and c.-283_-297[3] variable number tandem repeat displayed a significantly increased promoter activity (P<0.01) of 150 and 300%, respectively, compared with that of the wild-type sequence. Patients with thiopurine-S-methyltransferase mutations presented a significantly increased probability of developing hematotoxicity (odds ratio=5.68, 95% confidence interval=1.45-22.23, P=0.00625). Moreover, among the 75 patients who did not develop hematotoxicity, there was a marginally overrepresentation of functional genetic polymorphisms of Rac1 (odds ratio=0.18, 95% confidence interval=0.02-1.49, P=0.079). CONCLUSION: This study constitutes the first report of a functional genetic polymorphism that could affect Rac1 expression and thus modulate the risk of adverse drug reaction in patients under thiopurine treatment. A larger scale (case-control) study should enable us to confirm or cancel these preliminary results.

Molecular analysis of the CYP2F1 gene: identification of a frequent non-functional allelic variant.

The CYP2F1 is a human cytochrome P450 that is selectively expressed in lung tissue and involved in the metabolism of various pneumotoxicants with potential carcinogenic effects. In the present study, we report the first systematic investigation of the genetic polymorphism of this enzyme. We analyzed the nucleotidic sequence of the CYP2F1 gene in DNA samples from 90 French Caucasians consisting in 44 patients with lung cancer and 46 control individuals, using single-strand conformation polymorphism analysis of PCR products (PCR-SSCP). We identified 24 novel mutations distributed in the promoter region of the gene, as well as in the coding regions and their flanking intronic sequences. In addition to the wild-type CYP2F1*1 allele, seven allelic variant, CYP2F1*2A, *2B, *3, *4, *5A, *5B and *6, were characterized. The most frequent allelic variant, CYP2F1*2A (25.6%), harbors a combination of 9 mutations, including 2 missense mutations (Asp218Asn and Gln266His) and a 1-bp insertion (c.14_15insC) that creates a premature stop codon in exon 2, probably leading to the synthesis of a severely truncated protein with no catalytic activity. The identification of around 7% of homozygotes for the frameshift mutation in our Caucasian population suggests the existence of an interindividual variation of the CYP2F1 activity and, consequently, the possibility of interindividual differences in the toxic response to some pneumotoxicants and in the susceptibility to certain chemically induced diseases. However, our preliminary results did not show any evidence that the CYP2F1 genetic polymorphism has implications in the pathogenesis of lung cancer.

Evidence for a functional genetic polymorphism of the human retinoic acid-metabolizing enzyme CYP26A1, an enzyme that may be involved in spina bifida.

BACKGROUND: CYP26A1, together with CYP26B1 and CYP26C1, are key enzymes of all-trans retinoic acid (RA) inactivation and their specific and restricted expression in developing embryos participate in the fine tuning RA levels. As RA is a critical regulator of gene expression during embryonic development, the imbalance between the synthesis and degradation of RA during embryogenesis could contribute to malformations and developmental defects. METHODS: A PCR-single strand conformation polymorphism (PCR-SSCP) strategy was developed to screen for CYP26A1 sequence variations that could affect the enzyme expression and/or activity and applied to DNA samples from 80 unrelated Caucasians, comprising 40 French healthy volunteers and 40 Italian patients with spina bifida. The consequence of the 1-bp deletion identified in the coding sequence was investigated by an in vitro functional assay using COS-7 cells. RESULTS: A total of 7 polymorphisms were identified, comprising 1 nucleotide deletion in the coding sequence (g.3116delT) that results in a frameshift and consequently in the creation of a premature stop codon. The g.3116delT mutation is of particular interest because it was identified in a patient with spina bifida and likely encodes a truncated protein with no enzymatic activity, as demonstrated by our preliminary in vitro data. CONCLUSIONS: Despite the fact that our findings could not show any evidence that the CYP26A1 genetic polymorphism has implications in the pathogenesis of spina bifida, this work represents the first description of a functional genetic polymorphism affecting the coding sequence of the human CYP26A1 gene.

Evidence for a functional genetic polymorphism of the human thiosulfate sulfurtransferase (Rhodanese), a cyanide and H2S detoxification enzyme.

Rhodanese or thiosulfate sulfurtransferase (TST) is a mitochondrial matrix enzyme that plays roles in cyanide detoxification, the formation of iron-sulfur proteins and the modification of sulfur-containing enzymes. Transsulfuration reaction catalyzed by TST is also involved in H(2)S detoxification. To date, no polymorphism of the human TST gene had been reported. We developed a screening strategy based on a PCR-SSCP method to search for mutations in the 3 exons of TST and their proximal flanking regions. This strategy has been applied to DNA samples from 50 unrelated French individuals of Caucasian origin. Eleven polymorphisms consisting in seven nucleotide substitutions in non-coding regions, two silent mutations and two missense mutations were characterized. The functional consequences of the identified mutations were assessed in vivo by measurement of erythrocyte TST activity and/or in vitro using heterologous expression in Saccharomyces cerevisiae or transient transfection assay in HT29 and Caco-2 cell lines. The P(285)A variant appears to encode a protein with a 50% decrease of in vitro intrinsic clearance compared to the wild-type enzyme. Additionally, the six polymorphisms located upstream the ATG initiation codon are responsible for a significant decrease (ranging from 40% to 73%) in promoter activity of a reporter gene compared to the corresponding wild-type sequence. This work constitutes the first report of the existence of a functional genetic polymorphism affecting TST activity and should be of great help to investigate certain disorders for which impairment of CN(-) or H(2)S detoxification have been suggested to be involved.

Evidence for a functional genetic polymorphism of the human mercaptopyruvate sulfurtransferase (MPST), a cyanide detoxification enzyme.

Mercaptopyruvate sulfurtransferase (MPST) plays a central role in both cysteine degradation and cyanide detoxification. Moreover, deficiency in MPST activity has been suggested to be responsible for a rare inheritable disorder known as mercaptolactate-cysteine disulfiduria (MCDU). To date, no mutation of the human MPST gene has been reported. We developed a screening strategy to search for mutations in the MPST gene of 50 unrelated French individuals. Two intronic polymorphisms (IVS1-110C>G and IVS2+39C>T) and a nonsense mutation (Tyr(85)Stop) were identified and their functional consequences were assessed in vivo by measurement of erythrocyte MPST activity and/or in vitro using heterologous expression or transient transfection assay. The nonsense mutation likely leads to the synthesis of a severely truncated protein without enzymatic activity, as supported by our in vitro data. This work constitutes the first report of the existence of a functional genetic polymorphism affecting MPST and should be of great help to investigate certain disorders such as MCDU.