Artemisia dracunculus L. extract ameliorates insulin sensitivity by attenuating inflammatory signalling in human skeletal muscle culture.

AIMS: Bioactives of Artemisia dracunculus L. (termed PMI 5011) have been shown to improve insulin action by increasing insulin signalling in skeletal muscle. However, it was not known if PMI 5011's effects are retained during an inflammatory condition. We examined the attenuation of insulin action and whether PMI 5011 enhances insulin signalling in the inflammatory environment with elevated cytokines. METHODS: Muscle cell cultures derived from lean, overweight and diabetic-obese subjects were used. Expression of pro-inflammatory genes and inflammatory response of human myotubes were evaluated by real-time polymerase chain reaction (RT-PCR). Insulin signalling and activation of inflammatory pathways in human myotubes were evaluated by multiplex protein assays. RESULTS: We found increased gene expression of monocyte chemoattractant protein 1 (MCP1) and TNFα (tumour necrosis factor alpha), and basal activity of the NFkB (nuclear factor kappa-light-chain-enhancer of activated B cells) pathway in myotubes derived from diabetic-obese subjects as compared with myotubes derived from normal-lean subjects. In line with this, basal Akt phosphorylation (Ser473) was significantly higher, while insulin-stimulated phosphorylation of Akt (Ser473) was lower in myotubes from normal-overweight and diabetic-obese subjects compared with normal-lean subjects. PMI 5011 treatment reduced basal phosphorylation of Akt and enhanced insulin-stimulated phosphorylation of Akt in the presence of cytokines in human myotubes. PMI 5011 treatment led to an inhibition of cytokine-induced activation of inflammatory signalling pathways such as Erk1/2 and IkBα (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha)-NFkB and moreover, NFkB target gene expression, possibly by preventing further propagation of the inflammatory response within muscle tissue. CONCLUSIONS: PMI 5011 improved insulin sensitivity in diabetic-obese myotubes to the level of normal-lean myotubes despite the presence of pro-inflammatory cytokines.

Dental Caries: The Way Forward.

KNOWLEDGE TRANSFER STATEMENT: It is evident that some progress in reducing ECC prevalence in children has been made, but these improvements are not equally distributed. Systemic inequities in oral health among the youngest, most vulnerable children must be reduced.

Pathways Linking Oral Bacteria, Nitric Oxide Metabolism, and Health.

Nitrate-reducing oral bacteria have gained a lot of interest due to their involvement in nitric oxide (NO) synthesis and its important cardiometabolic outcomes. Consortia of nitrate-metabolizing oral bacteria associated with cardiometabolic health and cognitive function have been recently identified. Longitudinal studies and clinical trials have shown that chronic mouthwash use is associated with increased blood pressure and increased risk for prediabetes/diabetes and hypertension. Concurrently, recent studies are beginning to shed some light on the complexity of nitrate reduction pathways of oral bacteria, such as dissimilatory nitrate reduction to ammonium (DNRA), which converts nitrite into ammonium, and denitrification, which converts nitrite to NO, nitrous oxide, and dinitrogen. These pathways can affect the composition and metabolism of the oral microbiome; consequently, salivary nitrate and nitrite metabolism have been proposed as targets for probiotics and oral health. These pathways could also affect systemic NO levels because NO generated through denitrification can be oxidized back to nitrite in the saliva, thus facilitating flux along the NO3--NO2--NO pathway, while DNRA converts nitrite to ammonium, leading to reduced NO. It is, therefore, important to understand which pathway predominates under different oral environmental conditions, since the clinical consequences could be different for oral and systemic health. Recent studies show that oral hygiene measures such as tongue cleaning and dietary nitrate are likely to favor denitrifying bacteria such as Neisseria, which are linked with better cardiometabolic health. A vast body of literature demonstrates that redox potential, carbon-to-nitrate ratio, and nitrate-to-nitrite ratio are key environmental drivers of the competing denitrification and DNRA pathways in various natural and artificial ecosystems. Based on this information, a novel behavioral and microbial model for nitric oxide metabolism and health is proposed, which links lifestyle factors with oral and systemic health through NO metabolism.

