Cytogenetic evaluation of extractable agents from airborne particulate matter generated in the city of Catania (Italy)

In order to document cytogenetic damage associated with air pollution and, possibly, with health effects in the city of Catania, Sicily (Italy), we analyzed the induction of chromosomal aberrations by extractable agents from airborne particulate matter in a Chinese hamster epithelial liver (CHEL) cells. These cells retain their metabolic competence to activate different classes of promutagens/procarcinogens into biologically active metabolites. Airborne particulate matter was obtained from two stationary samplers (stations I and II) in two areas endowed by an elevated car transit in the centre of Catania. The results obtained clearly indicated that airborne particulate matter from both stations I and II proved to be clastogens in CHEL cells but not in Chinese hamster ovary (CHO) cells without metabolic activation, indicating that airborne particulate mixtures need to be metabolically converted before exerting their genotoxic potential. On the basis of these results we can assert that the test system employed to identify the cytogenetic potential of airborne particulate matter is useful and profitable for environmental control, and helpful to plan specific actions aimed at reducing the hazards derived from exposure to polluted air.     

Application of a two-stage Syrian hamster embryo cell transformation assay to cigarette smoke particulate matter

The induction of transformation in Syrian hamster embryo (SHE) cells is a multifactorial process, in comparison to endpoints induced in in vitro genotoxicity assays such as Ames, mouse lymphoma and cytogenetics [Y. Berwald, L. Sachs, In vitro cell transformation with chemical carcinogens, Nature (London) 200 (1963) 1182-1184]. Furthermore, a number of non-genotoxic carcinogens and promoters such as clofibrate and diethylhexylphthalate, have been positively identified in this assay, while giving false negative results in traditional genotoxicity assays [H. Yamasaki, J. Ashby, M. Bignami, W. Jongen, K. Linnainmaa, R.F. Newbold, G. Nguyen-Ba, S. Parodi, E. Rivedal, D. Schiffmann, J.W.I.M. Simons, P. Vasseur, Nongenotoxic carcinogens: development of detection methods based on mechanisms: a European project, Mutat. Res. 353 (1996) 47-63]. A high concordance between results obtained in this assay when compared with rodent carcinogenesis bioassays has also been noted [R.J. Isfort, G.A. Kerckaert, R.A. LeBoeuf, Comparison of the standard and reduced pH Syrian hamster embryo (SHE) in vitro cell transformation assays to predict the carcinogenic potential of chemicals, Mutat. Res. 356 (1996) 11-63]. Carcinogenesis is known to be a multistage process, with agents potentially acting at each stage. Specifically, mouse skin painting experiments established that tumour induction could be mechanistically divided into two distinct phases, termed initiation and promotion. Initiation, is defined as the stage at which a normal cell is converted to a latent tumour cell, followed by promotion where the latent tumour cell progresses to a tumour [W.F. Friedwald, P. Rous, The initiating and promoting elements in tumour production: analysis of the effects of tar, benzpyrene and methylcholanthrene on rabbit skin, J. Exp. Med. 80 (1944) 101-125]. A protocol for the pH 6.7 SHE transformation assay has been developed which allows separation of cell transformation process into two phases, potentially analogous to initiation and promotion in vivo. This allows chemicals found to be positive in the traditional SHE cell transformation assay to be further classified as initiators or promoters. Following validation with known initiators, benzo(a)pyrene and N-methyl-N'-nitro-N-nitrosoguanidine and promoters, 12-O-tetradecanoyl-phorbol-13-acetate and phenobarbitone, the two-stage model was applied to cigarette smoke particulates which was found to act both at the initiation and promotion stage of cell transformation.     

Biochemical activities of low molecular weight chitosans derived from squid pens

Chitosans were prepared by H₂O₂ oxidative depolymerization from squid pens with low molecular weights (LMW) of 13,025, 7011, 4169, 2242 and 963 Da. The bile acid binding capacities and antioxidant properties of LMW chitosans were studied in vitro. LMW chitosans exhibited stronger bile acid binding capacities than that of chitosan. The scavenging ability of LMW chitosans against DPPH radicals improved with increasing concentration, and EC₅₀ values were below 1.3 mg/mL. The EC₅₀ values of LMW chitosans against hydroxyl radicals ranged from 0.93 to 3.66 mg/mL. All LMW chitosans exhibited a strong ferrous ion chelating effect and reducing power. At 1 mg/mL, the scavenging ability of chitosan-963 towards superoxide radicals was 67.76%. These results indicated that LMW chitosans which have stronger bile acid binding capacity and antioxidant activities may act as potential antioxidants in vitro.     

