Thin-layer chromatography combined with surface-enhanced Raman scattering for rapid detection of benzidine and 4-aminobiphenyl in migration from food contact materials based on gold nanoparticle doped metal-organic framework.

In this work, a rapid and sensitive thin-layer chromatography combined with surface-enhanced Raman spectroscopy method was established for rapid detection of benzidine and 4-aminobiphenyl in migration from food contact materials based on Au nanoparticle doped metal-organic framework. Benzidine and 4-aminobiphenyl were firstly separated by thin-layer chromatography to solve the limitation of their overlapping Raman peaks. Then the target molecules were monitored by adding AuNPs/MIL-101(Cr) on the sample spots. Under the optimum conditions, the concentration of benzidine and 4-aminobiphenyl can be quantitatively measured in the range of 2.0-20.0 and1.0-15.0 µg/L, respectively with good linear relationship, and the limits of detection were 0.21 and 0.23 µg/L, respectively. Furthermore, the developed method was applied to analyze benzidine and 4-aminobiphenyl in migration of different food contact materials. The recoveries of benzidine and 4-aminobiphenyl for migration of food contact materials, including paper cups, polypropylene food containers, and polyethylene glycol terephthalate bottles, were 80.6-116.0 and 80.7-118% with relative standard deviations of 1.1-9.1 and 3.1-9.9%, respectively. Surface-enhanced Raman scattering detection was performed conveniently in the on-plate mode without additional elution process. The method shows great potential in rapid monitoring of hazardous substances with overlapping characteristic Raman peaks in food contact materials.

Strong impact of sulfotransferases on DNA adduct formation by 4-aminobiphenyl in bladder and liver in mice.

Bladder cancer risk is 3-4 times higher in men than women, but the reason is poorly understood. In mice, male bladder is also more susceptible than female bladder to 4-aminobiphenyl (ABP), a major human bladder carcinogen; however, female liver is more susceptible than male liver to ABP. We investigated the role of sulfotransferase (Sult) in gender-related bladder and liver susceptibility to ABP. Sulfation reactions of aromatic amine bladder carcinogens catalyzed by Sult may generate highly unstable and toxic metabolites. Therefore, liver Sult may decrease bladder exposure to carcinogens by promoting their toxic reactions in the liver. Notably, the expression of several liver Sults is suppressed by androgen in male mice. Here, we show that two Sults are critical for gender-related bladder susceptibility to ABP in mice. We measured tissue level of N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP), a principal ABP-DNA adduct, as readout of tissue susceptibility to ABP. We identified Sutl1a1 and to a lesser extent Sult1d1 as Sults that promote dG-C8-ABP formation in hepatic cells. In mice, gender gap in bladder susceptibility to ABP was narrowed by knocking out Sult1a1 and was almost totally eliminated by knocking out both Sutl1a1 and Sult1d1. This was accompanied by dramatic decrease in ABP genotoxicity in the liver (>97%). These results show the strong impact of the Sults on bladder and liver susceptibility to a human carcinogen. Because liver expression of both Sult1a1 and Sutl1d1 is suppressed by androgen in male mice, our results suggest that androgen renders bladder more exposed to ABP in male mice by suppressing Sult-mediated ABP metabolism in liver, which increases bladder delivery of carcinogenic metabolites.

Recent technical and biological development in the analysis of biomarker N-deoxyguanosine-C8-4-aminobiphenyl.

4-Aminobiphenyl (4-ABP) which is primarily formed during tobacco combustion and overheated meat is a major carcinogen responsible for various cancers. Its adducted form, N-deoxyguanosine-C8-4-aminobiphenyl (dG-C8-4-ABP), has long been employed as a biomarker for assessment of the risk for cancer. In this review, the metabolism and carcinogenisity of 4-ABP will be discussed, followed by a discussion of the current common approaches of analyzing dG-C8-4-ABP. The major part of this review will be on the history and recent development of key methods for detection and quantitation of dG-C8-4-ABP in complex biological samples and their biological applications, from the traditional 2P-postlabelling and immunoassay methods to modern liquid chromatography-mass spectrometry (LC-MS) with the latter as the focus. Many vital biological discoveries based on dG-C8-4-ABP have been published by using the nanoLC-MS with column switching platform in our laboratory, which has also been adopted and further improved by many other researchers. We hope this review can provide a perspective of the challenges that had to be addressed in reaching our present goals and possibly bring new ideas for those who are still working on the frontline of DNA adducts area.