Tumor invasion induced by oxidative stress is dependent on membrane ADAM 9 protein and its secreted form.

Oxidative stress plays a role in the regulation of cancer cell metastasis which involves cell invasion and adhesion that could be supported by ADAM proteins through the activities of their metalloprotease and disintegrin domains. We hypothesized that oxidative stress could act through the induction of ADAM9 protein in some cancer cells. Indeed, Western blot analysis for ADAM9 performed on A549 cells exposed to H(2) O(2) reveals a dose-dependent induction of two proteins (80 and 68 kDa) correlated with a sharp increase of the ADAM protease activity measured in supernatant while the activity measured on the cell layer was slightly affected. The 80kDa protein corresponds to the mature form of ADAM9. Immunoprecipitation analysis performed on concentrated supernatants revealed that the 68 kDa protein is a secreted form of ADAM9. When exposed to H(2) O(2) , A549 cells cocultured with confluent endothelial vascular cells resulted in a 5.5 fold (p < 0.001) increase in the number of adherent cells. Similarly, matrigel assay revealed a 3.25 fold (p < 0.01) increase in the number of invasive cells. The suppression of ADAM9 expression by specific small interfering RNA reduced oxidative stress-induced invasiveness and adhesiveness. These functions could be mediated by an interaction between ADAM9 and ß1 integrin because each of them were inhibited when the experiment is performed in presence of mAbs targeting ADAM9 ectodomain or ß1-integrin. These results emphasize the importance of oxidative stress in the regulation of cancer cell metastasis and suggest that ADAM9 and its secreted isoform can be important determinants in the ability of cancer cells to disseminate.

Differentiation of yeasts growing on dry-cured Iberian ham by mitochondrial DNA restriction analysis, RAPD-PCR and their volatile compounds production.

The efficiency of mitochondrial DNA (mtDNA) restriction analysis, RAPD-PCR and volatile compounds analysis to differentiate yeast biotypes involved in flavour development of dry-cured Iberian ham throughout the ripening process is evaluated. For this purpose, 86 yeasts isolated from Iberian hams in the main ripening stages at different industries of the four Protected Designations of Origin of this product, were used. The combination of mtDNA restriction analysis and RAPD-PCR using the primer (GACA)4 showed a higher variability in the yeast species detected than obtained using only mtDNA restriction analysis. Only two species, Debaryomyces hansenii and Candida zeylanoides, were identified throughout the whole ripening process and a wide diversity of biotypes was found in these two species, with those of D. hansenii predominating. Clear differences between biotypes were detected in the generation of volatile compounds, with the biotype C2-2 of D. hansenii showing the highest concentrations of volatiles. The combined use of mtDNA restriction analysis and RAPD-PCR distinguishes yeast biotypes with different production of volatile compounds. In addition, analysis of the production profile of volatile compounds is needed to differentiate yeast strains of the same biotype recovered at different stages of ripening. Thus, the combination of these three methods could be very useful to select or monitor yeasts as starter cultures in dry-cured meat products.

Oral Bacterial Acid-Base Metabolism in Caries Screening: A Proof-Of-Concept Study.

