Characterization of cytochrome P450 enzymes in human breast tissue from reduction mammaplasties.

Cytochrome P450 (CYP) enzymes in the breast may have an important role in regulating the capacity of individual cells to metabolize hormones and environmental carcinogens. Very little is known about the P450 expression pattern in human breast because of the limited amount of accessible tissue and the difficulties associated with detection of low P450 levels. Breast tissue from reduction mammaplasties is the only tissue available in relative abundance.

Mutations in the CDKN2A (p16INK4a) gene in microdissected sporadic primary melanomas.

The role of the CDKN2A (p16INK4a) gene in sporadic primary melanomas has remained unclear due to the inadequate number of mutational studies. In the present study, we analyzed the entire coding region of the CDKN2A gene in microdissected sporadic primary melanomas, for the presence of mutations and polymorphisms, using 2 independent methods of mutation detection, SSCP and CMC. We found 11 intragenic mutations in 8 melanomas out of 31 (26%) and the majority of mutations were located in exon 1, with 2 cases harbouring multiple mutations.

DNA adducts in human nasal mucosa and white blood cells from smokers and non-smokers.

The goal of the present study was to measure the levels of DNA adducts in human nasal mucosa cells and in total white blood cells in relation to smoking. DNA was isolated from samples of 20 healthy volunteers (six smokers and 14 non-smokers). The levels of DNA adducts were measured by 32P-postlabelling assay.

32P-Postlabelling of DNA adducts formed by allyl glycidyl ether in vitro and in vivo.

32P-Postlabelling analysis of allyl glycidyl ether-treated DNA after adduct enrichment on anion-exchange cartridges revealed two major and one minor DNA adducts. The major adducts were shown to originate from alkylation at N-7-guanine and N-1-adenine, respectively, while the minor adduct was at N-3-cytosine. In addition, rearrangement products of the 1-adenine and 3-cytosine adducts to N6-adenine and 3-uracil were indicated.

Lipoprotein metabolism in the fat Zucker rat: reduced basal expression but normal regulation of hepatic low density lipoprotein receptors.

Hyperlipoproteinemia is one of the phenotypic characteristics of the fat Zucker rat that carries a mutation in the leptin receptor gene. In the present study, we studied the regulation of hepatic low density lipoprotein (LDL) receptor expression in lean and fat Zucker rats. Compared with lean rats, the fat ones had a pronounced (approximately 60%) reduction in hepatic LDL receptor expression, whereas the levels of receptor messenger RNA (mRNA) were not reduced.

Somatic mutations in the human homologue of Drosophila patched in primitive neuroectodermal tumours.

The naevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterized by multiple developmental defects and cancer susceptibility, in particular to basal cell carcinomas (BCCs). Medulloblastomas, primitive neuroectodermal tumours (PNETs) arising in childhood, occur in about 3-5% of NBCCS patients and a subset of PNETs was previously found with allelic imbalance at 9q22-q23, the region containing the gene for NBCCS (PTCH). We have analysed tumour DNA samples from 37 unrelated patients with sporadic PNETs and five medulloblastoma cell lines for PTCH mutations using an exon-by-exon single strand conformation polymorphism assay.

Exon skipping and circular RNA formation in transcripts of the human cytochrome P-450 2C18 gene in epidermis and of the rat androgen binding protein gene in testis.

The cytochrome P-450 2C18 gene was found by reverse transcription-PCR to represent the most abundantly expressed gene of the P-450 2C subfamily in human epidermis. However, in addition to the canonical mRNA of nine exons, transcripts that have skipped exon 4 or 5, exons 4, 5, and 6, or exons 4, 5, 6, and 7 were also identified in this tissue. Remarkably, circular RNA transcripts synthesized by the joining of the donor and acceptor splice sites of the same exon were detected in human epidermis for exons 4 and 5.

Cytochrome P450 in the breast and brain: role in tissue-specific activation of xenobiotics.

