The human cytochrome P450 3A locus. Gene evolution by capture of downstream exons.

Using a bacterial artificial chromosome (BAC) clone, we have mapped the human cytochrome P450 3A (CYP3A) locus containing the genes encoding for CYP3A4, CYP3A5 and CYP3A7. The genes lie in a head-to-tail orientation in the order of 3A4, 3A7 and 3A5. In both intergenic regions (3A4-3A7 and 3A7-3A5), we have detected several additional cytochrome P450 3A exons, forming two CYP3A pseudogenes.

Specific DNA adducts induced by some mono-substituted epoxides in vitro and in vivo.

Alkyl epoxides are important intermediates in the chemical industry. They are also formed in vivo during the detoxification of alkenes. Alkyl epoxides have shown genotoxicity in many toxicology assays which has been associated with their covalent binding to DNA.

(32)P-postlabelling/HPLC analysis of various styrene-induced DNA adducts in mice.

Styrene oxide (SO), a reactive metabolite of styrene, modifies DNA at several nucleophilic sites. In the present work we have determined the SO-DNA adducts in vitro and in vivo by two different versions of (32)P-postlabelling/HPLC assays. When anionexchange cartridges were used for adduct enrichment the β-isomer of 7-substituted guanines was detected in in vitro SO-treated DNA as well as in mice lungs exposed to styrene at 750 and 1500 mg m(-3) for 21 days (6 h day(-1), 7 days week(-1)).

Hedgehog signalling in cancer.

Hedgehog signalling is a key regulator of embryonic development controlling proliferation and/or cell fate determination. With identification of the Hedgehog receptor PTCH1 as a tumour suppressor gene that underlies the human nevoid basal cell carcinoma syndrome (NBCCS), the Hedgehog signalling pathway was firmly linked to cancer. It now appears that constitutive activation of Hedgehog signalling, by inactivating mutations in PTCH1 or activating mutations in the coreceptor SMOH, is required and possibly sufficient for basal cell carcinoma development and also contributes to the formation of a variety of other tumour types, including medulloblastoma and rhabdomyosarcoma.

Allelic losses demonstrate monoclonality of multifocal bladder tumors.

The clonality of multifocal bladder tumors has been studied over the years with some controversial results. We have examined 5 patients with 2-11 low-grade superficial multifocal bladder tumors for loss of heterozygosity (LOH) at 87 loci on 9 chromosomes. When LOH was detected at a given marker, the tumors consistently showed deletion of a specific allele, suggesting the monoclonality of the patients' tumors.

Initiation-development modelling of allelic losses on chromosome 9 in multifocal bladder cancer.

Multiple low-grade, low-stage superficial tumours were analysed for loss of heterozygosity (LOH) on chromosome 9 with 29 markers. Three consensus regions were found, one at 9p (9p21-22) and two at 9q (9q21-31 and 9q32-34). Phylogenetic trees were calculated for each patient using both designated chromosome 9 regions and, separately, using individual microsatellite data.

Styrene oxide-induced 2'-deoxycytidine adducts: implications for the mutagenicity of styrene oxide.

The reaction between 2'-deoxycytidine and styrene 7,8-oxide (SO) resulted in alkylation at the 3-position and at the O(2)-position through the alpha- and beta-carbons of the epoxide but at the N(4)-position only through the alpha-carbon. The 3-alkylated adducts were found to deaminate to the corresponding 2'-deoxyuridine adducts (37 degrees C, pH 7.4) with half-lives of 6 min and 2.

32P-post-labelling of 7-(3-chloro-2-hydroxypropyl)guanine in white blood cells of workers occupationally exposed to epichlorohydrin.

Epichlorohydrin (ECH) is a simple 3-carbon epoxide of industrial importance. It has been shown to be genotoxic in several systems and carcinogenic in experimental animals. The aim of the present investigation was to study DNA adducts of ECH as a biomarker of occupational exposure to this chemical.

(32)P-postlabelling analysis of 1,3-butadiene-induced DNA adducts in vivo and in vitro.

