7-Alkyldeoxyguanosine adduct detection by two-step HPLC and the 32P-postlabeling assay

7-Alkyldeoxyguanosine DNA adducts may be a marker for some N-nitrosocompound exposures and subsequent human cancer risk. A sensitiveand highly specific assay for the detection of 7-methyl-2'-deoxyguanosine-3'-monophosphate(7-methyldGp) and 7-ethyl-2'-deoxyguanosine-3'-monophosphate(7-ethyldGp) has been developed by combining two different HPLCpurification steps with the ³²P-postlabeling assay. We previouslyreported that ion-pair reverse-phase (IP) chromatography coupledwith the ³²P-postlabeling assay detects 7-methyldGp in humanlung, but have found that other nucleotides and unknown adductsco-elute. Thus, weak anion exchange (AE) HPLC was added in tandemwith IP HPLC prior to the ³²P-postlabeling assay. 2'-Deoxyguanosine-3'-monophosphate(dGp) is incorporated into the assay as an internal standardfor the assessment of enzyme labeling efficiency and adductrecovery. The methodology was validated using radiolabeled DNAand liquid scintillation counting, which accounts for adductloss from enzymatic digestion to detection. Levels of 7-ethyldGpalso were correlated with accelerator mass spectrometry. Theoverall adduct recovery with this method was 58% for 7-methyldGpand 98% for 7-ethyldGp. The detection limit for both assaysusing 100 µg of DNA was one adduct in 10⁸ unmodified dGp.7-MethyldGp and 7-ethyldGp levels were determined in ten humanlung samples at levels of 1.4–5.4 and 0.6–3.1 adductsper 10⁷ dGp respectively, and in five human lymphocyte samplesat levels of 5.0–8.3 and 0.3–1.4 adducts per 10⁷dGp respectively. Combining the two HPLC purification stepsand the ³²P-postlabeling assay attains chemical specificity,retains sufficient quantitative sensitivity and should be usefulin human biomonitoring studies.     

Rapid and reliable genotyping for the Toll-like receptor 4 A896G polymorphism using fluorescence-labeled hybridization probes in a real-time polymerase chain reaction assay

BACKGROUND: The Toll-like receptor 4 (TLR4) is involved in immune response to endotoxin as well as the pathogenesis of atherosclerosis. The TLR4 gene was shown to carry a single-nucleotide polymorphism (A896G). We developed a rapid-cycle polymerase chain reaction (PCR) which allows for rapid genotyping and, therefore, may be useful in clinical risk assessment. METHODS: Fluorescently labeled oligonucleotide hybridization probes were designed for the LightCycler instrument. A PCR product was generated in a single reaction followed by melting point analysis. Ninety-three German Caucasians were genotyped. The interleukin-1beta (IL-1beta) response to endotoxin was assessed after whole blood stimulation with endotoxin according to TLR4 genotypes. RESULTS: The method suggested by us is a time-saving technique requiring no additional manual steps. Frequencies of the A and G allele were 0.95 and 0.05, respectively. The study population was in Hardy-Weinberg equilibrium. The specificity of the method was confirmed by sequencing. The IL-1beta levels were lower in carriers of the G allele. CONCLUSIONS: This PCR assay is a rapid and reliable technique for TLR4 genotyping.     

Arylalkylamine N-acetyltransferase (AANAT) genotype as a personal trait in melatonin synthesis

The melatonin rhythm is arguably the best marker for the phase of the endogenous "biological clock." Arylalkylamine N-acetyltransferase (AANAT) is known to catalyze the acetylation of serotonin, a rate-limiting process in melatonin synthesis. Different single-nucleotide polymorphisms (SNPs) in the AANAT gene were identified recently in the Japanese population, and one of the genes was significantly associated with the delayed sleep phase syndrome. Thus, 54 healthy Caucasian males were genotyped to investigate whether these SNPs in the AANAT gene affected melatonin levels. The endogenous melatonin levels were analyzed in saliva under standardized experimental conditions ("constant routines") by radioimmunoassay. Despite the broad temporal variation of the human nocturnal melatonin profiles, none of the investigated SNPs were found in the AANAT gene in this study. These findings point to ethnic differences with respect to these SNPs, rather than time of day termed "morningness." In summary, SNPs in the AANAT gene identified thus far cannot explain the observed interindividual differences for nocturnal melatonin profiles in the subjects investigated.     

