Profile of select hepatic insulin signaling pathway genes in response to 2-aminoanthracene dietary ingestion.

Some genes that regulate various processes such as insulin signaling, glucose metabolism, fatty acid, and lipid biosynthesis were profiled. The objective of the current investigation is to examine the mRNA expression of some genes that mediate insulin signaling due to 2AA toxicity. 2AA is a polycyclic aromatic hydrocarbon (PAH) that has been detected in broiled food and tobacco smoke. Twenty-four post-weaning 3-4-week-old F344 male rats were exposed to 0 mg/kg-diet, 50 mg/kg-diet, 75 mg/kg-diet, and 100 mg/kgdiet 2AA for 2 weeks and 4 weeks. The mRNA expression of AKT1, G6PC, GCK, GLUT4, INSR, IRS1, PP1R3C, PAMPK, SOCS 2, and SREBF1 was determined by qRTPCR followed by the quantification of G6PC and AMPK via ELISA. The results suggest that 2AA modulates these genes depending on the length of exposure. Up-regulation of AMPK and SOCS2 genes in animals treated with 100 mg/kg-diet and 50 mg/kg-diet, respectively, during 14 days of feeding was noted. G6PC expression was inhibited in the 2-week group while being dose-dependently increased in the 4-week group. Hepatic activity of G6PC was enhanced significantly in the livers of rats that ingested 2AA. It appears that 2AA intoxication leads to the activation of irs1 and akt1 genes in the liver. Quantified AMPK amounts increased significantly in the short-term treatment group. Dose-dependent rise of AMPK in animals treated to 2AA showed an increased production of hepatic AMPK in response to the toxicity of 2AA in order to maintain cellular homeostasis. In contrast, the reduction in AMPK concentration in treated animals within the 4-week set indicated an adaptive recovery.

Induction of CYP1A mRNA in Carp (Cyprinus carpio) from the Kalamazoo River polychlorinated biphenyl-contaminated superfund site and in a laboratory study.

The Kalamazoo River Superfund site in Michigan is contaminated with polychlorinated biphenyls (PCBs), which were heavily discharged into the river from several paper companies as part of the deinking process in the 1950s through 1970s. We characterized biomarkers of chronic PCB exposure in a resident fish population using real-time reverse transcriptase-polymerase chain reaction to examine mRNA expression levels of multiple genes in carp (Cyprinus carpio) liver from PCB contaminated and reference sites in the Kalamazoo River. We also measured these same genes in juvenile carp exposed to dietary PCBs for 4 months. Kalamazoo River carp had significantly increased levels of cytochrome P450 1A (CYP1A) mRNA as did carp fed PCBs in the laboratory. No significant mRNA upregulation occurred in the specific oxidative stress genes (gamma-glutamylcysteine synthetase and magnesium superoxide dismutase) and metabolic genes (phosphoenolpyruvate carboxykinase and nucleolin) examined. These data are consistent with the idea that carp from the Kalamazoo River Superfund Site are responding to PCB exposure via upregulation of CYP1A independent of activation of the oxidative stress response genes normally thought to be co-regulated with CYP1A.

Lack of involvement of mitochondrial factors in caspase activation in a Drosophila cell-free system.

Although mitochondrial proteins play well-defined roles in caspase activation in mammalian cells, the role of mitochondrial factors in caspase activation in Drosophila is unclear. Using cell-free extracts, we demonstrate that mitochondrial factors play no apparent role in Drosophila caspase activation. Cytosolic extract from apoptotic S2 cells, in which caspases were inhibited, induced caspase activation in cytosolic extract from normal S2 cells. Mitochondrial extract did not activate caspases, nor did it influence caspase activation by cytosolic extract. Silencing of Hid, Reaper, or Grim reduced caspase activation by apoptotic cell extract. Furthermore, a peptide representing the amino terminus of Hid was sufficient to activate caspases in cytosolic extract, and this activity was not enhanced by addition of mitochondria or mitochondrial lysate. The Hid peptide also induced apoptosis when introduced into S2 cells. These results suggest that caspase activation in Drosophila is regulated solely by cytoplasmic factors and does not involve any mitochondrial factors.

Suppression of arylamine toxicity in the Fischer-344 rat following ingestion of a complex mixture.