Butadiene monoepoxide (BMO), epoxybutanediol (EBD) and diepoxybutane (DEB) are reactive metabolites of 1,3-butadiene (BD), an important industrial chemical classified as a probable human carcinogen. The covalent interactions of these metabolites with DNA lead to the formation of DNA adducts which may induce mutations or other types of DNA damage, resulting in tumour formation. In the present study, two pairs of diastereomeric N-1-BMO-adenine adducts were identified in the reaction of BMO with 2´-deoxyadenosine-5´-monophosphate (5´-dAMP).

Human DNA adducts of 1,3-butadiene, an important environmental carcinogen.

The N-1-(2,3,4-trihydroxybutyl)adenine (N-1-THB-Ade) adducts induced by 1,3-butadiene (BD) were analysed from lymphocytes of 15 workers occupationally exposed to BD and 11 controls by (32)P-post-labelling using HPLC with radioactivity detection. The difference in the adduct levels between the BD-exposed workers (4.5 +/- 7.

Comparison of (32)P-postlabeling and high-resolution GC/MS in quantifying N7-(2-Hydroxyethyl)guanine adducts.

This study compares (32)P-postlabeling and high-resolution gas chromatography/mass spectrometry (GC/MS) in the quantification of N7-(2-hydroxyethyl)guanine adducts (7-HEG) in DNA obtained from the same tissue samples of control rats and rats exposed to ethene. The samples were obtained from two independent studies. In one study, male Sprague-Dawley rats were exposed to 300 ppm ethene for 12 h/day for 3 days ("Euro samples").

32P-postlabelling of propylene oxide 1- and N(6)-substituted adenine and 3-substituted cytosine/uracil: formation and persistence in vitro and in vivo.

Propylene oxide, a widely used monofunctional alkylating agent, has been shown to be genotoxic in in vitro test systems and induces tumors in the nasal tissues of experimental animals. Propylene oxide, like related alkylating agents, forms several different adducts with DNA bases, but predominantly at the 7-position of guanine. We have previously described the in vitro and in vivo formation and stability of this major adduct.

Cis-acting sequences from the rat cytochrome P450 2B1 gene confer pulmonary and phenobarbital-inducible expression in transgenic mice.

Specific cytochrome P450 enzymes show tissue-specific induction, and different regulatory units for expression of these enzymes have been identified. The regulation of the phenobarbital (PB)-inducible P450 genes has been relatively well characterized in terms of PB induction, but less so with regard to tissue-specific expression. CYP2B2 is not expressed in the rat lung, whereas cytochrome P450 2B1 (CYP2B1) is a dominating enzyme in the same tissue.

Endogenous histamine reduces plasma insulin-like growth factor I via H1 receptor-mediated pathway in the rat.

Endotoxin has been recently shown to reduce plasma insulin-like growth factor I. As it was reported that histamine can induce gut-derived endotoxemia, we wanted to determine whether histamine has a similar effect on plasma insulin-like growth factor I. Compound 48/80 (a histamine releaser) was injected subcutaneously into rats, then blood was taken for plasma insulin-like growth factor I assay and the livers were assayed for insulin-like growth factor I mRNA.

RNA molecules containing exons originating from different members of the cytochrome P450 2C gene subfamily (CYP2C) in human epidermis and liver.

Reverse transcription-PCR analysis in human epidermis, using primers from CYP2C18 and CYP2C19, revealed products containing combinations between canonically defined exons of these two genes. The major RNA species identified contained 2C18 exon 8 spliced with 2C19 exon 2. However, the terminal exons 1 and 9 were never detected in any of these composite molecules.

Formation and persistence of DNA adducts during and after a long-term administration of 2-nitrofluorene.

2-Nitrofluorene (NF) is an environmental pollutant. Our previous studies have shown that NF is a carcinogen, primarily targeting the liver, kidney and forestomach in rats. NF-induced DNA adducts were also shown higher levels in the tumor-targeting tissues compared to non-tumor targeting organs.

Endogenous and background DNA adducts by methylating and 2-hydroxyethylating agents.