The objective of this cross-sectional study was to clinically validate an array of biochemical tests for oral acid/alkali generation as caries screening instruments. 185 adult subjects (mean 33.6±10.6 years) were examined clinically for dental caries using the ICDAS criteria. Bitewing radiographs were used to confirm interproximal surfaces of posterior teeth. For the purposes of this study, subjects were classified as "caries-active" if they had at least one untreated caries lesion with ICDAS 4 or higher. Pooled supragingival plaque and unstimulated saliva samples were collected and assayed for pH changes from sucrose and urea metabolism using colorimetric tests. The validity of each test to discriminate between "caries-inactive" and "caries-active" subjects was assessed and compared to a commercial bacteriological caries-screening test using roc regression and logistic regression models. The AUCs of the plaque-urea (PU: 0.59 (0.51, 0.67)), plaque-urea-glucose (PUG: 0.59 (0.51, 0.67)) and saliva-urea-glucose (SUG: 0.59 (0.51, 0.67)) tests did not differ significantly from the bacteriological tests (CRT-mutans: 0.62 (0.54, 0.70); CRT-lactobacillus: 0.63 (0.56, 0.71) (P>0.05), but the plaque-glucose (SG), saliva-glucose (SG), saliva-urea (SU) and saliva-plaque-glucose (SPG) tests had significantly smaller AUCs (P<0.05). The AUCs for the PU, PUG, SUG, and the CRT-mutans tests were higher in subjects who had no existing dental restorations (PU: 0.90 (0.77, 1.04); PUG: 0.90 (0.79, 1.01); SUG: 0.89 (0.69, 1.08); CRT-mutans: 0.90 (0.73, 1.08)). The incorporation of the biochemical tests into a multidimensional bacteriological/psychosocial caries screening model significantly increased its diagnostic values (Se+Sp: 160.6, AUC: 0.846). In conclusion, as a proof of concept, the results of this study indicate that measuring the ability of dental plaque and saliva to metabolize urea together with the ability to generate acid from sugars may have a promising role in caries screening either independently, or as part of a multidimensional biological test.

Use of 5-azacitidine for therapy-related myeloid neoplasms in patients with concomitant active neoplastic disease.

BACKGROUND: Patients diagnosed with therapy-related myeloid neoplasms (TRMN) with concomitant active neoplastic disorder (CAND) are usually proposed for best supportive care (BSC). We evaluated the feasibility of using 5-azacytidine (AZA) in this setting. METHODS: All patients referred to Gustave Roussy between 2010 and 2015 for TRMN diagnosis (less than 30% blast) and eligible for AZA treatment were included. Patients with CAND proposed for BSC were also described. Patient's outcomes were analyzed based on the presence or not of a CAND. RESULTS: Fifty-two patients with TRMN were analyzed, including 19 patients with CAND (14 eligible for AZA) and 33 without CAND eligible for AZA. The 5 patients with CAND ineligible for AZA had a worst performance status (p=0.016) at diagnosis and a shorter overall survival (OS) (0.62 months). Baseline characteristics of patients eligible for AZA were similar in the 2 groups except a trend for best performance status in patients with CAND (p=0.06). Overall response rate (71.4% vs 60.3%), transfusion independence (50.0% vs 45.5%) and OS (12.7 months vs 10.8 months) were similar between patients with and without CAND respectively (p=ns). CONCLUSION: Here we report the feasibility and efficacy of AZA for selected patients with TRMN and a CAND.

Trisomy of rat chromosome 1 associated with mesothelial cell transformation.

Identification of specific chromosomal aberrations in transformed mesothelial cells is an important step in elucidating the mechanism of transformation of these cells which are targets for occupational and environmental carcinogens, such as asbestos fibers. Cytogenetic analysis of normal rat mesothelial cell lines revealed that at late passage (p20-p34), trisomy of chromosome 1 was present in greater than 80% of the cells in four spontaneously immortalized lines examined, whereas at early passage (p8-p10), only 15-44% of the cells had trisomy 1. Trisomy of chromosome 1 had increased in the population as a function of passage, suggesting that cells with trisomy 1 had a selective growth advantage under in vitro culture conditions and that this alteration was associated with transformation. A commercially available rat mesothelial cell line (4/4 RM4, ATCC), was also found to have a duplication of a portion of the long arm of chromosome 1. To determine if chromosome 1 alterations have relevance to the transformed phenotype in vivo, a neoplastic cell line was established from a spontaneous rat mesothelioma. At passage 15, trisomy of chromosome 1 was observed in 26% of the metaphases in this line. However, when these cells were injected into nude mice, 99% of the cells from the resulting tumor contained an additional copy of chromosome 1. Therefore, trisomy 1 also conferred a selective growth advantage in vivo and/or was associated with the malignant subpopulation in the tumor derived cell line. These studies suggest that chromosome 1 contains a gene(s) involved in transformation of rat mesothelial cells.

p53 mutations in formaldehyde-induced nasal squamous cell carcinomas in rats.

