DNA adducts in human breast tissue: association with N-acetyltransferase-2 (NAT2) and NAT1 genotypes.

The etiology of human breast cancer is poorly understood, but circumstantial evidence points toward exogenous genotoxins as causative agents; they are believed to exert their carcinogenic action by binding to DNA. Because this binding is often preceded by metabolic activation, it is dependent on the expression and activities of metabolic enzymes of the host. Human mammary tissue samples from 42 women undergoing surgery for breast cancer or reduction mammoplasty were analyzed for DNA adducts by 32P-postlabeling analysis. With the butanol extraction method of DNA adduct enrichment, adduct levels were determined to be 0-414.6 adducts per 10(9) nucleotides, with considerable interindividual variation. To characterize the DNA adducts, we reanalyzed the adduct spots by reversed-phase high-performance liquid chromatography. Of two major adduct spots detected on TLC that accounted for up to 70% of the DNA modification, one eluted as a single peak on high-performance liquid chromatography, whereas the other was resolved into two distinct peaks of radioactivity. These major adducts were highly lipophilic in character. The N-acetyltransferase-1 (NAT1) and NAT2 genes were analyzed for common mutations using random RFLP analysis. An association between NAT2 acetylator status and adduct levels was observed; significantly elevated adduct levels occurred in the mammary DNA from women who were designated slow acetylators for NAT2 [median adduct level = 83.0 adducts per 10(9) nucleotides (range, 9.0-414.6)], as compared with the levels in individuals designated rapid acetylators for NAT2 [median adduct level = 39.7 adducts per 10(9) nucleotides (range, 0-91.0; P = 0.0053)]. On the other hand, NAT1 genotypes were not significantly associated with adduct levels. Although the agents responsible for the DNA modifications in the human breast are not known, this pilot study supports the hypothesis that DNA adduct formation in the human breast may be influenced by the NAT2 genotype.

Kinetic studies on epiphyseal growth cartilage in the normal mouse.

The synthesis of DNA was studied in the proximal tibial growth plate of 25-day-old healthy NMRI mice by using the thymidine analog bromodeoxyuridine (BrdUrd), which is incorporated into cells in the S-phase. Such cells were found only in the upper three fifths of the morphologically defined proliferating zone. This zone was therefore subdivided into a functional proliferating zone (the S-phase zone) where most, if not all, chondrocytes proliferate, and a remaining maturation zone. The BrdUrd containing immunoreactive cells could then be followed at different intervals and they were found at the chondro-osseous junction after only 36 h. By using double-labeling with BrdUrd and iododeoxyuridine (IdUrd) the duration of cell cycle components could be estimated; that is, the time for DNA synthesis (S-phase), second gap and mitosis (G2 + M-phase), and remaining first gap (G1). We determined an S-phase time of 7.1 h and an average cell-cycle duration of 36 h. The G2 + M-phase was estimated as 3.5-4 h, leaving an average G1-phase time of 25 h, which probably varies considerably between chondrocytes. By combining these data with morphometrical data regarding distances between cells, we calculated a total growth rate of 9.0 microm/h. Of this rate, 80% was entirely related to the process of hypertrophy--that is, longitudinal expansion without any corresponding increase in cell number--and 75 % was the result of processes outside the S-phase zone. Five percent of the growth was due to the expansion of cell distances within the S-phase zone. In this way longitudinal expansion can be studied at different levels in the growth plate and the data permit calculation of changes in volumes of the extracellular matrix. The largest increases in matrix volume occurred in the hypertrophic zone. These data may serve as a basis for further studies on matrix turnover in relation to growth.

Optimization of a battery using nine immunocytochemical variables for distinguishing between epithelial mesothelioma and adenocarcinoma.

A battery of immunocytochemical analyses, previously established to distinguish between malignant mesothelioma and metastatic adenocarcinoma, was extended by analysing the same cases with three other commercially available antibodies. Altogether, 11 antibodies were studied in mesotheliomas diagnosed by other means, using 14 different immunocytochemical parameters. Logistic regression analysis indicated that the following parameters were of importance for this diagnostic problem: vimentin reactivity in epithelial cells (1), cytokeratin (CAM 5.2) reactivity in spindle-shaped (fibrous) cells (2), cell membrane-associated reactivity of EMA (3), HBME-1 (4) and thrombomodulin (5), and absence of reactivity to CEA (6), CD15 (7), BerEp4 (8) and Sialyl-TN (9). The analysis gave an algorithm with which a specific diagnosis of mesothelioma could be made in 80% of the cases-i.e., some improvement compared to the 55% sensitivity using the previous battery.

Immunoreactivity of pleural malignant mesotheliomas to glutathione S-transferases.