Mutagenicity in Salmonella typhimurium mutants of the benzene-soluble organic matter derived from air-borne particulate matter and its five fractions.

Air-borne particulate matter was collected on a filter, then extracted with benzene. The benzene-soluble material was separated into 5 fractions, namely acidic, basic, alipathic, polyaromatic and oxygenated fractions. The mutagenic activities of these fractions were examined with a set of Salmonella typhimurium mutants. The 6 mutants were from the TA1535 series, deep rough strains without excision repair, namely TA100 and TA98 (having a resistance-transfer factor) and the standard strain TA1535, TA1536, TA1537 and TA1538. Linear dose-response curves were obtained for the benzene-soluble organic matter, and its acidic, polyaromatic and oxygenated fractions with strain TA98 and a 9000 X g liver supernatant from both phenobarbital(PB)- and dibenz(a,h)anthracene(DBA)-treated rats. Among the 5 fractions tested, 3 fractions, namely the acidic, polyaromatic and oxygenated, played an important role in the mutagenicity of the benzene-soluble organic matter derived from air-borne particulate matter. The 9000 X g rat-liver supernatant was not required to make the acidic fraction mutagenic.     

Ectoenzymatic ratios in relation to particulate organic matter distribution (Ross Sea,Antarctica)

The results of a study on ectoenzymatic activity (the enzyme activity bound to particles larger than 0.2 micro m) and its relation to organic particle concentration are reported here. The sampling was carried out during the 1994 Antarctic spring, at a fixed station (Station 11) in the polynya of the Ross Sea, an area characterized by quick changes in sea ice cover. The sampling was repeated 4 times over a 20-day time period. The particulate organic matter distribution followed the physical structure of the water column, which depends on ice dynamics and is mainly determined by salinity. In the mixed-water surface layer (0-50 m) the concentrations were higher (on average 65.6 micro gC/L) than in the deeper water layer (50 m-bottom) (on average 19.1 micro gC/L). This distribution and quality, expressed by the protein:carbohydrate ratio, linked the particulate organic matter to the phytoplanktonic bloom which was in progress in the area. We determined the kinetic parameters of the glycolytic and proteolytic ectoenzymes and also the total activity for the proteolytic enzyme, in order to evaluate the contribution of the particle-bound activity. We observed higher values in the surface layer than in the deeper layer. b-Glucosidase activity ranged between 0.03 and 0.92 nmol L(-1) h(-1); b-N-acetylglucosaminidase activity was in the range of 0.04-0.58 nmol (L-1) (h-1). The total proteolytic activity (leucine aminopeptidase) ranged between 0.85 and 33.71 nmol L(-1) (h-1). The ectoproteolytic activity was about 35-60% of the total. The Km values were slightly higher for the proteolytic activity (on average 0.43 micro M for ectoproteolytic activity and 0.58 micro M for total proteolytic activity) than for the b-glucosidase (on average 0.36 micro M) and b-N-acetylglucosaminidase (on average 0.17 micro M), showing no remarkable variations in the water column. The ectoenzymatic ratios and their relationship with particulate organic substrates confirm the close link between organic substrate availability and degradation system response. The significant and positive correlations are not specific and suggest a prompt and efficient systemic response to the input of trophic resources. Nevertheless, changes in ectoenzyme activity and synthesis may act as adaptive responses to changing features of the ecosystem. In particular, variations in the proteolysis:glycolysis ratio depend on the functional features of the ecological system. In our study area this ratio is higher (about 10 or more) during production (particularly autotrophic) and lower (about 5 or less) during degradation/consumption events. The analysis of previous data, collected over a larger area characterized by different environmental conditions due to the changes of the pack ice cover, during the same cruise, confirms the existence of a significant relationship. Furthermore, the analysis of enzyme-uptake systems, expressed as Vmax:Km ratio, suggests that glycolytic ectoenzymes, although poorly expressed, may encourage microconsumers to grow rapidly on a wide range of organic substrates, including the refractory ones such as cellulose and chitin. However, low ectoenzyme potential exploitation rates of available organic substrates (on average about 5% for glycolytic and 12% for proteolytic ectoenzymes) would suggest that, during spring, zooplankton grazing or vertical and lateral transport are likely to play an important role in the removal of organic materials from the system.     

