Carbonic anhydrase integrated into a multimarker approach for the detection of the stress status induced by pollution exposure in Mytilus galloprovincialis: A field case study.

The work was addressed to study the sensitivity of the enzyme carbonic anhydrase (CA) to chemical pollution in the hepatopancreas of the bioindicator organism Mytilus galloprovincialis in the context of a multimarker approach in view of ecotoxicological biomonitoring and assessment application. The study was carried out by means of a transplanting experiment in the field, using caged organisms from an initial population exposed in the field in two areas of interest: Augusta-Melilli-Priolo, an heavy polluted industrial site (eastern Sicily, Italy), and Brucoli (eastern Sicily, Italy) an area not affected by any contamination and selected as a reference site. Mussels in Augusta presented a significant increase in the digestive gland CA activity and gene expression compared to the animals caged in the control site of Brucoli. The CA response in animals from the polluted site was paralleled by proliferation/increase in the size of lysosomes, as assessed by Lysosensor green charged cells, induction of metallothionein, up-regulation of hif-α (hypoxia-inducible factor), metabolic changes associated with protein metabolism, and changes in the condition factor. Biological responses data were integrated with information about sediment chemical analysis and metal residue concentration in animal soft tissues. In conclusion, obtained results highlighted the induction of CAs in the hepatopancreas of Mytilus galloprovincialis following to pollution exposure, and demonstrated its suitability to be integrated into a multimarker approach for the detection and characterization of the stress status induced by pollution exposure in this bioindicator organism.

Selective destabilization of tumor cells with pulsed electric and magnetic sequences: a preliminary report.

BACKGROUND: Various studies in vitro suggest that low electric and magnetic fields may modify cancer cell growth and recent studies in vivo have revealed anti-tumoral effects. After screening different tumor cell lines, we identified specific sequences of localized magnetic and electric fields (MESQ) that reduce cancer cell survival in vitro. This finding led us to design an experiment to determine the actual efficacy of above sequences in selectively destabilizing tumor cells and their effect on healthy cells. MATERIALS AND METHODS: We exposed the MCF7 cancer cell line and normal fibroblasts to MESQ for 1, 2, 3 and 6 hours, evaluating cell survival and induction of apoptosis. RESULTS: Exposure to MESQ reduced MCF7 survival, inducing apoptosis in a timedependent way, whereas fibroblasts were completely unaffected. CONCLUSION: These results have promising implications for the treatment of cancer and warrant further research.

Fine structure of the midgut and Malpighian papillae in Campodea (Monocampa) quilisi Silvestri, 1932 (Hexapoda, Diplura) with special reference to the metal composition and physiological significance of midgut intracellular electron-dense granules.

The fine structure of the midgut and the Malpighian papillae in Campodea (Monocampa) quilisi Silvestri, 1932 (Hexapoda, Diplura) specimens was described. We observed the presence of electron-dense granules (EDGs) in the midgut epithelial cells, similar in genesis, structure and aspect to the type A spherocrystals described in the midgut epithelium of Collembola and Diplopoda. Energy-dispersive X-ray microanalysis was used to detect the chemical composition of the granules and to relate it to the concentrations of some potential toxic heavy metals (Pb, Cu, Zn) in soil and litter. Chemical composition of the granules seems strongly influenced by the presence and bioavailability of heavy metals in the external environment. Specimens from a contaminated abandoned mining and smelting area (Colline Metallifere, southern Tuscany) were able to accumulate Fe, Mn, Zn, Pb and Cu in their midgut EDGs. In addition, we observed that C. (M.) quilisi was able to excrete the metal-containing granules into the external medium by the moulting of the intestinal epithelium. This confirms that the process of ionic retention of midgut cells is particularly significant in animals lacking Malpighian tubules.

Multi-trial biomarker approach in Meganyctiphanes norvegica: a potential early indicator of health status of the Mediterranean "whale sanctuary.".

The aim of this study was to propose a suite of biomarkers (BPMO activity, NADPH-cytocrome c reductase, NADH-ferricyanide reductase. esterases, porphyrins, vitellogenin and zona radiata proteins) and residue levels (organochlorines, PAHs and heavy metals) in the zooplanktonic euphausiid Meganyctiphanes norvegica as a potential multi-disciplinary diagnostic tool for assessment of the health status of the Mediterranean "whale sanctuary". Very little difference in BPMO was detected between sites, with values ranging from 0.75 to 2.68 U.A.F./mg prot/h. On the other hand larger differences between sites were found for reductase activities. Esterases (AChE), porphyrins (Copro-, Uro-, Proto-porphyrins) vitellogenin and zona radiata proteins were also detectable in this zooplanctonic species. Hg showed mean levels of 0.141 ppm d.w., Cd 0.119 ppm d.w. and Pb 0.496 ppm d.w. Total PAHs ranged from 860.7 to 5,037.9 ng/g d.w., carcinogenic PAHs from 40.3 to 141.7 ng/g d.w., HCB from 3.5 to 11.6 ng/g d.w., DDTs from 45.3 to 163.2 ng/g d.w. and the PCBs from 84.6 to 210.2 ng/g d.w.