It is still an open question as to whether, upon administration of procarcinogens to rodents, development of cancers in extrahepatic tissues is due to activation of these chemicals in the liver or to in situ activation within the tissue. The low level of P450 in many tissues means that it is very difficult to demonstrate the formation of significant amounts of reactive metabolites when these tissues are incubated with procarcinogens in vitro. It is our contention that the importance of tissue-specific activation of procarcinogens can best be decided when the cells which harbour P450 have been identified and the isozyme profile in the cells defined.

Analysis of tamoxifen-induced DNA adducts by 32P-postlabelling assay using different chromatographic techniques.

DNA isolated from livers of rats receiving tamoxifen was analysed by the 32P-postlabelling method. The postlabelled DNA hydrolysis mixture was analysed both by reversed-phase HPLC with 32P on-line detection and by TLC on polyethyleneimine plates followed by autoradiography. Using the HPLC method, five well separated adduct peaks could be detected, while by the TLC method, two groups of adduct spots were observed.

32P-postlabelling analysis of isomeric 7-alkylguanine adducts of styrene oxide.

Styrene 7,8-oxide, which reacts preferentially at the N-7 position of guanine, yielded two pairs of diastereomers 7-(1'-hydroxy-2'-phenylethyl)-dGMP (the alpha-isomer) and 7-(2-hydroxy-2-phenylethyl)-dGMP (the beta-isomer) on reaction with deoxyguanosine-3'-monophosphate (3'-dGMP). The alpha- and beta-isomers were formed in the ratio 32:68. T4 polynucleotide kinase preferentially mediated labelling of diastereomers corresponding to the beta-isomer.

cDNA cloning of a novel WD repeat protein mapping to the 9q22.3 chromosomal region.

To identify expressed sequences from the candidate genomic region of the nevoid basal cell carcinoma syndrome (9q22.3), the CpG island rescue PCR methodology was employed on the yeast artificial chromosome (YAC) ICI-8AD8 that contains microsatellite marker D9S180. A positive clone (IR10, size 350 bp) was isolated by screening a human epidermal cDNA library with the island rescue PCR products and mapped back to the 9q22.

Mutations in the human homologue of Drosophila patched (PTCH) in basal cell carcinomas and the Gorlin syndrome: different in vivo mechanisms of PTCH inactivation.

The nevoid basal cell carcinoma (Gorlin) syndrome (NBCCS) is an autosomal dominant disorder characterized by multiple developmental defects and cancer susceptibility, in particular to basal cell carcinoma. The human homologue of Drosophila patched (PTCH) was recently identified, mapped to the NBCCS locus on chromosome 9q22.3, and found mutated in patients with NBCCS and also in sporadic basal cell carcinomas.

Assay of different photoproducts after UVA, B and C irradiation of DNA and human skin explants.

A 32P-HPLC method was applied to study the induction of UVB- and UVC-induced DNA lesions (cyclobutane dimers, 6-4 photoproducts and Dewar isomers) in human skin explants. The employed technique was sensitive enough to detect the lesions at a dose of 10 J/m2 UVB. Comparison of photoadduct formation under UVC and UVB indicated the importance of photosensitization pathways in DNA damage.

Aromatic DNA adducts in lymphocytes of humans working at high and low traffic density areas.

Aromatic DNA adduct levels were determined by the 32P-postlabelling assay in lymphocytes isolated from newspaper vendors working at urban high traffic areas (n = 31) and suburban low traffic areas (n = 22) in Milan, Italy. The DNA adduct levels ranged from 0.7 to 6.

DNA adduct formation by allyl glycidyl ether.

Glycidyl ethers are reactive epoxides used as components of a variety of epoxy materials. These compounds are known to cause allergic reactions, but since they are generally also genotoxic it would be of interest to evaluate the risk for induction of such effects. Reaction products of allyl glycidyl ether with nucleic acid components were therefore studied.

Circular RNAs from transcripts of the rat cytochrome P450 2C24 gene: correlation with exon skipping.