Rapid determination of six carcinogenic primary aromatic amines in mainstream cigarette smoke by two-dimensional online solid phase extraction combined with liquid chromatography tandem mass spectrometry.

A fully automated, rapid, and reliable method for simultaneous determination of six carcinogenic primary aromatic amines (AAs), including o-toluidine (o-TOL), 2, 6-dimethylaniline (2, 6-DMA), o-anisidine (o-ASD), 1-naphthylamine (1-ANP), 2-naphthylamine (2-ANP), and 4-aminobiphenyl (4-ABP), in mainstream cigarette smoke was established. The proposed method was based on two-dimensional online solid phase extraction combined with liquid chromatography tandem mass spectrometry (SPE/LC-MS/MS). The particulate phase of the mainstream cigarette smoke was collected on a Cambridge filter pad and pretreated via ultrasonic extraction with 2% formic acid (FA), while the gas phase was trapped by 2% FA without pretreatment for determination. The two-dimensional online SPE comprised of two cartridges with different absorption characteristics was applied for sample pretreatment. Analysis was performed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) under multiple reaction monitoring mode. Each sample required about 0.5h for solid phase extraction and analysis. The limit of detections (LODs) for six AAs ranged from 0.04 to 0.58ng/cig and recoveries were within 84.5%-122.9%. The relative standard deviations of intra- and inter-day tests for 3R4F reference cigarette were less than 6% and 7%, respectively, while no more than 7% and 8% separately for a type of Virginia cigarette. The proposed method enabled minimum sample pretreatment, full automation, and high throughput with high selectivity, sensitivity, and accuracy. As a part of the validation procedure, fifteen brands of cigarettes were tested by the designed method.

Scientific assessment of the use of sugars as cigarette tobacco ingredients: a review of published and other publicly available studies.

Sugars, such as sucrose or invert sugar, have been used as tobacco ingredients in American-blend cigarettes to replenish the sugars lost during curing of the Burley component of the blended tobacco in order to maintain a balanced flavor. Chemical-analytical studies of the mainstream smoke of research cigarettes with various sugar application levels revealed that most of the smoke constituents determined did not show any sugar-related changes in yields (per mg nicotine), while ten constituents were found to either increase (formaldehyde, acrolein, 2-butanone, isoprene, benzene, toluene, benzo[k]fluoranthene) or decrease (4-aminobiphenyl, N-nitrosodimethylamine, N-nitrosonornicotine) in a statistically significant manner with increasing sugar application levels. Such constituent yields were modeled into constituent uptake distributions using simulations of nicotine uptake distributions generated on the basis of published nicotine biomonitoring data, which were multiplied by the constituent/nicotine ratios determined in the current analysis. These simulations revealed extensive overlaps for the constituent uptake distributions with and without sugar application. Moreover, the differences in smoke composition did not lead to relevant changes in the activity in in vitro or in vivo assays. The potential impact of using sugars as tobacco ingredients was further assessed in an indirect manner by comparing published data from markets with predominantly American-blend or Virginia-type (no added sugars) cigarettes. No relevant difference was found between these markets for smoking prevalence, intensity, some markers of dependence, nicotine uptake, or mortality from smoking-related lung cancer and chronic obstructive pulmonary disease. In conclusion, thorough examination of the data available suggests that the use of sugars as ingredients in cigarette tobacco does not increase the inherent risk and harm of cigarette smoking.

Comparison of exposure to selected cigarette smoke constituents in adult smokers and nonsmokers in a European, multicenter, observational study.