In vitro study of methylmercury in blood of bottlenose dolphins (Tursiops truncatus).

The biochemical behavior of methylmercury (MeHg) in dolphin blood was investigated in vitro. MeHg distribution between plasma and erythrocytes and its release from erythrocytes into plasma or medium without SH group was determined. At the subcellular level its distribution among different thiol-containing molecules was also investigated in erythrocytes and plasma. When blood was treated with 0.1 mM MeHg, about 98.1% was found in red cells and 1.9% in plasma; only 0.6% of MeHg present in the cellular compartment was bound to membranes. Hemoglobin (Hb) and albumin, principal proteins containing SH groups (PSH), and glutathione (GSH) appeared to be the main targets of MeHg in dolphin blood. Gel filtration of stroma-free hemolysate of treated red blood cells (RBCs) revealed that MeHg was almost equally present in high (52.5%) and low (47.5%) molecular weight fractions, whereas in plasma it only eluted with proteins (high molecular weight fractions). Hemoglobin was identified as the main intracellular protein binding MeHg. The exchange reaction of MeHg between GSH and dolphin hemoglobin was also evaluated and the equilibrium constants calculated.

Porphyrin levels in excreta of sea birds of the Chilean coasts as nondestructive biomarker of exposure to environmental pollutants.

In this preliminary study on sea birds we propose the use of porphyrins in excreta as a biomarker of exposure to contaminants. Samples of excreta were obtained from colonies of brown pelicans (Pelecanus occidentalis thagus), neotropic cormorants (Phalacrocorax olivaceus), and kelp gulls (Larus dominicanus) in three areas of the south coast of Chile with different human impact (Tubul, Talcahuano, and Valdivia). They were analyzed for porphyrin content (copro-, uro-, and protoporphyrins and total porphyrins) by a rapid fluorimetric method and by HPLC. The main outcomes of the study were: (a) kelp gulls and neotropic cormorants living in areas with high human impact showed a clear capacity to accumulate and eliminate porphyrins in the excreta; (b) species-related accumulation capacities are likely, as shown by the different levels found in different species living in the same area; (c) the porphyrin profile obtained by fluorimetry and HPLC showed a higher percentage of protoporphyrin than the other porphyrins; (d) although the fluorimetric method of Grandchamp is semiquantitative, it was found to be sensitive enough to detect differences in samples from field studies. The positive results of this preliminary study make it possible to propose this nondestructive method for a variety of field applications.

Trace elements in striped dolphins (Stenella coeruleoalba) from the western Mediterranean.

Tissues obtained from Stenella coeruleoalba stranded along the Spanish and Italian Mediterranean coasts from 1987 to 1994 were analysed for cadmium (Cd), copper (Cu), mercury (Hg), selenium (Se) and zinc (Zn). The age, length and weight of the dolphins were recorded. Hg levels were also assayed in skin biopsies from dolphins of the same species in the waters off northeastern Spain and in the Tyrrhenian and Ligurian Seas. Levels of all elements differed in muscle of stranded dolphins from the two areas. Hg was higher in tissues from animals stranded on the Italian coasts and in skin biopsies obtained in the Tyrrhenian and Ligurian Seas, than in the respective Spanish samples. This is probably related to Hg pollution from the natural weathering of cinnabar ores in central Italy. Se and Cd levels had similar accumulation patterns to those of Hg. Accumulation of Hg and Se is explained by the existence of a detoxification pathway involving both elements, however the reason for the similar Cd trend is unclear. Geographical differences in the accumulation pattern of these elements may reflect the existence of two different populations of Stenella coeruleoalba in the western Mediterranean.

Chlorinated hydrocarbons in the tissues of South Polar Skuas (Catharacta maccormicki) and Adélie Penguins (Pygoscelis adeliea) from Ross Sea, Antarctica.