Formaldehyde induces squamous cell carcinomas in the nasal passages of rats following chronic inhalation exposure at concentrations of > or = 10 ppm. We have examined the complementary DNA of the tumor suppressor gene p53 from 11 primary formaldehyde-induced tumors for mutation using DNA sequence analysis. A polymerase chain reaction-amplified fragment of the rat p53 complementary DNA containing the evolutionarily conserved regions II-V was directly sequenced from each tumor. Point mutations in the p53 complementary DNA sequence were found in 5 of 11 of the tumors analyzed. These data demonstrate p53 point mutations in formaldehyde-induced squamous cell carcinomas and indicate a common alteration in certain rat and human squamous cell carcinomas of the respiratory tract.

Characterization of platelet-derived growth factor and platelet-derived growth factor receptor expression in asbestos-induced rat mesothelioma.

Although altered expression of platelet-derived growth factor (PDGF) is a hallmark of human mesothelioma, expression of PDGF receptors has not been characterized in this cell type. In addition, the expression of this growth factor and its cognate receptor in rodent mesothelioma has not been investigated. In this study, examination of transformed mesothelial cells derived from asbestos-induced rat mesotheliomas revealed that these cells expressed high affinity PDGF receptors (Kd = 0.5 nM) and receptor number was 1.6 x 10(5)/cell. Western analysis using antibodies specific for either the alpha-type or beta-type PDGF receptor and Northern analysis using probes specific for alpha- and beta-type receptor RNA transcripts indicated that these cells expressed beta-type PDGF receptors but that alpha-type receptors could not be detected. However, when the mesothelioma-derived cells were examined for the expression of PDGF, no expression of this growth factor could be detected. The transformed cells expressed no detectable A- or B-chain PDGF RNA transcripts; and using a competitive enzyme immunoassay specific for isoforms containing the B chain of PDGF and a sandwich enzyme-linked immunosorbent assay specific for A-chain-containing isoforms, neither AA, nor AB, nor BB isoforms of this growth factor could be detected in medium conditioned by these cells. The absence of alterations in PDGF expression in rat mesothelioma, in contrast to the data for the human disease, suggests that the production of this growth factor by transformed mesothelial cells may be species specific.

Expression of insulin-like growth factor II in spontaneously immortalized rat mesothelial and spontaneous mesothelioma cells: a potential autocrine role of insulin-like growth factor II.

Insulin-like growth factors (IGFs) are polypeptides that play an important role in cellular proliferation and differentiation. The present study examines the role of IGFs in the growth of mesothelial cells. Cell lines derived from normal rat mesothelium as well as lines derived from spontaneous rat mesotheliomas were found to express RNA transcripts for IGF-II. In contrast, cell lines derived from asbestos-induced rat mesotheliomas did not express this growth factor. All cell lines expressed receptors for IGF-I and IGF-II, as well as insulin receptors. Coexpression of IGF-II and its cognate receptor suggested that IGF-II was acting as an autocrine growth factor in the spontaneously immortalized cells and the cells derived from the spontaneous tumors. The biological activity of IGF-II secreted by the cell lines into conditioned medium could be neutralized using an IGF-II-specific antibody. Growth was inhibited in a dose-dependent manner; at the highest antibody concentration used (100 micrograms anti-IGF-II/ml), cell growth was decreased to 47% of control values. This inhibition was partially reversible by treatment of the cultures with IGF-II (91% of the control). These data suggest that IGF-II expression may be involved in the spontaneous alteration of rat mesothelial cells and may function as an autocrine or paracrine growth factor to modulate the growth of these cells in vitro and in vivo. Ubiquitous expression of IGF-II by cells that have not been exposed to asbestos and the lack of IGF-II expression by asbestos-transformed cells suggest that the mechanisms of changes in growth factor expression differ in mesothelial cells transformed by different pathways.