BACKGROUND: This multicenter, observational study was conducted in three European countries (Germany, Switzerland, and the United Kingdom) to determine the exposure of adult cigarette smokers and nonsmokers to selected cigarette smoke constituents: 1,3-butadiene, 2-naphthylamine, 4-aminobiphenyl, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), acrolein, benzene, carbon monoxide, nicotine, pyrene, and o-toluidine. METHODS: Smokers were grouped by tar category (TC) according to the tar yield of their regular cigarette brand: TC1: ≤4 mg tar, TC2: 5-7 mg tar, and TC3: ≥8 mg tar [to the legal tar yield ceiling in the respective countries (10 or 12 mg tar)]. Levels of biomarkers of exposure to the aforementioned cigarette smoke constituents were compared between smokers and nonsmokers, and within smokers across tar categories. RESULTS: The full population consisted of 1,631 subjects (1,223 smokers and 408 nonsmokers). Biomarkers of exposure were analyzed for 1,558 subjects (valid case population) as follows: 1,159 smokers (TC1: n = 402, TC2: n = 379, TC3: n = 378), and 399 nonsmokers. Exposure levels were higher in smokers than nonsmokers and increased with increasing tar yield and cigarette consumption. An association of tar category and exposure level was observed for all smoke constituents, except pyrene, 4-aminobiphenyl, and o-toluidine, whereas only NNK exposure was different in all three tar categories. CONCLUSIONS: Smoking status and, among smokers, daily cigarette consumption and tar yield were observed to affect biomarker of exposure levels. IMPACT: This research provides a comprehensive evaluation of smoke constituent exposure of adult cigarette smokers and nonsmokers in three European countries.

DNA adducts of ortho-toluidine in human bladder.

BACKGROUND: 4-Aminobiphenyl (4-ABP) and o-toluidine are known human bladder carcinogens, but only 4-ABP-releasing DNA adducts are known. METHODS: Determination of 4-ABP and o-toluidine-releasing DNA adducts in epithelial and submucosal bladder tissues of sudden death victims (SDV: n=46), and bladder tumours (n=12) by gas chromatography/mass spectrometry. RESULTS: Above background, 4 and 11 of 12 tumour samples contained adducts of 4-ABP (0.057 ± 0.125 fmol/µg DNA) and o-toluidine (8.72 ± 4.49 fmol/µg DNA), respectively. Lower adduct levels were present in both epithelial and submucosal bladder tissues of SDV (4-ABP: 0.011 ± 0.022 and 0.019 ± 0.047 fmol/µg DNA; o-toluidine: 0.24 ± 0.63 and 0.27 ± 0.70 fmol/µg DNA). CONCLUSION: Detection of o-toluidine-releasing DNA adducts support the carcinogenicity of o-toluidine in the human bladder.

Identification of carcinogen DNA adducts in human saliva by linear quadrupole ion trap/multistage tandem mass spectrometry.

DNA adducts of carcinogens derived from tobacco smoke and cooked meat were identified by liquid chromatography-electrospray ionization/multistage tandem mass spectrometry (LC-ESI/MS/MS(n)) in saliva samples from 37 human volunteers on unrestricted diets. The N-(deoxyguanosin-8-yl) (dG-C8) adducts of the heterocyclic aromatic amines 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-9H-pyrido[2,3-b]indole (AalphaC), 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (MeIQx), and the aromatic amine, 4-aminobiphenyl (4-ABP), were characterized and quantified by LC-ESI/MS/MS(n), employing consecutive reaction monitoring at the MS(3) scan stage mode with a linear quadrupole ion trap (LIT) mass spectrometer (MS). DNA adducts of PhIP were found most frequently: dG-C8-PhIP was detected in saliva samples from 13 of 29 ever-smokers and in saliva samples from 2 of 8 never-smokers. dG-C8-AalphaC and dG-C8-MeIQx were identified solely in saliva samples of three current smokers, and dG-C8-4-ABP was detected in saliva from two current smokers. The levels of these different adducts ranged from 1 to 9 adducts per 10(8) DNA bases. These findings demonstrate that PhIP is a significant DNA-damaging agent in humans. Saliva appears to be a promising biological fluid in which to assay DNA adducts of tobacco and dietary carcinogens by selective LIT MS techniques.

Detoxification of benzidine-based azo dye by E. gallinarum: time-course study.