In vitro cytokinin binding to a particulate fraction of tobacco cells

At least two types of cytokinin-binding sites are present in a particulate fraction of tobacco (Nicotiana tabacum L.) cells that sediments at 80,000 x g. The major binding component has a low affinity towards cytokinins, is resistant to heating at 100°C, and is not specific for biologically active cytokinin analogues. The second site occurs in much lower frequency, is heat labile, shows high affinity towards cytokinins, and is specific for biologically active analogs of the hormone. The testing for binding specificity was mainly performed with a series of halogenated benzyladenine derivatives having a wide range of biological activities. The low-affinity binding site shows some of the same features as talcum powder, a non-biological material which binds cytokinins in a non-specific fashion. The properties of the high-affinity binding site are consistent with the expected characteristics of a cytokinin receptor. However, the role of the observed high-affinity binding site with regard to the biological action of cytokinins is not yet known.Abbreviations BA N ⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - K_d equilibrium dissociation constant - R_t total concentration of binding sites In partial fulfillment of the requirements for the Ph.D. degree in the Department of Botany and Plant Pathology, Michigan State University     

Enzymic and chemical analysis of particulate organic matter from a boreal river

SUMMARY. 1. Benthic particulate organic matter (POM) was collected from a shallow pool of a fourth order boreal stream and sorted into seven size fractions ranging from 63 to >4000μm. Each size fraction was analysed for fibre, total phosphorus, and total Kjeldahl nitrogen. Microbial activity was measured by oxygen consumption and characterized by assaying for eleven classes of exoenzymes including cellulase, phenol oxidase, peroxidase, phosphatase and sulphatase.
2. Indices of detritus quality such as C/N, C/P, percent lignin, and microbial respiration showed improvement with decreasing particle size. Three covarying exoenzyme groups were identified: a carbohydrase-phosphatase group that included eight enzymes, a phenol oxidase-peroxidase group, and sulphatase. The activity of the carbohydrase-phosphatase group, was significantly correlated with microbial respiration and the carbohydrate content of the POM. Phenol oxidase-peroxidase activity was correlated with lignin content for POM greater than 250 μm, but activity increased markedly in the two smallest size fractions even though the lignin content of the POM continued to decline, Sulphatase activity was inversely related to particle size over the entire range.
3. The changes in microbial activity with particle size were attributed to the increasing surface area to volume ratio of smaller particles and to an ecological succession in the microbial community.     

Mass loss and qualitative change in stream-incubated fine particulate organic matter derived from leaves differing in rate of processing

The initial quantitative breakdown of fine particulate organic matter (FPOM) was investigated by measuring the loss (over 73 days) of substrate mass of particles of known size ranges (53–125 µm, 125–250 µm, 250–500 µm, 500 µm-1 mm) and derived from known organic sources (Alnus rubra, Acer macrophyllum, Polystichum munitum). Qualitative examinations (organic content, C : N ratio) also were made. Particles ranging from 500 µm to 1 mm in diameter differed greatly from particles in other size ranges, and results of studies with these particles closely resembled results of coarse particulate (CPOM) leaf pack studies. Despite variation, alder particles generally exhibited the greatest mass loss, those of sword-fern, the least, and mass loss of bigleaf maple particles was intermediate. Organic contents of all particle substrates decreased over time. In general, the C : N ratios of alder particles increased, those of bigleaf maple decreased, and those of sword-fern exhibited little change. All particle substrates were incubated in the field in vials, which allowed for influx of natural detritus of unknown source and period of residence. Given the overall abundance and prevalence of the FPOM resource in lotic systems, standardization of a procedure such as that used in this investigation would be useful in extending understanding of stream system processes, including detrital processing and decomposition.     