Detection of 7-alkylguanine DNA adducts is useful to assess human exposure to and the resulting DNA damage caused by simple alkylating agents. The background 7-methylguanine (7-MG) and 7-hydroxyethylguanine (7-HEG) adduct levels were determined in human and rat tissues, using thin-layer chromatography (TLC) combined with high pressure liquid chromatography (HPLC). In addition, these two adduct levels were also compared in various tissues between smokers and non-smokers.

PTCH2, a novel human patched gene, undergoing alternative splicing and up-regulated in basal cell carcinomas.

By a combination of cDNA library screening, rapid amplification of cDNA ends analysis, and BAC sequencing, a novel human patched-like gene (PTCH2) has been cloned and sequenced. The genomic organization is similar to PTCH1 with 22 exons and, by radiation hybrid mapping, PTCH2 has been localized to chromosome 1p33-34, a region often lost in a variety of tumors. Several alternatively spliced mRNA forms of PTCH2 were identified, including transcripts lacking segments thought to be involved in sonic hedgehog binding and mRNAs with differentially defined 3' terminal exons.

32P-postlabelling of N6-adenine adducts of epoxybutanediol in vivo after 1,3-butadiene exposure.

Epoxybutanediol is one of the epoxide metabolites of butadiene (BD). A pair of diastereomeric N-1-adenine adducts were formed by reacting epoxybutanediol with deoxyadenosine 5'-monophosphate (5'-dAMP). These two N-1-adenine adducts rearranged in a base-catalysed reaction to an N6-trihydroxybutyl-adenine adduct, which was characterized by UV and mass spectroscopy.

Tissue distribution of DNA adducts in male Fischer rats exposed to 500 ppm of propylene oxide: quantitative analysis of 7-(2-hydroxypropyl)guanine by 32P-postlabelling.

7-(2-Hydroxypropyl)guanine (7-HPG) constitutes the major adduct from alkylation of DNA by the genotoxic carcinogen, propylene oxide. The levels of 7-HPG in DNA of various organs provides a relevant measure of tissue dose. 7-Alkylguanines can induce mutation through abasic sites formed from spontaneous depurination of the adduct.

Non-homologous recombination mediated by Thermus aquaticus DNA polymerase I. Evidence supporting a copy choice mechanism.

RT-PCR amplification of P450 2C6 from rat liver, using primers in opposite orientations of exon 6, resulted in PCR products containing segments of exons joined at non-consensus splice sites. Moreover, many of the PCR products identified were composed of not only a single region containing exonic segments joined at non-consensus splice sites but, instead, of several repeats of the non-canonically joined region. To investigate whether these PCR products represent pre-existing molecules or are generated during the amplification process, the liver cDNA template was replaced by a plasmid containing the P450 2C6 cDNA.

Mechanisms of pre-mRNA splicing: classical versus non-classical pathways.

Expression of genetic information proceeds through two major biological events, transcription and translation. However, in eukaryotic cells, the primary transcript (pre-mRNA) is not the template that the translational apparatus scans through, in order to produce the corresponding protein. Pre-mRNAs undergo several modifications (cap site addition, poly A+ tail addition) prior to becoming mature mRNAs, with the most important one being the excision (splicing) of the intronic sequences.

Sensitive 32P-HPLC technique shows base sequence dependent differences in photolesion repair in human keratinocytes.

Understanding the basis for individual susceptibility to skin cancer requires an understanding of the factors contributing to tumorigenesis. One such factor is the ability of the cell to repair DNA lesions induced following insult to the genome. Currently, research in this field is hampered by the lack of a suitably sensitive and specific method for the detection of DNA lesions.

High levels of dipyrimidine dimers are induced in human skin by solar-simulating UV radiation.

UV light is considered an important contributor to skin cancer, but methods have been lacking to quantify specific UV-induced lesions in human skin in situ. We applied a newly developed 32P-postlabeling technique to measure specific UV-induced cyclobutane dimers and 6-4 dipyrimidine lesions in the skin of healthy volunteers. At a dose of 400 J/m2, solar-simulated radiation caused at least 20 cyclobutane dimers/10(6) nucleotides, which is much higher than any known DNA adducts induced by specific external chemical exposure in human target tissues.