Direct black 38 (DB38) dye is a well-established toxic and carcinogenic compound. Present investigation reports isolation of an Enterococcus gallinarum strain capable of decolorizing and degrading it. Changes in toxicity and mutagenicity of DB38 and its metabolites were also determined using a battery of carefully selected tests (cytotoxicity, respiration inhibition test and Ames test). Toxicity assays were carried out on E. gallinarum itself as this also gave information about suitability of this strain for the dye decolorization operation. The strain was found to reduce both toxicity and mutagenicity of DB38 metabolites. Benzidine and 4-aminobiphenyl (4-ABP) were identified as the DB38 metabolites, responsible for its toxic and mutagenic properties, by HPLC-MS analysis. Further degradation of benzidine and 4-ABP was found to result in the decrease in toxicity and mutagenicity.

Voltammetric determination of aminobiphenyls at a boron-doped nanocrystalline diamond film electrode.

Voltammetric behavior of 2-aminobiphenyl, 3-aminobiphenyl, and 4-aminobiphenyl at a boron-doped nanocrystalline diamond film electrode was investigated using cyclic voltammetry and differential pulse voltammetry. Optimum conditions have been found for the determination of those genotoxic substances by differential pulse voltammetry at the above given electrode in the concentration range of 2 x 10(-7) to 1 x 10(-5) mol/L.

Quantification of N-(deoxyguanosin-8-yl)-4-aminobiphenyl adducts in human lymphoblastoid TK6 cells dosed with N-hydroxy-4-acetylaminobiphenyl and their relationship to mutation, toxicity, and gene expression profiling.

Gene expression profiles that are anchored to phenotypic endpoints may lead to the identification of signatures that predict mutagenicity or carcinogenicity. The study presented here describes the analysis of DNA adducts in the human TK6 lymphoblastoid cell line after exposure to N-hydroxy-4-aminobiphenyl, a mutagenic metabolite of 4-aminobiphenyl. A validated nano-LC microelectrospray mass spectrometry assay is reported for the detection and quantification of N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP), the principal DNA adduct of 4-aminobiphenyl. Limits of quantification, based on a signal-to-noise ratio of 10:1, are determined to correspond to approximately 27 fg of dG-C8-ABP injected on-column. The assay has been used to measure the steady-state levels of the adduct in the human TK6 lymphoblastoid cell line as a function of dose (0.5, 1.0, and 10.0 microM) and time (2, 6, and 27 h) after exposure to N-hydroxy-4-aminobiphenyl. The levels of dG-C8-ABP adducts in the cells, ranging from 18 to 500 adducts in 10(9) nucleotides, were then correlated to cell toxicity, induced mutation at the TK (thymidine kinase) and HPRT loci, and gene expression profiling through microarray analysis. Cell cultures were evaluated for toxicity by growth curve extrapolation, mutation assays were performed on the HPRT and TK loci, and gene expression profiles were generated by analyses using microarray technology. In the mutation assay analysis, as the toxicant concentration increased, there was an increase in mutation fraction, indicating a direct correlation to metabolite dosing level and mutations occurring at these two loci. Statistical analysis of the gene expression data determined that a total of 2250 genes exhibited statistically significant changes in expression after treatment with N-OH-AABP (P < 0.05). Among the genes identified, 2245 were up-regulated, whereas 5 genes that had functions in cell survival and cell growth and, hence, could be indicators of toxicity, were down-regulated relative to controls. The results demonstrate the value of anchoring gene expression patterns to phenotypic markers, such as DNA adduct levels, toxicity, and mutagenicity.

Identification of aminobiphenyl derivatives in commercial hair dyes.