High rates of egg infertility and embryo death in a colony of South Polar Skuas breeding in the Antarctic were similar to those in polluted North Atlantic populations of the Great Skua. Such loss could not be linked to factors such as organochlorine pollutants, as levels of DDE and PCBs in the contents of skua eggs from the population were only a small fraction of those in polluted skua populations from the Northern Hemisphere. Average eggshell thickness for skuas nesting on Ross Island has shown no significant change since the introduction of DDT. Concentrations of DDE and PCBs in South Polar Skuas were 13 and 22 times higher, respectively, than those in the eggs of sympatric Adélie Penguins, and this probably reflects the greater exposure of skuas to pollution when they migrate north of the Antarctic Convergence in winter. Residues in liver tissue showed a similar trend, and a higher rate of mixed function oxidase induction in skua liver compared to that of penguins is consistent with the trends seen in pollutant levels. The same PCB congener predominated in both skua and penguin samples. Comparisons with historical residue data suggest that global levels of DDT residues are declining.

Porphyrins as biomarkers of methylmercury and PCB exposure in experimental quail.

Chemicals such as heavy metals and polyhalogenated hydrocarbons have a high capacity to interfere with the enzymatic processes responsible for haem biosynthesis. These compounds can produce accumulation in tissues and organs and increased elimination of porphyrins in excreta (Andrew et al, 1990). The development of fast and easy analytical methods and the wide variety of biological media in which porphyrins can be detected have suggested their use as biomarkers of environmental pollution (Akins et al, 1993; De Matteis and Lim 1994). The analysis of porphyrins in the excreta is of special interest because it enables nondestructive monitoring of wild animals in the assessment of threatened or endangered species (Fossi et al, 1994). Methylmercury and PCBs are ubiquitous global pollutants and there is evidence they accumulate in terminal consumers, particularly those belonging to marine trophic chain (Renzoni et al, 1986; Yamashita et al, 1993). There have been some reports on methylmercury-induced (e.g. Woods et al, 1991; Bowers et al, 1992; Miller and Woods 1993) and PCB-induced porphyria (e.g. Vos and Pennings, 1971; Miranda et al, 1987; Elliot et al, 1990; Miranda et al, 1992) but little data on their combined effect. In order to investigate the quality of porphyrins as biomarkers we performed an experiment in which Japanese quail were fed a diet containing methylmercury and polychlorobyphenyls (PCBs as Arochlor 1260) individually or combined in different ratios. The present study aims to provide preliminary data on liver and fecal levels of porphyrins in response to methylmercury and PCB administration, and on whether the indicator is sensitive to synergism or antagonism between the two compounds, administered simultaneously.

Persistent organochlorine residues in fish and water birds from the Biobio river, Chile.

Concentrations of polychlorinated biphenyls (PCBs), DDT and its metabolites, HCH isomers and hexachlorobenzene (HCB) were determined in fish and birds from different locations in the Biobio river basin (central Chile). Samples collected near the mouth of the river contained high concentrations of PCBs, reflecting the massive use of these xenobiotics in the urban and industrial areas of Concepcion and Talcauano. Samples collected in the central part of the basin contained very high concentrations of lindane that coincide with the widespread use of lindane-based pesticides (purified γ-HCH) in this area. DDT was distributed homogeneously throughout the basin, except at Laguna Icalma, the source of the river in the Andes. Most PCB residues in fish and birds consisted of congeners between penta- and hepta-chlorobiphenyls. In fish, the predominant congeners were the pentachlorobiphenyl 23'44'5 (IUPAC number 118) and the hexachlorobiphenyl 22'344'55' (PCB-153); in birds 22'44'55' (PCB-180) prevailed.

Multiresponse biomarker evaluation of interactions between methylmercury and Arochlor 1260 in quail.

The single and combined effects of methylmercury and Arochlor 1260 were investigated in experimental quail treated chronically with the two compounds at low and high doses. A series of metabolic and biochemical biomarkers were evaluated together with mercury and PCB accumulation to pinpoint the effects of treatment with one or both chemicals. Methylmercury alone was associated with a decrease in serum cholesterol. Less PCBs were accumulated in tissues when Arochlor 1260 was combined with methylmercury than when the former was administered alone. Liver monooxygenase (MFO) activity was depressed 50% more in the presence of methylmercury than with Arochlor 1260 alone. Single or combined treatment with high doses of the two compounds resulted in similar degrees of DNA damage. This approach was found to provide a good picture of the interaction between environmental contaminants.

Interspecies differences in mixed function oxidase activity in birds: Relationship between feeding habits, detoxication activities and organochlorine accumulation.