Autocrine growth stimulation by transforming growth factor alpha in asbestos-transformed rat mesothelial cells.

Although the association between asbestos exposure and mesothelioma development has been established for decades, very little is known regarding the molecular mechanism(s) by which asbestos fibers induce this disease. In this series of experiments, the potential for transforming growth factor alpha (TGF-alpha) to act as an autocrine growth factor in transformed mesothelial cells was examined in rats, a model system frequently used to assess the tumorigenic potential of fibrous particulates. Both asbestos-transformed cells and spontaneously transformed cells expressed functional EGF receptors, although only the asbestos-transformed cells expressed TGF-alpha. Expression of TGF-alpha transcripts was correlated with secretion of picogram amounts of growth factor into conditioned medium by the asbestos-transformed cells. In addition, whereas TGF-alpha inhibited the growth of spontaneously transformed mesothelial cells, it stimulated the growth of asbestos-transformed cells. Neutralizing antibody that recognized TGF-alpha secreted by the asbestos-transformed cells was able to inhibit the growth of these cells. Taken together, these data indicate that TGF-alpha acts as an autocrine growth factor for asbestos-transformed rat mesothelial cells. Therefore, in asbestos-transformed mesothelial cells, altered production and responsiveness to TGF-alpha distinguish these cells from spontaneously transformed mesothelial cells. These data suggest that differences in mesothelioma etiology may be reflected in differences in the molecular alterations present in these tumors.

Body composition analysis by dual-energy X-ray absorptiometry in young preschool children.

BACKGROUND/OBJECTIVES: Dual-energy X-ray absorptiometry (DXA) is considered a specific method for measuring body composition to assess obesity and osteoporosis, although few studies have been conducted in preschool children. The aim of this study was to provide sex - and age-specific references for bone mineral density (BMD), bone mineral content (BMC), fat mass (FM) and fat-free mass (FFM) normative data for children aged 2 to <6 years. SUBJECTS/METHODS: One hundred and eighty seven healthy white children from Buenos Aires City suburbs, Argentina, were studied by the Lunar DPX-L DXA, pediatric software: BMC less head (g), BMD (g/cm2), FM (%) and FFM (g). RESULTS: BMD and BMC increased significantly with age (P<0.0001), but only BMD was significantly different between boys and girls of similar age, being greater for boys (P=0.013). FM was not significantly different among the various age groups of boys and girls. However, the FFM/height was higher in boys and the BMC/FFM was higher in girls. The Z-scores and centile curves were derived separately for each sex and age. Q-Q detrended plots and LMS curves produced robust, unbiased fits that generated references for the 3rd, 50th and 97th percentiles for BMD, BMC, FM and FFM data, respectively. CONCLUSIONS: These DXA scans add to the scarcity of accurate measurements of body composition of white young children. The data analyses provided greater accuracy, particularly at the upper and lower ends of the distribution, which is important in clinical settings for identification of children with impaired body composition.

Novel enzyme activities and functional plasticity revealed by recombining highly homologous enzymes.

BACKGROUND: Directed evolution by DNA shuffling has been used to modify physical and catalytic properties of biological systems. We have shuffled two highly homologous triazine hydrolases and conducted an exploration of the substrate specificities of the resulting enzymes to acquire a better understanding of the possible distributions of novel functions in sequence space. RESULTS: Both parental enzymes and a library of 1600 variant triazine hydrolases were screened against a synthetic library of 15 triazines. The shuffled library contained enzymes with up to 150-fold greater transformation rates than either parent. It also contained enzymes that hydrolyzed five of eight triazines that were not substrates for either starting enzyme. CONCLUSIONS: Permutation of nine amino acid differences resulted in a set of enzymes with surprisingly diverse patterns of reactions catalyzed. The functional richness of this small area of sequence space may aid our understanding of both natural and artificial evolution.

Aqueous extracts of cigarette tar containing the tar free radical cause DNA nicks in mammalian cells.