Malignant mesotheliomas show a highly variable aggressiveness, but it is difficult to predict the outcome in the individual case at the time of diagnosis. Glutathione S-transferases are detoxification enzymes that have been correlated with the prognosis in some tumours. We have therefore assessed the value of GST expression as a prognostic parameter in mesotheliomas. The reactivities to GST-pi, -alpha and -mu antibodies were studied in histological sections from altogether 88 cases. Most of them showed distinct cytoplasmic reactivity to one or more of the GST antibodies tested. This high prevalence is in good agreement with the low responsiveness of mesotheliomas to chemotherapy. However, there was no prognostic value in detecting GST immunoreactivity, and it gave no information of value for distinguishing between neoplastic and reactive mesothelium.

An optimized battery of eight antibodies that can distinguish most cases of epithelial mesothelioma from adenocarcinoma.

An immunocytochemical battery comprising 9 antibodies specifically distinguishes 80% of the epithelial malignant mesotheliomas from adenocarcinomas. The discriminatory power of antibodies to calretinin was tested together with this battery to determine whether the performance thereby could be improved. The study comprises 119 mesotheliomas of epithelial or mixed phenotype and 57 adenocarcinoma metastases in the pleural cavity. The differences between the 2 groups were highly significant for all recorded parameters, but typical reactivity for all parameters was seen in only 6 (5.0%) of the 119 mesotheliomas. An algorithm based on stepwise logistic regression was used to interpret divergent reaction patterns. Most diagnostic information was obtained with 8 of the parameters studied. The resulting algorithm identified almost 90% of the mesotheliomas with high specificity. The battery can be performed in 2 steps: several adenocarcinomas first are diagnosed with a few antibodies, applying the rest of the battery on the remaining unresolved cases.

Pancreatic DNA adducts formed in vitro and in vivo by the food mutagens 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA alphaC).

Genotoxic heterocyclic amines have been detected in grilled or fried meat and tobacco smoke. Among these, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA alphaC) have been shown to induce tumours in rodents in several organs. Here we report on the DNA adduct formation by PhIP and MeA alphaC in vitro and in vivo, both in rat hepatic and rat pancreatic tissues or cells. Using 32P-postlabelling analysis both compounds were shown to induce a dose-dependent DNA modification in primary rat hepatocytes that was correlated with cytotoxicity in these cells. In explanted rat pancreas maintained in dynamic short-term organ culture MeA alphaC was shown to induce covalent DNA adducts. No DNA adducts were observed with PhIP in this assay. DNA adducts were observed in the liver and the pancreas of F344 rats treated with PhIP, with a 36-times higher level of adducts in the pancreas, confirming data reported earlier. DNA adduct levels induced by feeding 32, 160 or 800 ppm MeA alphaC in the diet were dose-dependent and higher in the liver compared with other organs including pancreas. While for PhIP the N2-(desoxyguanin-8-yl)-derivative was accounting for more than 90% of DNA adducts detected, in the case of MeA alphaC the N2-(desoxyguanin-8-yl) adduct was predominant in vitro and determined in vivo as one of up to 5 DNA adducts. MeA alphaC had been reported to induce preneoplastic foci and tumours in the liver and tumours and atrophy in the pancreas. In the case of MeA alphaC, the DNA adduct formation and cytotoxicity observed by us in vitro and in vivo correlate with the organ specificity of the reported pathological lesions. In the case of PhIP our in vitro data in pancreas and liver and the low adduct levels in liver in vivo also reflect the reported lack of pathological effects in these organs. In contrast, in pancreas, in vivo extraordinarily high adduct levels induced by PhIP were observed confirming studies published earlier, in spite of the fact that this compound does not cause pancreatic lesions. This enigmatic observation is discussed and the relevant literature is reviewed.

Lectin-binding pattern of neuroepithelial and respiratory epithelial cells in the mouse nasal cavity.

Sections from the nasal cavity of 12-day-old Swiss albino mice (NMRI strain) were subjected to lectin histochemistry. A panel of biotinylated lectins (Con A, WGA, s-WGA, PNA, SBA, DBA and UEA I) and a horseradish peroxidase-conjugated lectin (GSA II) showed marked differences in binding to the respiratory and the neuroepithelial cells. SBA (affinity for galactose and N-acetylgalactosamine), PNA (galactose) and WGA (sialic acids and N-acetylglucosamine) labelled the receptor neurons in the olfactory and vomeronasal epithelium. DBA (N-acetylgalactosamine) labelled a subgroup of about 5% of the olfactory receptor neurons, but most neurons in the vomeronasal organ. UEA I (fucose) and s-WGA (N-acetylglucosamine) intensely labelled the entire nerve cell population in the vomeronasal organ, but in the olfactory epithelium the labelling with these lectins was stratified. In the respiratory epithelium the ciliated cells were labelled with WGA and s-WGA, while the secretory cells bound most of the lectins. Thus different sugars are exposed on the surface of the different types of epithelia in the nasal cavity, providing a basis for selectivity in microbial attacks on these areas.