The relationship between feeding habits and interspecies differences in the detoxication ability of the mixed function oxidase (MFO) system was investigated in birds. The role of MFO (particularly aldrin epoxidase activity) in the detoxication/bioaccumulation of organochlorines was also investigated. Euriphagic (yellow-legged herring gull (Larus cachinnans), black-headed gull (Larus ridibundus), jackdaw (Corvus monedula), magpie (Pica pica) and stenophagic (cormorant (Phalacrocorax carbo), coot (Fulica atra) and sparrow (Passer italiae)) species were collected in northern and central Italy. The following liver microsomal monooxygenase activities were measured: aldrin epoxidase, ethoxyresorufin dealkylation (EROD), benzyloxyresorufin dealkylation (BROD) and pentoxyresorufin dealkylation (PROD). NADPH-and NADH-cytochrome c reductase (NAD(P)H-CYT-CRED) and NADH-ferricyanide reductase (NADH-FERRIRED) activities were also measured in the liver microsomal fraction. Glutathione concentration (GSH) was measured in the liver cytosolic fraction. As a marker of liver damage gamma-glutamyl transpeptidase (GGTP) activity was assayed in the serum. The residues of organochlorines (HCB, pp'DDTs and PCBs) were determined in muscle samples. Omnivorous species, particularly the yellow-legged herring gull, had the highest aldrin epoxidase activities. In the yellow-legged herring gull the activity was approximately twice as high as in the jackdaw (p < 0.05), black-headed gull (p < 0.05) and coot (p < 0.05). Values five times lower were detected in the specialist fish-eater, the cormorant (p < 0.001). The lowest values of aldrin epoxidase activity were detected in the sparrow. Feeding habits were found to be related to evolutionary interspecies differences in MFO activity. A significant statistical correlation (r = 0.656) was found between the 'omnivore index' and MFO detoxication activity expressed as aldrin epoxidation. Organochlorines, and particularly PCBs, were higher in cormorants and yellow-legged herring gulls from a polluted lagoon than in the other species analysed (black-headed gull p < 0.01; sparrow p < 0.001; jackdaw p < 0.01; coot p < 0.001). Levels of pp'DDE were much higher in the cormorant and sparrow than in the other birds. Knowledge of the different species-specific detoxication abilities was found to be a useful tool for the identification of species potentially at risk in environments with high pollution by chlorinated hydrocarbons, or in general contaminants metabolized by the MFO system.

Blood esterase inhibition in birds as an index of organophosphorus contamination: field and laboratory studies.

: With the aim of developing a nondestructive biomarker (serum 'B' esterases) for monitoring bird populations exposed to azamethiphos S((6-chloro-2-oxooxazolo(4,5-b)pyridin-3(2H)-yl)methyl) 0,0-dimethyl phosphorothioate (9Cl), parallel laboratory and field studies were performed. In japanese quail (Coturnix coturnix japonica) treated with azamethiphos at 10 mg kg(-1), serum butyrylcholinesterase (BChE) and carboxylesterase (CE) activities were inhibited by 88% and 35%, respectively, after 24 h. A gradual recovery was observed after 48 and 72 h. With a higher dose (50 mg kg(-1)), the birds died, or were killed 3 h after dosing. Serum BChE and brain acetylcholinesterase (AChE), in birds that were sampled at 3 h, were inhibited by 98% and 92%, respectively. Hepatic microsomal and serum CEs were also inhibited. There was a statistically significant correlation (r=0.9808, p<0.001) between the activities of serum BChE and brain AChE. Swallows (Hirundo rustica) nesting in a stable treated with azamethiphos showed a drastic reduction in BChE (56%) and CE (36%) 24 h after treatment of the stable.

The differential modulation of the enzymes of glutathione metabolism. Indication of overlapping effects of toxicity and repair in mouse liver and kidney after dietary treatment with methyl mercury and sodium selenite.

The effect of methylmercury (MM) and MM plus sodium selenite (SE) on the activity of various GSH-dependent enzymes was studied in the liver and kidney of mice. Ten groups of mice were fed diets containing graded proportions of MM, alone or with graded quantities of SE. GST, GSH-Px, and GSSG-RED were assayed in the cytosolic fraction of liver and kidney homogenates. After treatment with MM, instead of the expected decrease in enzyme activities, an increase was observed in the kidney and a small decrease in the liver with no dose-response relation in either organ. In protected groups, a general pattern of induction was observed in both organs, but again there was little evidence of dose-response relationships. Detailed analysis of the results suggests that the effects observed were not directly caused by MM or SE but are the resultant of complex interactions presumably related to contemporaneous mechanisms of damage and repair.

Serum esterase inhibition in birds: a nondestructive biomarker to assess organophosphorus and carbamate contamination.

