A comparative in vitro study of the toxic potency of five inorganic lead compounds on a rat liver epithelial cell line (REL).

Relative insolubility of inorganic Pb compounds is one of the major problems in the evaluation of the toxicological profile of this metal. Different characteristics of Pb-containing solutions may, in fact, alter the biological properties of Pb compounds and influence their toxic potency. To investigate these aspects, we used selected experimental conditions to evaluate and compare the specific biological effects of five inorganic Pb compounds (soluble salts and oxide) on the viability and proliferation rate of a rat liver-derived cell line (REL cells). The study was performed according to classical toxicological criteria (dose- and time-response, reversibility/transience of the effect). Each Pb compound was accurately solubilised and the quantification of the real concentration of Pb(II) ions was performed either on the culture media used for each treatment, or on the extracts of exposed cells. Our study shows that four, out of the five Pb compounds we tested, induce the same dose- and time-related anti-proliferative effects on REL cells, being these effects also reversible, transient and directly related to the intracellular content of the metal. Since the intracellular concentration of the metal and, consequently, its biological effects on REL cells, directly depends on the bioavailability of the Pb(II) cation present in the treatment solutions, our results indicate that, in the experimental procedures aimed to assess the toxic potency of this metal, the solubility of each Pb compound should be carefully evaluated and taken into account.

[Inorganic lead stimulates endothelin secretion in the MDCK cell line]. / Il piombo inorganico stimola la secrezione di endoteline nella linea cellulare MDCK.

BACKGROUND: Lead (Pb) is an environmental toxin whose acute intoxication causes haematological, gastrointestinal and neurological dysfunctions. Moreover it is well-established that prolonged exposure to low levels of inorganic Pb compounds is closely related to hypertension in experimental animals and occupationally exposed humans. Previous reports have suggested that endothelins (ETs), a family of peptides with potent vasoconstrictive properties, might be involved in the pathogenesis of lead-induced hypertension. In vivo studies demonstrated that rats chronically exposed to Pb low levels exhibited blood pressure elevation coupled with an increase of ET-3 concentration in plasma and urine in comparison with control animals. OBJECTIVE: Since kidney is one of the target organs of lead injury, as well as the site of production/action of ETs, we investigated the effects of an inorganic Pb compound (Pb chloride) on the synthesis and secretion of these peptides, using, as in in vitro model, a renal-derived cell line (MDCK). METHODS: The ETs assays in culture media of sub-confluent cell cultures exposed to different concentration of PbCl2 were performed by Enzyme linked Immunoassay (EIA), using two experimental procedures: a) cultures were exposed to 1100 and 200 microM PbCl2 for 30 min, next cells received Pb-free culture medium up to 24 h (pulse/chase experiment); b) cultures were fed continuously up to 24 h with treatment media containing the same PbCl2 concentrations (pulse experiment). Concomitantly, the Pb influence on cell viability was evaluated by different cytotoxicity assays (LDH release, DAPI staining and cell density assays). The mRNA expression of ET-1 was evaluated in pulse experiments by RT-PCR analysis before and after cell exposure to PbCl2. The Pb2+ cellular content of parallel MDCK cell cultures was assessed by AAS analysis. RESULTS: In our experimental conditions, the administration of PbCl2 to sub-confluent MDCK cell cultures did not significantly affect cell viability. Either in pulse or in pulselchase experiments, the ETs content, evaluated in culture media of cells exposed to 100 microM PbCl2, significantly increased. On the contrary, cell treatment with 1 or 200 microM PbCl2 did not modify the ETs secretion. Because the amounts of ETs released in culture media were similar in both kinds of experiment, our results suggest that the metal induces the ETs secretion already after 30 min of cell exposure to the toxicant. Moreover, the ET-3 EIA specific assay did not reveal any immunoreactivity, excluding the involvement of this isoform in the Pb-induced secretion of ETs. Additionally, our results seem to exclude any Pb-induced up-regulation of ET-1 transcripts. The Pb2+ quantification in cell extracts demonstrated that the uptake of the metal is dose- and time-dependent and, in pulse experiments, it was maximum after six hours from the beginning of treatments, then the intracellular Pb2+ content decreased. This last phenomenon suggests the involvement of an ATP-dependent transporter in the mechanism of Pb cell excretion. Moreover, the ETs cell release in culture media of MDCK cells appears to depend on the intracellular content of Pb ions reached within 30 min of treatment. CONCLUSION: Our results indicate that there is a range of PbCl2 doses (100 microM) at which MDCK cells enhance their ETs secretion. Lower doses (1 microM) of Pb salt seem to be ineffective to stimulate ETs release, while, doses equivalent to 200 microM seem to inhibit this phenomenon.

