Trace metals in sediments of two estuarine lagoons from Puerto Rico.

Concentrations of As, Cd, Cu, Fe, Hg, Pb and Zn were evaluated in surface sediments of two estuaries from Puerto Rico, known as San José Lagoon (SJL) and Joyuda Lagoon. Significantly higher concentrations in microg/g dw of Cd (1.8 vs. 0.1), Cu (105 vs. 22), Hg (1.9 vs. 0.17), Pb (219 vs. 8), and Zn (531 vs. 52) were found in sediment samples from SJL when compared to Joyuda Lagoon. Average concentrations of Hg, Pb, and Zn in some sediment samples from SJL were above the effect range median (ERM) that predict toxic effects to aquatic organisms. Enrichments factors using Fe as a normalizer, and correlation matrices showed that metal pollution in SJL was the product of anthropogenic sources, while the metal content in Joyuda Lagoon was of natural origins. Sediment metal concentrations found in SJL were comparable to aquatic systems classified as contaminated from other regions of the world.

Identification by differential display of the IF1 inhibitor peptide of ATP synthase/ATPase as a gene inducible in rat liver by pregnenolone 16alpha-carbonitrile.

The synthetic steroid, pregnenolone-16alpha-carbonitrile (PCN), activates hepatic metabolism and elimination of xenobiotics mediated by its interaction with the PXR, a nuclear receptor that binds PCN and such glucocorticoids as dexamethasone (Dex). We used mRNA differential display to define further the domain of genes under the control of PCN/PXR. We found 76 cDNA fragments representing mRNAs differentially expressed in the liver of rats treated with PCN or Dex. Sequence analysis of one of these revealed a PCN induced cDNA fragment as IF1, an inhibitor peptide of ATP synthase/ATPase complex. Northern blot analysis revealed that IF1 was detectable in untreated liver and was induced 2-3 fold following treatment with PCN. IF1 mRNA was not detected in lung, heart, kidney, or testes of control or PCN treated rats. We conclude that IF1 inhibitor peptide is a novel representative of an apparently large set of previously unrecognized genes coordinately controlled by the PCN/PXR system to maintain homeostasis during toxic stress.

Toxic evaluation of organic extracts from airborne particulate matter in Puerto Rico.

In recent years, several hypotheses have emerged to explain the toxicologic activity of particulate matter. Organic compounds, ultrafine particles, biologic components, and transition metals are some of the constituents that reportedly exert some type of adverse effect on human health. A considerable fraction of the urban particulate matter consists of carbon compounds, which originate mostly from anthropogenic sources. The toxicity of organic fractions from particulate matter have been mainly evaluated by considering their mutagenic activity. This research expands on the toxicologic profile of organic compounds adsorbed to particulate matter, specifically in Puerto Rico, by using the cytotoxic neutral red bioassay (NRB). The NRB uses normal human epidermal keratinocytes or other types of cells to measure the effect on cell viability when exposed to organic compounds associated to the particles in the air. We validated the NRB for particulate matter by using a standard reference material (SRM 1649). We used the NRB to determine toxicologic differences of extracts between an urban industrialized site with anthropogenic activity versus a coastal region with less human activity. The cytotoxicity associated with organic compounds in particulate matter collected at the urban industrialized site was detected in both the particulate matter (3/4) 10 microm in aerodynamic diameter (PM(10)) and particulate matter (3/4) 100 microm in aerodynamic diameter (PM(100)). Greater toxic effects were observed in PM(10) extracts than in PM(100) extracts, but PM(10) toxic effects were not significantly different from those in PM(100). The extracts from the industrialized site were more cytotoxic than the extracts from coastal reference site, although in the summer, extracts from both sites were significantly cytotoxic to normal human epidermal keratinocytes. In addition, the nonpolar extracts of both PM(10) and PM(100) exerted the greatest cytotoxicity, followed by the polar, and, finally, the moderately polar extract. This study demonstrates that extracts from the Guaynabo industrialized site were more toxic than similar extracts obtained from a reference coastal site in Fajardo, Puerto Rico.

Insulin-induced gene 33 mRNA expression in Chinese hamster ovary cells is insulin receptor dependent.