32P-post-labelling analysis of DNA adducts formed by food-derived heterocyclic amines: evidence for incomplete hydrolysis and a procedure for adduct pattern simplification.

Food-derived aminoimidazoazarenes have been shown to be mutagenic and carcinogenic and to form covalent DNA adducts. 32P-Post-labelling analysis of DNA modified with these heterocyclic amines (HA), including 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,4-dimethylimidazo [4,5-f]quinoline (MeIQ), 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline (7,8-DiMeIQx) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) has resulted in considerable interlaboratory variation in the characteristic patterns of DNA adduct spots, with up to six being detected for each compound. Similar complex patterns were observed when azido-derivatives of HA were photoreacted with calf thymus DNA. When deoxyguanosine 3'-monophosphate was modified with the azido derivatives and analysed using the 32P-post-labelling procedure, one major spot was observed for IQ, 4,8-DiMeIQx, 7,8-DiMeIQx or PhIP and two major spots for MeIQ or MeIQx. In each case, these adducts were chromatographically indistinguishable from the major adducts formed with DNA. No major adduct spots were observed when 3'-phosphate derivatives of deoxyadenosine, deoxycytidine or thymidine were reacted with the azido-derivatives of HA. In an attempt to identify the additional spots, azido derivatives of PhIP or IQ were reacted with the synthetic homopolymer poly(dG).poly(dC), the alternating copolymer poly(dC-dG) or a synthetic oligonucleotide (TTT-GTTTTTTCTTTCCCT): in each case a reduced number of adduct spots were detected. The introduction of an additional nuclease P1 hydrolysis step following the labelling reaction further reduced the number of adduct spots to only one or two major spots. Reversed-phase HPLC analysis showed that the number of peaks of radioactivity was also reduced to one or two, presumably corresponding to the [32P]-5'-monophosphate deoxyguanosine adducts. We suggest that many of the additional spots commonly observed in conventional 32P-post-labelling analysis of HA-modified DNA are adducted oligonucleotides that are partly resistant to hydrolysis by micrococcal nuclease and spleen phosphodiesterase but are susceptible to hydrolysis by nuclease P1.

Characterization of the major DNA adduct formed by the food mutagen 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAalphaC) in primary rat hepatocytes.

Cooking of proteinous food results in the formation of heterocyclic amines. Among these, 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAalphaC) has been identified as a mutagenic pyrolysis product of soya protein and has been detected in grilled or pan fried meat. It was subsequently proven to be carcinogenic in mice and, recently, in rats and to form covalent DNA adducts in vitro and in vivo. The corresponding nitro compound, 2-nitro-3-methyl-9H-pyrido[2,3-b]indole (MeNalphaC), was prepared and shown to be a direct acting mutagen in the Ames Salmonella reversion assay. When MeNalphaC was chemically reduced in the presence of DNA a major DNA adduct was detected using the 32P-postlabelling assay. This major adduct was characterized by UV spectroscopy and mass spectrometry as N-(deoxyguanosin-8-yl)-2-amino-3-methyl-9H-pyrido[2,3-b]indole. This structure was corroborated by identification of the modified base as a guanine moiety modified at the C8 position as judged by chromatographic and spectral comparison with a standard synthesized from acetylated guanine-N3-oxide and MeAalphaC was characterized by UV/Vis and 1H NMR spectroscopy and mass spectrometry. Treatment of primary rat hepatocytes with MeAalphaC (100 microM, 24 h) resulted in adduct levels of 9.8 fmol/microg DNA as determined by 32P-postlabelling analysis. Using HPLC analysis, two major 32P-labelled adducts were observed accounting for 80 and 13% of total binding respectively. The major adduct was chromatographically indistinguishable from the synthetic deoxyguanosine-C8 adduct using both ion-exchange thin layer or reversed-phase HPLC. Although MeAalphaC is formed only in low p.p.b. levels in cooked food, the contribution to the human carcinogenic risk that might be imposed by heterocyclic amines is not to be neglected.

Growth parameters in the epiphyseal cartilage of brachymorphic (bm/bm) mice.