[In vitro study of the nephrotoxic mechanism of mercuric chloride]. / Studio in vitro dei meccanismi di nefrotossicità del cloruro di mercurio.

OBJECTIVES: Mercury (Hg), one of the most diffused and hazardous organ-specific environmental contaminants, exists in a wide variety of physical and chemical states, each of which with unique characteristics of target organ specificity. Exposure to Hg vapour and to organic mercurials specifically affects the CNS, while the kidney is the target organ for inorganic Hg compounds. Despite the increasing number of studies, the molecular bases of the nephrotoxic potential of Hg has not, up to now, been clarified, even if there is evidence suggesting that the ability of the metal to interact with proteins (thiol groups) or to generate oxygen radicals may play a major role. Within this context, the aim of the present study was to investigate, in vitro, the mechanism(s) of the early nephrotoxic potential of mercury chloride (HgCl2), one of the most diffused and biologically active mercury (Hg2+) compounds. For this purpose, two kidney-derived in vitro systems (the MDCK and the LLC-PK1 cell lines) were tested for their sensitivity to the salt, and MDCK was chosen as the most suitable in vitro model for our study. As possible biological markers of the organ-specific toxicity of the metal we analysed: i) critical biochemical parameters related to oxidative stress conditions (effect of Hg2+ on the anti-oxidant status of the cell), and ii) gap-junctional function (GJIC). METHODS: Classical toxicity tests (MTT and NR) were used for assessing the sensitivity (IC50) of LLP-CK1 and MDCK cell lines to the mercuric salt. Complete solubilisation of the salt in the culture media was verified by inductively coupled plasma mass spectrometry (ICP-MS). The influence of the metal on cell growth rate and viability were evaluated by conventional proliferation assays. For the following mechanistic studies, cells were exposed for different time periods (4 to 72 hours) to non-cytotoxic (0.1-50 microM) HgCl2 concentrations. The biochemical analysis of the pro-oxidant properties of the mercuric compound was performed by the measurement of anti-oxidant cellular defences against H2O2 [catalase (Cat), glutathione peroxidase (Gpx), and total glutathione (GSH)]. The influence of the metal on the GJIC capacity of MDCK cells was assessed by the "microinjection/dye-coupling" assay. RESULTS: Among the two kidney-derived in vitro systems, MDCK cell line was the most specifically sensitive to the toxic effect of HgCl2: it was, consequently, chosen as a "tubular cell model" for the following experimental steps. Tested for various time periods at increasing concentrations, the HgCl2 effect on MDCK cell proliferation and viability was found to be time- and dose-related. For concentrations < or = 50 microM, HgCl2 inhibits MDCK cell growth rate, being this effect significant (> 50% in respect to untreated controls) from the 24th from the beginning of the treatment, while, for concentrations > 50 microM, the metal causes cell death. Concerning the influence of HgCl2 on MDCK anti-oxidant defences, the most interesting results were obtained by analysing the influence of the mercury salt on the GSH cell content and Gpx activity. Both were, in fact, significantly affected by the presence of the mercury ion. HgCl2 also induced a rapid, dose- and time-related inhibitory effect on the GJIC capacity of the cells. CONCLUSIONS: Even if further investigations are needed to better clarify the possible causal relationship between our findings, they indicate that: a) MDCK cells represent a suitable in vitro model for the study of Hg nephrotoxicity; b) GJIC function is, among those considered in our study, one of the most sensitive biological endpoints for investigating the mechanism(s) of Hg2+ specific toxicity.

Antioxidant potential and gap junction-mediated intercellular communication as early biological markers of mercuric chloride toxicity in the MDCK cell line.