The ability of aqueous extracts of cigarette tar to nick DNA was investigated using viable mammalian cells. Tar extracts contain a radical with a stable electron spin resonance (ESR) signal at g = 2.0036 characteristic of a semiquinone. The association of the tar component that carries the ESR signal with DNA was demonstrated using viable rat alveolar macrophages. The formation of single-strand DNA breaks caused by cigarette tar extracts in viable rat thymocytes follows saturation kinetics, indicating a tar component associates with DNA and then nicks it. These studies support our hypothesis that tar components that contain the cigarette tar radical can enter cells, associate with, and then nick DNA.

Environmental tobacco smoke is just as damaging to DNA as mainstream smoke.

This study demonstrates the ability of tar isolated from environmental tobacco smoke (ETS) to nick DNA in mammalian cells. Solutions of ETS tar behave similarly to aqueous solutions of cigarette tar from mainstream smoke. Both solutions contain the tar semiquinone radical, and this radical associates with the DNA in viable rat alveolar macrophages. Solutions of tar from ETS cause single-strand DNA breaks in rat thymocytes in proportion to the amount of tar present, until a plateau is reached. ETS tar solutions, like mainstream tar solutions, produce hydrogen peroxide. Hydrogen peroxide appears to be an essential component of the mechanism by which both ETS tar and mainstream tar cause DNA damage in rat thymocytes, as catalase substantially protects against DNA damage. Glutathione also protects against DNA nicking by both ETS and mainstream tar solutions by scavenging radicals and/or oxidants. The chelator diethylenetriamine pentaacetic acid also provides partial (40%) protection. The studies demonstrate that the water-soluble components of ETS tar can enter cells, associate with, and then nick DNA.

Comparison of pleural responses of rats and hamsters to subchronic inhalation of refractory ceramic fibers.

In the present subchronic study, we compared pleural inflammation, visceral pleural collagen deposition, and visceral and parietal pleural mesothelial cell proliferation in rats and hamsters identically exposed to a kaolin-based refractory ceramic fiber, (RCF)-1 by nose-only inhalation exposure, and correlated the results to translocation of fibers to the pleural cavity. Fischer 344 rats and Syrian golden hamsters were exposed to 650 fibers/cc of RCF-1, for 4 hr/day, 5 days/week for 12 weeks. Following 4 and 12 weeks of exposure, and after a 12-week recovery period, pleural lavage fluid was analyzed for cytologic and biochemical evidence of inflammation. Visceral and parietal pleural mesothelial cell proliferation was assessed by immunocytochemical detection of bromodeoxyuridine incorporation. Pleural collagen was quantitated using morphometric analysis of lung sections stained with Sirius Red. Fiber-exposed rats and hamsters had qualitatively similar pleural inflammation at each time point. Mesothelial cell proliferation was more pronounced in hamsters than in rats at each time point and at each site. In both species, the mesothelial cell labeling index was highest in the parietal pleural mesothelial cells lining the surface of the diaphragm at each time point. Hamsters but not rats had significantly elevated collagen in the visceral pleura at the 12-week postexposure time point. Fibers were found in the pleural cavities of both species at each time point. These fibers were generally short and thin. These results suggest that mesothelial cell proliferation and fibroproliferative changes in the pleura of rodents following short-term inhalation exposure are associated with fiber translocation to the pleura and may be predictive of chronic pleural disease outcomes following long-term exposure.

Comparison of pulmonary and pleural responses of rats and hamsters to inhaled refractory ceramic fibers.