We studied kinetics in the epiphyseal cartilage of the brachymorphic (bm/bm) mouse, combining morphometry and labeling with halogenated nucleotides. The defective synthesis of the sulfate donor PAPS in these homozygous mutants is evident in tissues with a large production of glycosaminoglycans; these compounds become undersulfated. Compared with their heterozygous siblings, the longitudinal growth of the mutant mice was reduced by two-thirds. This was mainly associated with (1) reduced height of the proliferating zone, (2) a substantial number of G0 cells in this zone, and (3) reduced hypertrophy which, in turn, may be related to premature mineralization and prevention of normal expansion of cells. No significant effects on cell-cycle parameters were detected, such as S-phase time or cell-cycle time, and the rate at which each cell increased the matrix volume seemed normal. An effect on matrix mineralization may be related to known changes in the structure of matrix PGs, whereas the effect on proliferation may be related to other factors. Candidates for such other effects of undersulfation are the cell surface PGs, which are important for binding of growth factors.

Identification of the major hepatic DNA adduct formed by the food mutagen 2-amino-9H-pyrido[2,3-b]indole (A alpha C).

2-Amino-9H-pyrido[2,3-b]indole (A alpha C) is among the most prevalent heterocyclic amines detected in grilled or panfried meat; it was shown to be carcinogenic in mice, to induce preneoplastic foci in rat liver, and to form covalent DNA adducts in vitro and in vivo. The corresponding nitro compound 2-nitro-9H-pyrido[2,3-b]indole (N alpha C) was prepared and shown to be a direct acting mutagen in the Salmonella assay, while the amino compound required external metabolic activation with rat liver homogenate (S9). When A alpha C was incubated with S9 in the presence of calf thymus DNA, one major DNA adduct spot was detected upon 32P-postlabeling analysis. This adduct comigrated on ion-exchange TLC and reversed-phase HPLC with the major adduct detected in primary hepatocytes treated with A alpha C. In DNA isolated from livers of male F344 rats treated with 800 and 160 ppm, the formation of the same major adduct was observed with relative adduct levels of 20.6 +/- 9.6 and 1.4 +/- 1.1 adducts/10(8), respectively, as determined with the butanol extraction variant of the 32P-postlabeling assay. No DNA adducts were detected in liver DNA from rats treated with 32 ppm A alpha C or control animals. The major adduct spot was eluted and hydrolyzed and the modified base characterized by chromatographic and UV spectral comparison with a synthetic standard synthesized from acetylated guanine N3-oxide and A alpha C. Electrospray mass spectrometry and 1H- and 13C-NMR spectroscopy provided further evidence for the major adduct as N2-(guanin-8-yl)-2-amino-9H-pyrido[2,3-b]indole. A alpha C is formed especially in high-temperature preparation of food and may contribute considerably to the human carcinogenic risk that might be imposed by heterocyclic amines.

Electrical impedance and other physical parameters as related to lipid content of human stratum corneum.

BACKGROUND/AIMS: In previous studies we have shown that variations in the properties of the stratum corneum are reflected by alterations in electrical impedance. The aim of this study was to explore the ability of the electrical impedance technique to detect changes in the lipid content of the stratum corneum, and to compare It with the other non-invasive methods, measurement of transepidermal water loss and of skin moisture. METHODS: Twenty-two healthy test subjects were recruited. Transepidermal water loss was measured at standard sites on the forearms and wrists, followed by skin moisture estimation by electrical capacitance, and finally by the recording of electrical impedance spectra in the frequency range 1 kHz to 1 MHz. Readings by all three methods were taken before the start of each series of test procedures, as well as after cyclohexane swabbing, a skin stripping procedure and lipid extraction, and also during the recovery process. A mixture of hexane:isopro-panol was used for lipid extraction of the skin, and the extracts were evaluated using HPLC/LSD and GC/MS/FID analysis. Biopsy samples for light and electron microscopy were obtained after lipid extraction. RESULTS: Electrical impedance results showed greater changes after lipid extraction than either transepidermal water loss or skin moisture content. Baseline values varied from the cubital fossa to the wrist, both for the non-invasive methods and for lipid composition. CONCLUSIONS: The electrial impedance is dependent on the lipid content of the stratum corneum, as studied by lipid extraction experiments.

Oral IgA-IgG treatment of chronic non-specific diarrhoea in infants and children.

The effect of orally administered immunoglobulin (IgAbulin) on chronic non-specific diarrhoea of infancy was studied in seven children, median age 26 (21-36) months and duration of diarrhoea 32 (18-84) weeks. Routine laboratory tests for malabsorption and small bowel biopsies were taken in all children before and after 3 weeks of IgAbulin treatment. The biopsy specimens were analysed with regard to histopathology, electronmicroscopy, immunohistochemistry and microbiology. The number of stools decreased from a median of 4.0 (3.0-5.0) to 1.5 (1.0 3.5) (p < 0.05) stools per day over the study period.