A recent epidemiological study suggested that aromatic amines present in hair dyes may contribute to an increased risk of bladder cancer (Gago-Dominguez, et al. (2003) Carcinogenesis 24, 483-489). Moreover, a preliminary study linked frequent hair dye usage with elevated levels of DNA adducts of 4-aminobiphenyl (4-ABP) in human epithelial breast cells (Gorlewska, et al. Proc. Am. Assoc. Cancer Res. 43, 1018-1019). Therefore, we sought to determine if 4-ABP, a recognized human urinary bladder carcinogen, is present in commercial hair dyes. 4-ABP was isolated from dyes by solvent extraction with hexane, followed by silica gel chromatography, either with or without chemical treatment of the extract with Zinc/HCl, and a final purification with a mixed cation exchange reversed-phase resin. The identity of 4-ABP was confirmed by both HPLC with electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) and gas chromatography with negative ion chemical ionization mass spectrometry (GC-NICI-MS) following chemical derivatization with pentafluoropropionic anhydride (PFPA). The levels of 4-ABP ranged from not detectable (<0.29 parts per billion (ppb)) up to 12.8 ppb. The noncarcinogenic isomer 2-aminobiphenyl (2-ABP) was also found at quantities up to 310 ppb. 4-ABP was detected in eight of the 11 hair dyes and found in black, red, and blonde hair dyes but not in brown hair dyes. 1,4-Phenylenediamine (PPD) is a key constituent for color development of many permanent hair dyes. Some batches of chemical research grade PPD were contaminated with 4-ABP (up to 500 ppb) and 2-ABP (up to 70 parts per million) and may be a source of ABP contamination in hair dyes. These analytical data demonstrate that 4-ABP is present in some hair dyes. Studies on dermal absorption and bioavailability of 4-ABP from hair dyes are required to determine if this aromatic amine contributes to the increased risk of bladder cancer reported in frequent users of hair dyes.

Analysis of arylamine isomers by micellar electrokinetic chromatography.

Analysis of carcinogenic substances is a high-priority area. Carcinogenic arylamines draw the analyst's attention because dyes and pigments are in production and used in large volumes. Identification of carcinogenic isomers of arylamines employing micellar electrokinetic chromatography (MEKC), a mode of capillary electrophoresis was studied as it offers better scope for separation science. Mixed micellar modes of MEKC techniques were employed to achieve acceptable analyses. Success of this analytical method was proved by real-sample analysis, which confirmed that this is a promising technique for the arylamine species.

Carcinogen-DNA adducts in human breast epithelial cells.

Diet and environmental exposures are often regarded as significant etiologic factors in human breast cancer. Chemicals that may be involved in these exposures include heterocyclic amines, aromatic amines, and polycyclic aromatic hydrocarbons, which also serve as strong mammary carcinogens in different animal models. In this study, we chose to quantify the major DNA adducts derived from one member of each of these classes of carcinogens, that is, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 4-aminobiphenyl (ABP), and benzo[a]pyrene (B[a]P), respectively, in DNA isolated from exfoliated ductal epithelial cells in human breast milk. Milk was collected from healthy, nonsmoking mothers. The isolated DNA was digested to 3' nucleotides and subjected to (32)P-postlabeling. Adduct enrichment was achieved using Oasis Sep-Paks and the analyses were conducted by HPLC using radiometric detection. Critical to the analyses were the syntheses of bis(phosphate) standards for the C8-dG adducts of PhIP and ABP, and the N(2)-dG adduct of B[a]P, which were added to each reaction as UV markers. Of the 64 samples analyzed, adducts were found in 31 samples. Thirty samples contained detectable levels of PhIP adducts, with a mean value of 4.7 adducts/10(7) nucleotides; 18 were positive for ABP adducts with a mean value of 4.7 adducts/10(7) nucleotides; and 13 were found to contain B[a]P adducts with a mean level of 1.9 adducts/10(7) nucleotides. These data indicate that women are exposed to several classes of dietary and environmental carcinogens and that these carcinogens react with DNA in breast ductal epithelial cells, the cells from which most breast cancers arise.

Does smoking reduction result in reduction of biomarkers associated with harm? A pilot study using a nicotine inhaler.

