Toxicity and metal content of organic solvent extracts from airborne particulate matter in Puerto Rico.

The importance of airborne particulate matter (PM) in causing increases in morbidity and mortality in humans has been confirmed by numerous epidemiological and laboratory studies. It has been proposed that PM might deliver transition metals to the airways were they react and generate reactive oxygen species (ROS), thus promoting the expression of inflammatory mediators, and cytotoxicity. In Puerto Rico (PR), the northern Guaynabo area is a US EPA non-attainment zone for PM10 (PM with a mass median aerodynamic diameter 10 microm), and a previous study found that organic PM10 extracts from this area were cytotoxic. The purpose of this research project is to compare the toxicity between organic PM extracts from Guaynabo (a coastal urban site) and Fajardo (a coastal rural town) based on their polarity, collection season, and geographical location. We will also evaluate if the metal content of such extracts is associated with their biological activity. PM10 filters from both locations were subjected to a sequential Soxhlet extraction using hexane and acetone. Normal and transformed bronchial epithelial cells were then exposed to the extracts. Using the neutral red assay to measure cell viability we found that coastal urban PM from PR generally exhibits higher cytotoxicity than coastal rural PM. However, this effect is dependent on the polarity of the extracts and the collection season (in winter hexane PM10 is more toxic, whereas during the summer acetone PM10 is more toxic). We also found that non-polar organic constituents in PM from PR are generally more toxic than the polar organic constituents. The main conclusion from this work is that the metal contents of the organic PM extracts from PR could play a minor role in the cytotoxicity observed. This is supported by the findings of elements such as As, V, Ni, and Cu in the most cytotoxic extracts. However, organic compounds probably play the major role. The presence of bioactive fractions of PM underscores the importance of conducting more detailed studies.

Ectopic expression of MHC class II genes (RT1.B(I) beta/alpha) in rat hepatocytes in vivo and in culture can be elicited by treatment with the pregnane X receptor agonists pregnenolone 16 alpha-carbonitrile and dexamethasone.

The synthetic steroid, pregnenolone-16-alpha-carbonitrile (PCN), has served for decades as a probe for a postulated series of hepatic defenses activated under situations of environmental "stress". PCN, an antiglucocorticoid, and also such glucocorticoids as dexamethasone (Dex) appear to stimulate hepatic metabolism and elimination of xenobiotics by binding to the nuclear pregnane X receptor (PXR) which then interacts with a distinct DNA response element associated with induction of cytochrome P450 3A genes. To explore the full domain of genes controlled by PCN/PXR, we used differential display to detect rat liver mRNA species selectively induced by PCN or by Dex. Sequence analysis identified one of many PCN induced cDNA fragments as RT1.B(I)beta, a member of the major histocompatability class II (MHC) gene family usually found only in antigen presenting cells. Northern blot analysis of RNA from rat liver or from cultured hepatocytes confirmed that amounts of RT1.B(I)beta mRNA and also of its companion gene, RT1.B(I)alpha mRNA, became readily detectable within 3-6 hours following treatment with PCN or Dex, whereas no induction was observed in spleen RNA. Induction by PCN of RT1.B(I)beta immunoreactive protein was localized to the hepatocytes as judged by immunofluorescence. We conclude that ectopic expression of MHC II genes, an unprecedented effect of steroids or drugs, is rapidly evoked by PCN acting on the liver, directly. The concept of a set of genes coordinately controlled to maintain homeostasis in parenchymal tissues during toxic stress must now be extended to include the immune system.

Plasma homocysteine concentrations in a population of rhesus monkeys (Macaca mulatta): reference ranges and accompanying plasma concentrations of folate and vitamin B12.

The circulating level of homocysteine (Hcy) is recognized as a major independent risk factor for cardiovascular disease in humans. Nonhuman primates are being investigated to see whether they will be accurate models for Hcy indicators of cardiovascular dysfunction. Normal reference values are available in humans for Hcy as classified by age, gender, ethnic origin, and biological factors, however similar information in nonhuman primates had not been published previously. The purpose of this report is to provide normal Hcy values in a large group of nonhuman primates in light of age, gender, and physiologic state (pregnancy and lactation) and to compare these values to the same parameters in humans to highlight similar and dissimilar trends. In addition, plasma levels of folic acid and vitamin B(12), which are determinants of Hcy status in humans, are presented. Samples obtained from a troop of 149 rhesus monkeys (Macaca mulatta) fed a high protein commercial diet were analyzed for Hcy by using high-performance liquid chromatography. Folate and vitamin B(12) levels were determined by using an autoanalyzer. Results (mean [95% confidence interval]) for the entire troop were: Hcy, 4.5 (4.2-4.9) micromol/L; folic acid, 8.6 (8.0-9.1) micromol/L; and vitamin B(12), 673 (611-741) pmol/L. Quantitative values are similar to published values for another species of wild-caught macaques. Similar to trends noted for humans, male monkeys had higher Hcy values than did female animals, pregnant animals had lower values than did nonpregnant ones, and Hcy levels were inversely proportional to plasma folate and vitamin B(12) concentrations. However, homocysteine levels in rhesus monkeys did not vary consistently with age, whereas they increase with age in humans.

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 ofmRNAg33 levels was maximal. 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 eceptor. 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