In this study, the early nephrotoxic potential of mercuric chloride (HgCl(2)) has been evaluated in vitro, by exposing a renal-derived cell system, the tubular epithelial Madin-Darby canine kidney (MDCK) cell line, to the presence of increasing HgCl(2) concentrations (0.1-100 microM) for different periods of time (from 4 to 72 h). As possible biological markers of the tubular-specific toxicity of HgCl(2) in exposed-MDCK cultures we analysed: (i) critical biochemical parameters related to oxidative stress conditions and (ii) gap-junctional function (GJIC). HgCl(2) cytotoxicity was evaluated by cell-density assay. The biochemical analysis of the pro-oxidant properties of the mercuric ion (Hg(2+)) was performed by evaluating the effect of the metal salt on the antioxidant status of the MDCK cells. The cell glutathione (GSH) content and the activity of glutathione peroxidase (Gpx) and catalase (Cat), two enzymes engaged in the H(2)O(2) degradation, were quantified. HgCl(2) influence on MDCK GJIC was analysed by the microinjection/dye-transfer assay. HgCl(2)-induced morphological changes in MDCK cells were also taken into account. Our results, proving that subcytotoxic (0.1-10 microM) HgCl(2) concentrations affect either the antioxidant defences of MDCK cells or their GJIC, indicate these critical functions as suitable biological targets of early mercury-induced tubular cell injury.

Metabolic fate of extracellular NAD in human skin fibroblasts.

Extracellular NAD is degraded to pyridine and purine metabolites by different types of surface-located enzymes which are expressed differently on the plasmamembrane of various human cells and tissues. In a previous report, we demonstrated that NAD-glycohydrolase, nucleotide pyrophosphatase and 5'-nucleotidase are located on the outer surface of human skin fibroblasts. Nucleotide pyrophosphatase cleaves NAD to nicotinamide mononucleotide and AMP, and 5'-nucleotidase hydrolyses AMP to adenosine. Cells incubated with NAD, produce nicotinamide, nicotinamide mononucleotide, hypoxanthine and adenine. The absence of ADPribose and adenosine in the extracellular compartment could be due to further catabolism and/or uptake of these products. To clarify the fate of the purine moiety of exogenous NAD, we investigated uptake of the products of NAD hydrolysis using U-[(14)C]-adenine-NAD. ATP was found to be the main labeled intracellular product of exogenous NAD catabolism; ADP, AMP, inosine and adenosine were also detected but in small quantities. Addition of ADPribose or adenosine to the incubation medium decreased uptake of radioactive purine, which, on the contrary, was unaffected by addition of inosine. ADPribose strongly inhibited the activity of ecto-NAD-hydrolyzing enzymes, whereas adenosine did not. Radioactive uptake by purine drastically dropped in fibroblasts incubated with (14)C-NAD and dipyridamole, an inhibitor of adenosine transport. Partial inhibition of [(14)C]-NAD uptake observed in fibroblasts depleted of ATP showed that the transport system requires ATP to some extent. All these findings suggest that adenosine is the purine form taken up by cells, and this hypothesis was confirmed incubating cultured fibroblasts with (14)C-adenosine and analyzing nucleoside uptake and intracellular metabolism under different experimental conditions. Fibroblasts incubated with [(14)C]-adenosine yield the same radioactive products as with [(14)C]-NAD; the absence of inhibition of [(14)C]-adenosine uptake by ADPribose in the presence of alpha-beta methyleneADP, an inhibitor of 5' nucleotidase, demonstrates that ADPribose coming from NAD via NAD-glycohydrolase is finally catabolised to adenosine. These results confirm that adenosine is the NAD hydrolysis product incorporated by cells and further metabolized to ATP, and that adenosine transport is partially ATP dependent.

Enzymatic activities affecting exogenous nicotinamide adenine dinucleotide in human skin fibroblasts.

The fate of nicotinamide adenine dinucleotide (NAD), AMP, and ADP-ribose supplied to intact human skin fibroblasts was monitored, and the concentrations of intra- and extracellular pyridine and purine compounds were determined by HPLC analysis. Two enzymatic activities affecting extracellular NAD were detected on the plasma membrane, one hydrolyzing the pyrophosphoric bond and yielding nicotinamide mononucleotide (nucleotide pyrophosphatase) and the other cleaving the glycoside link and releasing nicotinamide (NAD-glycohydrolase). No AMP or ADP-ribose was found in the extracellular medium of cells incubated with NAD, the former being completely catabolized to hypoxanthine and the latter degraded to adenine and hypoxanthine.

Uptake and metabolism of a fluorescent sulfatide analogue in cultured skin fibroblasts.

The sulfatide fluorescent analogue N-lissamine rhodaminyl-(12-aminododecanoyl) cerebroside 3-sulfate was administered in the form of albumin complex to normal human skin fibroblasts and its metabolic fate was investigated. Ceramide, galactosylceramide, glucosylceramide, sphingomyelin and free acid, all containing the fluorophore lissamine rhodamine, have been synthesized as reference standards for the identification of the metabolic products. Ceramide appeared to be the main metabolic product present both in cell extract and medium, followed by galactosylceramide and sphingomyelin. Fluorescence microscopy of cells showed a marked perinuclear fluorescence.