The aim of the study was to determine if smoking reduction using a nicotine inhaler in heavy cigarette smokers who wanted to reduce but not stop smoking results in decreased levels of known biomarkers of harm. The study design was a one-sample within-subject comparative open-label study of 23 (10 male and 13 female) subjects using a nicotine inhaler to reduce smoking, with follow-up at 24 weeks. A structured protocol was used with a smoking-reduction schedule from 40 or more cigarettes per day to 10 cigarettes per day by week 9. Behavioral counseling was provided by a research assistant and ad lib use of the nicotine inhaler for 12 weeks was permitted. Blood thiocyanate, cotinine, 4-aminobiphenyl hemoglobin adducts; urine NNAL and NNAL-glucuronide; and expired air carbon monoxide were measured. On average, the subjects were able to reduce their smoking by over 50% at week 12, but only two were able to reduce to 10 cigarettes per day. The reported reduction in smoking was not associated with a consistent reduction in the biomarkers. There was no reduction in the NNAL, 4-aminobiphenyl hemoglobin adducts nor carbon monoxide levels of expired air. There was a significant reduction of NNAL-glucuronide and the sum of NNAL and NNAL-glucuronide but only at week 24. Thiocyanate levels increased. Before widely promoting harm reduction as a treatment strategy for heavy smokers, more research needs to be performed to prove conclusively that such smokers who want to reduce but not stop can actually reduce and maintain their smoking rate at a level which is likely to reduce harm. It also needs to be determined whether a reduction in the smoking rate translates into reduction of harm. At the present, for heavy smokers, an abstinence approach seems to be more scientifically sound.

Determination of some aromatic amines in finger-paints for children's use by supercritical fluid extraction combined with gas chromatography.

A test method based on supercritical fluid extraction (SFE) and gas chromatography has been developed for some aromatic amines, such as 4-chloro-o-toluidine, beta-naphthylamine and 4-aminobiphenyl. A two-level factor design was used as the optimization procedure. Four main variables were considered: CO2 pressure, extraction temperature, static extraction time and volume of modifier (methanol). Results obtained for 4-chloro-o-toluidine, indicated that the volume of modifier was the variable with the most important influence on extraction, CO2 pressure had a negative effect and temperature and time were less significant. For the other amines, static time was the most important variable in both cases, followed by CO2 pressure and volume of modifier, with no influence of temperature. SFE was compared with Soxhlet extraction, and was found to give higher recoveries in all cases. Other commercial finger-paints were tested for the presence of aromatic amines.

The use of 4-aminobiphenyl hemoglobin adducts and aromatic DNA adducts in lymphocytes of smokers as biomarkers of exposure.

Two biomarkers of exposure to cigarette smoke, 4-aminobiphenyl-hemoglobin (Hb) adducts and aromatic DNA adducts in lymphocytes, were determined from a population of 55 smokers and 4 nonsmokers. The levels of these adducts were related to daily cigarette consumption and also to (calculated) tar and nicotine intake. The Hb adduct levels seemed to correspond best to the number of cigarettes (cig) smoked, but at a cigarette consumption of >30 cig/day, a saturation effect was observed. Lymphocytic DNA adducts also correlated well with cigarette and tar consumption; for this type of adduct, a saturation level was reached at a dose of approximately 15-20 cig/day. From a subpopulation, a second sample was obtained after 2 months, and the adduct levels were compared with their initial adduct levels. Strong correlations were found between the first and second DNA adduct measurements (r = 0.84). In another subpopulation, resampling was performed after 6 months. No correlation between DNA adduct levels in the first and last samples was found, but 4-aminobiphenyl Hb adduct levels were strongly correlated (r = 0.78), the absolute quantities measured being comparable (paired t test: t = -1.27, P = 0.22, n = 15). We found no influence of GSTM1 and NAT2 polymorphisms on Hb adduct formation and of GSTM1 polymorphism on aromatic DNA adduct formation. A significantly lower aromatic DNA adduct level was observed for intermediate acetylators when compared to slow acetylators.

Immunohistochemical analysis of 4-aminobiphenyl-DNA adducts in oral mucosal cells of smokers and nonsmokers.