Serum, plasma and paraffin-embedded tissues as sources of DNA for studying cancer susceptibility genes

The ability to isolate DNA from archived human serum, plasma and paraffin-embedded human tissues enhances opportunities to study breast, lung and other cancer risk factors. We report herein a simple and fast protocol for the extraction of genomic DNA from these sources. Using a phenol-based extraction method, the recovery for DNA is quantitative and reproducible. DNA yields in serum (250 microl) were between 162 and 1060 ng (n = 18 subjects), in plasma (250 microl) were between 165 and 375 ng (n = 5 subjects) and in embedded tissues (5-microm thick sections for ethanol fixed, and between 5- and 20-microm sections for formaldehyde fixation) were between 1 microg and 11.7 microg (n = 32 subjects). The extraction method was combined with newly designed PCR- based assays for cancer susceptibility marker genes such as CYP1A1 (exon 7), CYP2E1 (Dra1, Rsa1), GSTM1 and NAT2 [NAT2*5A (C481T), NAT2*6A (G590A), NAT2*7A (G857A)]. Genotyping results from the serum and paraffin-embedded tissues compared favorably to results from archived freshly frozen tissues, where concordance was 98% for serum, 100% for ethanol-fixed embedded tissues, and 97% for formaldehyde-fixed and paraffin-embedded tissues. This facile method will allow for the use of archived tissue samples of prospective cohort and other studies where intact DNA was not previously available.      

Expression of N-acetyltransferase in monocyte-derived dendritic cells

Dendritic cells (DCs) are known to internalize, process, and present low-molecular-weight chemicals to T cells in the course of the sensitization and elicitation phase of allergic contact dermatitis. Thus, DCs may be involved in metabolic activation and detoxification of haptens and thereby influence the quantity of immunogens inducing sensitization. Recently, the cytochrome P-450 enzymes expressed in monocyte-derived dendritic cells (MoDCs) were characterized. In the present study, N-acetyltransferase 1 and 2 (NAT-1 and -2) mRNA expression and N-acetylation capacities of these cells were investigated. Monocytes from healthy donors were incubated with granulocyte-monocyte colony-stimulating factor (GM-CSF) and interleukin (IL)-4 for 6 d and the resulting immature MoDCs were characterized by flow cytometry. Total RNA from MoDCs was isolated, reverse transcribed, and polymerase chain reaction (PCR) for NAT-1 and NAT-2 mRNA was performed. Data showed the presence of mRNA for NAT-1 (9 of 10 donors) and NAT-2 (8 of 10 donors) in these cells. NAT-1 enzyme activities were achieved through acetylation of para-aminobenzoic acid (PABA) by MoDC cell lysates and activities varied between 23.4 and 26.6 nmol/mg/min. In addition, complete cell acetylation of para-phenylenediamine (PPD), estimated via analysis of monoacetyl-PPD (MAPPD) and diacetyl-PPD (DAPPD) in cell culture supernatants, confirmed that in vitro generated MoDCs (4 of 6 donors) express metabolic active N-acetyltransferase (NAT-1). In the case of PPD, our results emphasize that N-acetylation status may influence the amounts of immunogens available for sensitization to PPD.     

N-acetyltransferase 1 in colon and rectal cancer cases from an industrialized area

Colon and rectal cancers are both associated with genetic as well as nutritional, occupational, and environmental factors. Aromatic amines and heterocyclic amines are established colorectal carcinogens. The polymorphic enzyme N-acetyltransferase 1 (NAT1) contributes to heterocyclic amine metabolism in the human colon. Thereby, NAT1 may influence the risk for development of colorectal cancer. The distribution of NAT1 genotypes was determined in 107 colon cancer cases, 77 rectal cancer cases, and 185 controls (suffering from nonmalignant diseases) by standard methods. In addition, possible occupational and nonoccupational risk factors were determined by a personal interview. Cancer cases and controls were derived from an area of former coal, iron, and steel industries, which is known for elevated colon cancer mortality. The proportions of NAT1*4/*4 genotype were 72% in controls, 75% in rectal cancer cases, and 72% in colon cancer cases. The proportions of the NAT1*4/*10 genotype were 17.8% in controls, 12.9% in rectal cancer cases, and 14% in colon cancer cases. Combinations of the determined NAT1 alleles *3/*3, *3/*10, *4/*3, *4/*11, *10/*10 and *11/*11 contributed to 10.2% of the genotypes in controls, 12.1% in rectal cancer cases, and 14% in colon cancer cases. In contrast to another study on healthy German volunteers, the NAT1*4/*4 genotype (wild type) is overrepresented. This might be due to the variation in the proportion of NAT1 alleles in the general population. The present study does not support a relevant impact of the NAT1 genotype on colorectal cancer risk development in the study area.     

Sensitization assays: monocyte-derived dendritic cells versus a monocytic cell line (THP-1)

Dendritic cells (DCs) play a critical role in the skin sensitization process of contact allergens. Many efforts have been made to develop in vitro sensitization tests that employ DCs, but more recently protocols were introduced that use cell lines other than DCs. The potential of the cell line THP-1 compared to monocyte-derived DCs (MoDCs) was evaluated using a known potent sensitizer 2,4-dinitrochlorobenzene (DNCB) and the terpenoid ascaridol (1,4-epodioxy-p-menth-2-ene), an ingredient present in oxidized tea tree oil. Activation of these cells was studied by estimation of the CD86 and CD54 cell surface expression. Overall, comparable results were found. The expression of CD86 was augmented by ascaridol in THP-1 and MoDCs, while the expression of CD54 was not reproducibly increased. These results encourage the further development of THP-1 cells as a short-term model for sensitization testing.