Gene 33 (g33) is a non-tissue-specific gene regulated in rat liver and hepatoma cells by insulin and other agents. It is thought to participate in the transition from quiescence to proliferation in mitogen-treated cells. The mechanism(s) by which insulin exerts its action on g33 are not totally understood; it is unclear whether a functional insulin receptor is required for this action. In this study, we evaluate the mechanism for insulin induction of g33 mRNA in Chinese hamster ovary (CHO) cells transfected with the neomycin-resistant plasmid (CHONeoB), human insulin receptor (CHONewIRa), and a kinase-defective insulin receptor mutated at the ATP-binding site (CHOK1018A). Transfected cells had higher levels of insulin binding than that of CHONeoB cells; insulin-induced phosphorylation of the insulin receptor and its intracellular substrates were impaired in CHOK1018A cells. Maximal insulin induction of mRNA(g33) occurred 3 h after hormonal exposure in all cell lines. The degree of insulin stimulation of g33 mRNA levels was four- to sixfold higher in CHONewIRa than in CHONeoB or CHOK1018A cells, which had minimal levels of insulin-stimulated g33 mRNA levels. Half-maximal stimulation of g33 mRNA levels was observed at 0.06 +/- 0.01 nM in CHONewIRa cells, consistent with insulin interaction with its own receptor. Wortmannin, an inhibitor of phosphatidyl inositol 3-kinase (PI3K), had some effects on insulin stimulation of g33 mRNA in CHO NewIRa cells. PD98059, an inhibitor of mitogen-activated kinase kinase (MAPKK), and rapamycin, a p70 S6 kinase inhibitor, had minimal effect on insulin stimulation of g33 mRNA in all cells tested. By contrast, hydroxy-2-naphthalenylmethyl)phosphonic acid triacetoxymethyl ester (HNMPA(AM)(3), a selective inhibitor of the insulin receptor tyrosine kinase, caused complete inhibition of insulin stimulation of g33 mRNA levels. These data indicate that the insulin receptor with intact kinase activity is required for insulin stimulation of g33 mRNA levels. They also suggest that AKT, a PI 3-kinase downstream effector molecule, could mediate insulin stimulation of g33 mRNA. The mechanism(s) of insulin regulation of g33 expression downstream of receptor do not seem to rely entirely on the classic insulin receptor transduction pathway, as a minor effect was observed upon inhibition of MAPKK, suggesting that multiple pathways may be involved.

Insulin and phorbol ester regulation of gene 33 expression in CHO cells.

Rat gene 33 (g33) mRNA has a widespread tissue distribution. Insulin and various agents such as glucocorticoids, phorbol esters and plant lectins regulate G33 expression in rat hepatoma cells. The regulation of g33 by insulin and a phorbol ester was examined in two Chinese Hamster ovary (CHO) cell lines, CHO-T cells (which overexpress human insulin receptors (hIR)) and wild type CHOwt cells. These cell lines were used to determine how expression of the hIR influences the capacity of g33 to respond to insulin and phorbol myristate acetate (PMA). Treatment of CHOwt and CHO-T cells with insulin increased mRNAg33 levels three to four-fold, with a maximum effect reached after three hours of treatment. PMA treatment of CHOwt and CHO-T cells caused a similar elevation of mRNAg33 levels after three hours. Insulin had no effect on mRNAg33 stability in both CHO cell lines. Additionally, the effects of insulin and PMA on mRNAg33 levels were additive only in CHO-T cells. Insulin or PMA-pretreated CHO-T cells were able to respond to both agents, but elevation of mRNAg33 levels was submaximal. In contrast, when insulin and/or PMA-pretreated CHOwt cells were exposed to insulin or PMA, g33 was able to respond maximally. These results suggest that insulin and phorbol esters act through different signaling mechanisms in CHOwt cells. Additionally, insulin's ability to stimulate g33 expression in CHOwt cells suggests that this insulin effect may be independent of the insulin receptor. There are differences in the regulation pattern of g33 by insulin and PMA in rat hepatoma and among the two CHO cell lines used in this study.