With the aim of proposing a nondestructive biomarker for monitoring the toxicological risk to birds of exposure to the organophosphorus insecticide azamethiphos and the carbamate insecticide methomyl, laboratory studies were performed on serum "B" esterases in Japanese quail (Coturnix coturnix japonica). The birds received two single dose treatments of each compound (azamethiphos and methomyl), i.e., 50 mg/kg and 250 mg/kg respectively. In the first treatment, serum butyrylcholinesterase (BChE) and carboxylesterase (CbE) were drastically inhibited in the azamethiphos-treated group, 24 h after the dose. No inhibition was detected for BChE and CbE activities in the methomyl-treated group, 24 h after the dose. In the second treatment, the birds died or were sacrificed 3 h after the dose. Serum BChE and brain acetylcholinesterase (AChE) were strongly inhibited after treatment with both insecticides. Serum CbE, hepatic microsomal CbE and 7-ethoxyresorufin dealkylation activities were also inhibited. A statistically significant correlation between serum BChE and brain AChE was found at lethal and sublethal doses of these xenobiotics. The experimental results indicate that the nondestructive biomarker BChE can give an early qualitative and semi-quantitative warning of the toxic effects of organophosphate and carbamate insecticides in birds.

Heavy metals and selenium in stranded dolphins of the northern Tyrrhenian (NW Mediterranean).

Heavy metal (Hg, Cd, Pb and Zn) and selenium levels were determined in striped (Stenella coeruleoalba) and bottle-nosed dolphins (Tursiops truncatus) stranded along the coast of Tuscany and Latium, Italy in the period 1987-1989. Lead and zinc concentrations were quite low and there was modest accumulation of cadmium in the kidney of both species. Mercury levels were very high, especially in the liver where they reached peaks of 4400 ppm (dry weight) in the striped dolphin and 13,150 ppm (dry weight) in the bottle-nosed dolphin. Selenium levels were also high and were significantly correlated with mercury levels in some organs and tissues. The toxicological significance of the selenium-mercury interaction is discussed.

Influence of cadmium on polychlorobiphenyl uptake, MFO activity, and serum lipid levels in Japanese quail.

The effect of cadmium on the metabolism of polychlorobiphenyls (PCBs) (Aroclor 1260) was investigated in quails fed diets containing 100 ppm Cd, 100 ppm PCBs (Aroclor 1260) and 100 ppm Cd + 100 ppm PCBs for a period of 45 days. The presence of Cd in the diet did not interfere with MFO activity (EROD and ALDE) and cytochrome P-450 enzymes. Quails fed simultaneously with Cd and PCBs, accumulated PCBs in muscle five times greater than quails fed PCBs only. Quails fed with Cd-containing diet showed a slight increase of cholesterol and triglycerides. These data suggest that a combined chronic exposure to Cd and PCBs could modify PCBs metabolism. The interaction seems due not to inhibition of drug-metabolizing enzymes but to the increase of circulating lipids that favor the accumulation of lipophilic compounds.

The relationship between gamma-glutamyl transpeptidase and Hg levels in Se/Hg antagonism in mouse liver and kidney.

The gamma-glutamyl transpeptidase (gamma-GT) activity and Hg concentrations were studied in Se/Hg antagonism in mouse liver and kidney after treatment with methyl mercury (MM) (MM group) and MM + sodium selenite (SE) (SM group). In acute treatment (dietary doses: MM = 250 p.p.m.; SE = 90 p.p.m.; length of treatment 11 days), hepatic gamma-GT activity increased in both protected and unprotected animals with respect to controls and reached a peak after 3 days with respect to controls, its value being relatively greater in the MM group. On the contrary, renal gamma-GT decreased with time with respect to controls and was higher in the SM group at 3 and 7 days. Liver and kidney accumulation of Hg increased and decreased respectively with time, and was higher in SM groups in most cases. In chronic treatment (dietary doses: MM = 12.5 p.p.m.; SE = 9 p.p.m.; length of treatment 12 months) hepatic gamma-GT activity in the MM group was higher than in the SM group at 1.5 and 7 months, whereas the renal activity was lower at 7 months and unchanged at 1.5 and 12 months. In comparison with the acute treatment, the trend of Hg accumulation was similar in liver and different in kidney; Hg concentrations of the SM group were always greater than those of the MM group. Glutathione (GSH) in liver and non-protein SH groups (NPSH) in kidney were also measured in acutely treated animals. On the first day GSH was about 50% of the control value in both the MM and SM groups; it subsequently remained constant in the MM group, but increased to a peak at 7 days, without reaching the control value, in the SM group. Unlike the liver, renal NPSH increased in both groups on the first day, and then decreased with time without reaching the control value, SM group values always exceeding those of MM group. The modulation of gamma-GT activity in liver and kidney caused by SE suggests that the enzyme plays a role in Hg accumulation.