Distribution and composition of particulate organic matter in the Ross Sea (Antarctica)

The biochemical composition and spatial distribution of particulate organic matter (POM) were studied in the Ross Sea (Antarctica) in summer 1989 to assess the quantitative role of organic carbon fractions in the cycling of organic matter in the water column. Large differences in chemical composition were observed between surface and deep layers. The results indicated that, despite large geographical differences, POM was quite homogeneous, of phytoplankton origin and mostly detrital. Different ratios were used to investigate the changes in biochemical composition of particulate organic matter in relation to the ice-melting: CN (organic carbonorganic nitrogen ratio) and C-POMPOC (sum of carbohydrate, protein and lipid carbontotal organic carbon ratio) were used to analyse the percentage of refractory organic material. PPRTPCHO (proteincarbohydrate ratio) were used to establish POM age and RNADNA ratios as a relative measure of particulate activity; POCChl a and N-PPRTChl a ratios were used to estimate the autotrophic contribution to the suspended particulate organic matter. Despite its low caloric value (5.3 Kcal g POM^–1), an high caloric content in the photic layer (1.6 Kcal m^–3 of POM and 2.5 Kcal m^–3 of POC) was found thus indicating that a large amount of food was available to higher trophic levels.     

Over-expression of a cold-induced plasma membrane protein gene (MpRCI) from plantain enhances low temperature-resistance in transgenic tobacco

The plasma membrane is the most direct target of low temperature injury in plants. We have cloned a cold-induced gene (MpRCI) from plantain (Musa paradisiaca L.; ABB Group). Expression of an MpRCI::GFP fusion protein in onion epidermal cells showed localization of the protein product in the plasma membrane. The expression profile of MpRCI was analyzed by RT-PCR and the results indicated that MpRCI is induced by low temperatures in leaves and leafstalk, but not in the shoot meristematic or roots. We also cloned a 1.2 kb fragment upstream of MpRCI, predicted to contain several elements related to abiotic stresses, and demonstrated that the sequence has characteristics of low temperature- and ABA-induced promoter activity. Furthermore, the results of the phenotypic espial and ion leakage assays, using transgenic tobacco containing the gene, indicated that over-expression of the cold-induced plasma membrane protein gene MpRCI enhanced low temperature-resistance in the these plants. These results suggest that MpRCI is involved in maintaining the stability of the plasma membrane at low temperatures.     

Oscillations of the internal CO(2) concentration in tobacco leaves transferred to low CO(2)

Measurement of the internal CO(2) concentration (Ci) in tobacco leaves using a fast-response CO(2) exchange system showed that in the light, switching from 350 microLL(-1) to a low CO(2) concentration of 36.5 microLL(-1) (promoting high photorespiration) resulted in the Ci oscillating near the value of CO(2) compensation point (Gamma*). The oscillations are highly irregular, the range of Ci varying by 2-4 microLL(-1) in substomatal cavities with a period of a few seconds. The statistical properties of the time series became stationary after a transient of approximately 100s following transfer to low CO(2). Attractor reconstruction shows that the observed oscillations are not chaotic but exhibit stochastic behavior. The period of oscillations is consistent with the duration of photorespiratory post-illumination burst (PIB). We suggest that the observed oscillations may be due to a similar mechanism to that which leads to PIB, and may play a role in switching mitochondrial operation between oxidation of the photorespiratory glycine and of the tricarboxylic acid cycle substrates.     

Extremely low frequency weak magnetic fields enhance resistance of NN tobacco plants to tobacco mosaic virus and elicit stress-related biochemical activities

Increasing evidence has accumulated concerning the biological effects of extremely low frequency magnetic fields (ELF-MFs) in different plant models. In the present study, effects of ELF-MFs in tobacco plants reacting to tobacco mosaic virus (TMV) with a hypersensitive response (HR) were evaluated. Plants were exposed for 8 or 24 h (either before or after TMV inoculation) to a static MF, at either -17 or 13 microT, combined with a 10 Hz sinusoidal MF with different intensities (25.6 or 28.9 microT). The working variables were the area and number of hypersensitive lesions in leaves. Following ELF-MFs exposure, an increased resistance was detected, particularly after an 8-h treatment, as shown by the decrease in lesion area and number. Moreover, two enzyme activities involved in resistance mechanisms were analyzed: ornithine decarboxylase (ODC) and phenylalanine ammonia-lyase (PAL). Uninoculated leaves previously exposed to ELF-MFs in general showed a significant increase relative to controls in ODC and PAL activities, in particular for 13 microT static MF plus 28.9 microT, 10 Hz sinusoidal MF (24 h) treatment. In conclusion, ELF-MFs seem to influence the HR of tobacco to TMV, as shown by the increased resistance and changes in ODC and PAL activities, indicating the reliability of the present plant model in the study of bioelectromagnetic interactions.     