Synthesis, spectral properties and enzymatic hydrolysis of fluorescent derivatives of cerebroside sulfate containing long-wavelength-emission probes.

Fluorescent derivatives of cerebroside sulfate (sulfogalactosyl ceramide, sulfatide) containing long-wavelength-emission fluorophores were synthesized. For this purpose a procedure was developed for preparing a cerebroside 3-sulfate derivative with an amino group on the terminal carbon atom of its fatty acyl residue. The latter compound has been used to prepare cerebroside 3-sulfate, coupled to lissamine-rhodamine, fluoresceine, eosine and NBD. The spectroscopic properties of these compounds, in different solvent systems and when incorporated into micelles of a non-ionic detergent or liposomes of a phospholipid, are reported. Incubation of these respective sulfatides with a human leukocyte preparation, resulted in the formation of the corresponding fluorescent cerebrosides.

Rapid preparation of a distinct lysosomal population from myelinating mouse brain using Percoll gradients.

To study the vesicular lysosome-associated transport and the metabolism of some brain macromolecules (in particular, sialoglycoconjugates), we developed a rapid procedure to obtain a distinct lysosomal population starting from myelinating mouse brain. This procedure is based on an initial differential centrifugation step producing a 1,000-17,500-g fraction (P2), followed by isopycnic centrifugation of fraction P2 on a self-generated colloidal silica gel (Percoll) gradient. The heaviest subfraction thus obtained is very rich in acid hydrolase activities like beta-galactosidase, arylsulfatase A, and acid phosphatase. The enrichment of these enzymes is approximately 100-fold as compared with the starting homogenate, whereas the markers of other subcellular organelles, such as mitochondria, plasma membranes, or the Golgi apparatus, are virtually absent. The lysosomal preparation contains approximately 12-14% of the total acid hydrolase activities, with a protein yield of approximately 0.12%. Electron microscopy shows that the lysosomal fraction is composed of an approximately 90% pure population of lysosomes. Therefore, the procedure described here is suitable for obtaining a highly purified lysosome preparation from myelinating mouse brain.

Hormonal aspects of human gout--excretion of adrenal hormone derivatives in gouty patients.

Forty-seven patients with gout, 28 of whom had not previously been treated with allopurinol, and 25 normal subjects, were examined for 24-h urinary excretion of the most important adrenal steroid derivatives. Results were submitted to statistical analysis and several variables have been taken in consideration. The untreated patients showed significantly higher values of uricemia, urinary uric acid, triglycerides, slightly higher values of androsterone, 11-oxo-androsterone + 11-oxo-etiocholanolone, dehydroepiandrosterone, and slightly lower values of 11-hydroxyandrosterone and pregnanetriol, in comparison to normal subjects. The different hormonal pattern seems to discriminate between patients with gout and normal subjects.

[The influence of fasting, of a hyperprotein diet and of nicotinamide on hepatic L-threonine deaminase]. / Influenza del digiuno, della dieta iperproteica e della nicotinamide sulla L-treonina deaminasi epatica.

The induction of L-threonine deaminase, following nicotinamide injection has been studied: the effect of fasting and of hyperproteic diet have been also taken in consideration. Maximal induction is observed after 5 days hyperproteic diet, and is additional only with nicotinamide treatment. Results are interpreted assuming a different hepatic content and behavior of multiple forms of the enzyme.

[Myelin]. / La mielina.

The lipid and protein composition and the metabolic turnover of myelin in the C.N.S., are briefly reported. For a better understanding, it seemed useful to introduce some historical data on the discovery of myelin and to give a morphological description at structural and ultrastructural level.

[Behavior of certain acid hydrolases in the central nervous systems of various mammals]. / Comportamento di alcune idrolasi acide nel sistema nervoso centrale di vari mammiferi.

The activity of several acid hydrolases in different sections of the C.N.S. of various mammalian has been evaluated. The enzymes which have been studied are: beta-glucosidase, beta-galactosidase, beta-N-acetylglucosaminidase, beta-N-acetylgalactosaminidase, beta-glucuronidase, alpha-mannosidase. The enzymes show a higher activity in the gray matter than in the white matter. The results are interpreted on the assumption that glyco-lipoprotein turnover is higher in the gray matter than in the white matter, since myelin, which is the major component of the white matter, is a relatively stable structures.