BACKGROUND: The "biologically effective dose markers", DNA and protein adducts, are a direct index of carcinogen induced cell damage and an indirect one of genetic susceptibility. This study aimed to examine the dose-response relationship for 4-Aminobiphenyl-DNA adducts in oral cells of smokers and non smokers. MATERIALS AND METHODS: An immunoperoxidase method with the monoclonal 3C8 antibody, which recognizes 4-Aminobiphenyl-DNA, has been used for detecting DNA damage in oral cells of 12 smokers and 12 non smokers. RESULTS: Higher staining for 4-Aminobiphenyl-DNA was detected in the cells of smokers (187 +/- 42) vs. non smokers (135 +/- 35) (p = 0.004), with a twofold range in relative staining for both groups, suggesting individual differences relevance in metabolizing carcinogens and/or repairing DNA damage. CONCLUSIONS: This non invasive method requiring small cell amounts is a tool for monitoring large groups of subjects at risk in primary prevention programs.

Immunoperoxidase quantitation of 4-aminobiphenyl- and polycyclic aromatic hydrocarbon-DNA adducts in exfoliated oral and urothelial cells of smokers and nonsmokers.

Immunoperoxidase methods using two antibodies were developed for detection and quantitation of DNA damage in single cells. A monoclonal antibody that recognizes 4-aminobiphenyl (4-ABP)-DNA adducts was initially tested on liver tissues of BALB/c mice treated with 4-ABP, then applied to the detection of adducts in oral mucosa and exfoliated urothelial cells of smokers and nonsmokers. Levels of 4-ABP-DNA in exfoliated urothelial cells were elevated in each of 20 smokers (mean relative staining intensity, 517 +/- 137) compared with age-, race-, and sex-matched nonsmokers (313 +/- 79; P < 0.0005). Significantly higher damage levels were also observed in oral mucosa cells of smokers compared with nonsmokers (552 +/- 157 versus 326 +/- 101; P < 0.0005). A polyclonal antiserum that recognizes benzo(a)pyrene and structurally related polycyclic aromatic hydrocarbon (PAH) diol epoxide-DNA adducts was also applied to the same study samples after validation by staining of 10T1/2 cells treated with (+/-)-trans-anti-benzo(a)pyrene diol epoxide. Smokers had higher levels of PAH-DNA damage in oral mucosa and exfoliated urothelial cells than nonsmokers (oral mucosa cells, 684 +/- 107 versus 370 +/- 83; P < 0.0005; urothelial cells, 689 +/- 72 versus 495 +/- 57; P < 0.0005). A similar 2-3-fold range in relative staining was found in smokers and nonsmokers for both 4-ABP- and PAH-DNA, suggesting the importance of individual differences in capacity to metabolize the carcinogens and/or repair damaged DNA. Significant correlations were found among the biomarkers in both cell types. This noninvasive method, requiring small numbers of cells and with a relatively low cost, will be useful for monitoring DNA damage in large-scale molecular epidemiology studies.

Assembling and evaluation of new dehydrogenase enzyme electrode probes obtained by electropolymerization of aminobenzine isomers and PQQ on gold, platinum and carbon electrodes.

Pt, Au and graphite electrodes have been coated by electropolymerization of 1,2-, 1,3-, 1,4-diaminobenzene (DAB) and 4-aminobiphenyl in the presence of PQQ using cyclic voltammetry. The activity of the modified electrodes for the oxidation of paracetamol, ascorbic and uric acid was reduced by approximately 90% as compared to the bare electrodes. Polymerization in the presence 4,5-dihydro-4,5-dioxo-1H-pyrrolo(2,3-f)quinoline-2,7,9-tricarboxilic+ ++ acid, pyrroloquinolinequinone (PQQ) led, after optimization, to electrodes capable of catalysing the electrooxidation of beta-nicotinamide adenine dinucleotide, reduced form (NADH), in the range 10(-4)-10(-2) mol/l with a detection limit of 5 x 10(-5) mol/l. Amperometric measurements of NADH have been carried out at +0.2 V and the efficiency of different electrodes based on different materials has been studied. By co-entrapment of dehydrogenase highly selective enzymes, electrodes for glucose, L-lactate and L-glutamate were obtained. Dehydrogenase substrates such as glucose, lactate and glutamate were measured in the range 5 x 10(-5)-1 x 10(-2) mol/l, with detection limits of 10(-5) and 5 x 10(-6) mol/l, respectively. Probe stability under non-dynamic conditions was evaluated over 2 months. All the probes showed a decrease of 10% over 1 month and a residual activity of 50% over 2 months.