Cytogenetic comparison of the sensitivity to mutagens in mice selected for high (HA) and low (LA) swim stress-induced analgesia

Sensitivity to mutagens was studied in mouse lines selectively bred for high analgesia (HA) and for low analgesia (LA) induced by 3-min swimming in 20 degrees C water. Apart from pain-related traits HA mice also manifest, as compared to the LA line, higher emotionality in various behavioural tests, and cope worse with the hypothermic challenge of swimming in cold water. In the present study HA mice appeared more susceptible to the mutagenic effect of whole-body gamma-radiation and mitomycin-C injection. Both treatments caused higher frequencies of chromosomal aberrations and micronucleus in bone marrow cells in the HA than in the LA line. The results are discussed in terms of a genetic correlation between animals' susceptibility to environmental stressors and the mechanism of mutagenesis. As shown by our recent molecular study, the selection for magnitude of swim analgesia has differentiated the parental outbred population into two distinct genotypes characterised by specific minisatellite and microsatellite sequences for each line, which may be genetic markers of particular traits. We conceive that the breeding strategy, along with the differentiation of stress-related phenomena, has altered the frequencies of genes controlling DNA repair in each line.     

Mutagenicity monitoring of airborne particulate matter (PM10) in the Czech Republic.

The mutagenic activities associated with inhalable airborne particulate matter (PM10) collected over a year in four towns (Czech Republic) have been determined. The dichloromethane extracts were tested for mutagenicity using the Ames plate incorporation test and the Kado microsuspension test both with Salmonella typhimurium TA98 and its derivative YG1041 tester strains in the presence and absence of S9 mixture. The aim of this study was to assess the suitability of both bacterial mutagenicity tests and to choose the appropriate indicator strain for monitoring purposes. To elucidate the correlation between mutagenicity and polycyclic aromatic hydrocarbons (PAHs), the concentration of PAHs in the air samples were determined by GC/MS. In general, the significant mutagenicity was obtained in organic extracts of all samples, but differences according to the method and tester strain used were observed. In both mutagenicity tests, the extractable organic mass (EOM) exhibited higher mutagenicity in the YG1041 strain (up to 97 rev/microg in the plate incorporation and 568 rev/microg in the microsuspension tests) than those in TA98 (up to 2.2 rev/microg in the plate incorporation and 14.5 rev/microg in the microsuspension tests). In the plate incorporation test, the direct mutagenic activity in YG1041 was on average 60-fold higher and in microsuspension assay 45-fold higher with respect to strain TA98. In the presence of S9 mix, the mutagenic potency in YG1041 declined (P<0.001) in summer, but increased in TA98 (P<0.05) in samples collected during the winter season. The microsuspension assay provided higher mutagenic responses in both tester strains, but in both strains a significant decrease of mutagenic potency was observed in the presence of S9 mix (P<0.001 for YG1041, P<0.05 for TA98 in winter). The mutagenic potencies detected with both indicator strains correlated well (r=0.54 to 0.87) within each mutagenicity test used but not (for TA98) or moderately (r=0.44 to 0. 66 for YG1041) between both of the tests. The mutagenic activity (in rev/m(3)) likewise the concentration of benzo[a]pyrene and sum of carcinogenic PAHs showed seasonal variation with distinctly higher values during winter season. A correlation between the PAH concentrations and the mutagenicity results for the plate incorporation, but not for the microsuspension tests was found. In samples from higher industrial areas, the higher mutagenicity values were obtained in plate incorporation test with TA98 and in both tests with YG1041 in summer season (P<0.05). According to our results, plate incorporation test seems to be more informative than microsuspension assay. For routine ambient air mutagenicity monitoring, the use of YG1041 tester strain without metabolic activation and the plate incorporation test are to be recommended.     

Elimination of antibacterial activities of non-peptide luteinizing hormone-releasing hormone (LHRH) antagonists derived from erythromycin A

Antibacterial SAR for a series of macrolides derived from erythromycin A that are potent LHRH antagonists was developed in an attempt to eliminate the antibiotic activities of these compounds. Increasing the size of the alkyl substituents on the desosamine 3'-amine resulted in potent LHRH antagonists that were inactive against staphylococcal bacteria strains, and were significantly (>10-fold) less active against streptococcal bacteria strains. Complete elimination of antibacterial activities could be achieved by replacement of one or both methyl groups on the 3'-amine with a large alkyl substituent.