The cytochrome P450 2C24 gene is characterized by the capability to generate, in rat kidney, a transcript containing exons 2 and 4 spliced at correct sites but having the donor site of exon 4 directly joined to the acceptor site of exon 2 (exon scrambling). By reverse transcriptase-PCR analysis, it is now shown that the only exons present in the scrambled transcript are exons 2, 3, and 4 and that this molecule lacks a poly(A)+ tail. Furthermore, the use of PCR primers in both orientations of either exon 2 or exon 4 revealed that the orders of the exons in the scrambled transcript are 2-3-4-2 and 4-2-3-4, respectively.

32P-postlabelling of diastereomeric 7-alkylguanine adducts of butadiene monoepoxide.

The reaction of 3,4-epoxy-1-butene (BMO) with deoxyguanosine-3'-monophosphate (3'-dGMP) resulted in the formation of two pairs of diastereomeric 7-alkyl-3'-dGMP derivatives corresponding to two isomers C¿-1 and C¿-2. The T4 polynucleotide kinase-mediated phosphorylation with [gamma-32P]-ATP showed preferential labelling of diastereo- mers of the C¿-1 isomer. The diastereomers 1 and 2 of the C¿-1 isomer had labelling efficiencies of 42%.

Future research directions in the use of biomarkers.

Many DNA adduct studies have been carried out in occupational groups that have been at a risk of cancer based on epidemiological results relating to exposure decades ago. Even new epidemiological publications on cancer cannot accurately address the effective exposures after about 1970. This is one justification for biomarker studies.

Determination of DNA adducts of malonaldehyde in humans: effects of dietary fatty acid composition.

The effects of dietary fatty acid composition on the endogenous formation of DNA adducts of malonaldehyde (MA), the major product of lipid peroxidation, were investigated in humans. A group of 59 healthy individuals of both sexes and different ages was initially fed a milk fat-based diet rich in saturated fatty acids for 14 days. Following this initial period, after which the group was considered homogeneous with respect to diet, 30 randomly chosen subjects were given a sunflower oil-based (rich in polyunsaturated fatty acids) (SO) diet and the remaining 29 individuals a low erucic acid rapeseed oil-based (rich in monounsaturated fatty acids) (RO) diet for 25 days.

Tobacco smoke-associated N7-alkylguanine in DNA of larynx tissue and leucocytes.

The presence of N7-alkylguanine adducts in DNA was analysed in a group of 46 patients with larynx tumours. All patients were subjected to laryngectomy and the tissues accessible for analysis by (32)P-post-labelling assay were larynx tumour, larynx non-tumour and peripheral blood leucocytes. N7-Alkylguanine adducts were detected in all the studied DNA samples.

Separation of 7-methyl- and 7-(2-hydroxyethyl)-guanine adducts in human DNA samples using a combination of TLC and HPLC.

We have used a combination of thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC) to achieve separation of 32P-postlabelled 7-methylguanine and 7-(2-hydroxyethyl)-guanine adducts. The level of these two adducts was determined in human total white blood cells (mean values 0.7 to 1.

32P-postlabelling of bulky human DNA adducts enriched by different methods including immunoaffinity chromatography.

DNA adducts in lymphocytes and granulocytes of men exposed occupationally and environmentally to high concentrations of aromatic compounds in air were measured by the 32P-postlabelling method. Adducts in the same samples were characterized using nuclease P1 enrichment, butanol extraction and immunoaffinity purification with an antiserum raised against benzo[alpha]pyrene diol epoxide (BPDE). Only part of the adducts found in human samples were extracted by butanol.

UV-induced photoproducts in human skin explants analysed by TLC and HPLC-radioactivity detection.

The objective of this study was to apply the 32P-postlabelling method for the detection of UV-induced dipyrimidine photoadducts in human skin explants. [32P]TLC and [32P]HPLC analysis were used. These techniques allow detection of different combinations of cyclobutane dimers and 6-4 photoproducts in human skin at low doses of irradiation.