The present study was designed to determine whether pleural fiber burdens or subchronic pleural fibroproliferative and inflammatory changes can help explain the marked interspecies differences in pleural fibrosis and mesothelioma that are observed following long-term inhalation of RCF-1 ceramic fibers by rats and hamsters. Fischer 344 rats and Syrian golden hamsters were exposed to RCF-1 for 4 h per day, 5 days per week, for 12 consecutive weeks. Lung and pleural fiber burdens were characterized during and after exposure. For all time points, approximately 67% of fibers associated with lung tissues from both rats and hamsters were longer than 5 microns in length. In comparison, fibers longer than 5 microns recovered from the pleural compartment, following a 12-week exposure and 12 weeks of recovery, accounted for 13% (hamsters) and 4% (rats) of the distribution. In the 12 weeks after the cessation of exposure, the number of fibers longer than 5 microns in length remained constant in the hamster at approximately 150 fibers per cm2 pleura. This was 2 to 3 times the corresponding fiber surface density in the rat. Significant pulmonary and pleural inflammation was detected at all time points and for both species. DNA synthesis by pleural mesothelial cells was quantified by bromodeoxyuridine uptake following 3 days of labeling. Labeling indices were higher in hamsters than in rats, both for RCF-1-exposed and filtered air-control animals and was highest for the parietal surface of the pleura. Significantly greater collagen deposition was measured in the visceral pleura of hamsters 12 weeks post-exposure but was not significantly elevated in rats. These findings demonstrate that subchronic inhalation exposure to RCF-1 induces pleural inflammation, mesothelial-cell turnover, pleural fibrosis, and an accumulation of fibers with a length greater than 5 microns in the hamster. The accumulation of long fibers in the pleural space may contribute to the pathology observed in the hamster following chronic inhalation of RCF-1, whereas the presence of short, thin fibers may play a role in the acute-phase biological response seen in both species.

Evaluation of the genotoxic potential of glutaraldehyde.

The cytotoxic and genotoxic effects of glutaraldehyde were studied in vitro in the human TK6 lymphoblast cell line and in primary cultures of rat hepatocytes. TK6 lymphoblasts were exposed to glutaraldehyde for 2 hr in serum-free GSH-free media. Cytotoxic effects were observed at concentrations as low as 10 microM with only 10% cell survival at 20 microM. Alkaline elution studies indicated that glutaraldehyde-induced DNA-protein crosslinking increased linearly over the concentration range from 0 to 25 microM. Glutaraldehyde-induced mutations were assessed at the thymidine kinase locus over the same concentration range and reached a plateau at 10 microM of about six times the background mutant frequency. At equivalent levels of DNA-protein crosslinks and cytolethality, glutaraldehyde was mutagenic at approximately a one-seventh lower concentration than the rodent nasal carcinogen formaldehyde (Craft et al.; Mutation Research 176:147-155, 1987). Glutaraldehyde induced a marginal increase in unscheduled DNA synthesis in the in vitro hepatocyte DNA repair assay, but only at the two highest concentrations of 50 and 100 microM, indicating the induction of some DNA excision-repair activity. These data demonstrate that glutaraldehyde exhibits DNA-reactive genotoxic activity that may involve, at least in part, DNA-protein crosslinking in these cell culture models. These findings suggest the need to examine the potential carcinogenic activity of glutaraldehyde in appropriate inhalation studies.

The ozonation of cholesterol: separation and identification of 2,4-dinitrophenylhydrazine derivatization products of 3 beta-hydroxy-5-oxo-5,6-secocholestan-6-al.

The ozonation products of cholesterol, which are of interest as possible biomarkers of O3 exposure, were studied by derivatization with 2,4-dinitrophenylhydrazine (DNPH). The DNPH derivatization of 3 beta-hydroxy-5-oxo-5,6-secocholestan-6-al (2) produces the expected trans (3b) and cis (3c) derivatives of 3 beta-hydroxy-5-oxo-5,6-secocholestan-6-al, and the unexpected DNPH derivative of 3,5-dihydroxy-B-norcholestane-6-carboxaldehyde (3a). The structures of 3a, 3b, and 3c were identified with 1H nuclear magnetic resonance (NMR), 13C NMR, DEPT, COSY, and H-C correlation two-dimensional NMR techniques, and by comparison with the spectra of known compounds. A possible mechanism involving an enamine functionality is proposed for the formation of 3a. The ratio of 3a/(3b + 3c) depends on the concentration of acid used and the reaction time.