The toxic effects of a mixture of 2-aminoanthracene (2-AA), benzanthracene (BA), and dinitropyrene isomers (DNP), and the toxic effects of these compounds individually, were investigated in the Fischer-344 rat following dietary exposure via a powdered basal diet. Animals were sacrificed at 14-, 30-, and 80-days of dietary exposure. Exposure to dietary 2-AA alone induced anorexia, cachexia, variable mortality, and altered serum chemistry profiles in the F-344 rat. Reduced lymphocyte counts were also shown in rats exposed to 2-AA. A temporal pattern of effect of 2-AA dietary exposure was observed in the progression of hepatic lesions in exposed animals. Dietary exposure to either DNP isomers or BA at a 10-fold higher concentration in the diet, relative to 2-AA, did not induce detectable toxic responses. However, exposure of rats to a mixture of 2-AA, BA, and DNP isomers (100 mg/kg, 1.0 g/kg, and 1.0 g/kg of diet, respectively) resulted in the attenuation of toxic effects when compared to exposure of F-344 rats to 2-AA alone. These results indicate that the toxic effects of 2-AA are suppressed by co-administration of DNP and BA and suggest that compound interactions need to be considered when predicting the toxic potential of specific environmental pollutants.

Hepatic toxicity and recovery of Fischer 344 rats following exposure to 2-aminoanthracene by intraperitoneal injection.

Humans may be exposed to 2-aminoanthracene (2-AA), a substituted polycyclic aromatic hydrocarbon, and a recognized mutagen and carcinogen, through oral and respiratory routes from contact with a variety of environmental sources. For the present study, we sought to evaluate hepatic damage and recovery in Fischer 344 rats following multiple i.p. injections of 5 mg of 2-AA. Rats were injected weekly for up to 5 weeks. Subgroups were then allowed to recover for 1, 5, or 9 weeks, and biochemical and pathologic changes were evaluated. We observed that weight gains were reduced relative to controls for all groups receiving > or = 2 injections. Serum enzyme levels indicative of liver damage were evident and included alterations in serum aspartate aminotransferase, alkaline phosphatase, total protein, albumin, and globulin. These alterations usually returned to normal by 5 weeks following cessation of 2-AA administration. In contrast, histologic liver changes, including hepatocyte hypertrophy, biliary hyperplasia with oval cell proliferation, altered foci, nodular hyperplasia, and one hepatocellular adenoma became more severe with time. This experiment demonstrates patterns of hepatic damage and recovery in rats exposed to 2-AA.

Synthesis of C8-adenosine adducts of arylamines using palladium catalysis.

We employed palladium-catalyzed coupling procedures for the synthesis of new C8-adenosine adducts of various arylamines (aniline, benzidine, 4-aminobiphenyl, and 2-aminofluorene).[reaction: see text]

Apoptosis, PCNA, and p53 in Fundulus grandis fish liver after in vivo exposure to N-methyl-N'-nitro-N-nitrosoguanidine and 2-aminofluorene.

Dysfunction in homeostatic mechanisms of cell death and proliferation are considered to be important in the pathogenesis of chemically induced neoplasia. p53 has been implicated in the regulation of cell death and proliferation. To determine whether expression of apoptosis, proliferating cell nuclear antigen (PCNA), and p53 differ between an alkylating agent and a polycyclic aromatic hydrocarbon, host response was measured through sequential immunohistochemical detection of apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling method), PCNA PC-10, and p53 (PAb 240) in livers of the fish Fundulus grandis. Nine hundred fish were randomly assigned to 3 groups of 300 fish each and kept in separate aquarium tanks. One group of fish was exposed to 6.7 microM N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 1 group was exposed to 6.9 mM 2-aminofluorene (2-AF), and the remaining group served as a control. A significant decrease (p = 0.005) in the level of apoptosis and a significant increase (p < 0.0001) in the level of p53 were found on experimental day 180 in the livers of MNNG-exposed fish. PCNA was significantly increased (p < 0.005) by day 9 of the experiment in both MNNG and 2-AF fish when compared with controls, but no significant differences existed between the 2 groups of treated fish. Response of fish liver cells to MNNG-mediated and 2-AF-mediated injury differs, at least initially, in the expression of p53, inhibition of apoptosis, and increased net cell proliferation. Concurrent use of a marker for cell death with a marker of proliferation greatly enhances the assessment of the effect of these compounds on liver cells.

Establishment and validation of an isolated rat lung model for pulmonary metabolism studies.

An isolated rat lung model was established and validated for use in pulmonary metabolism studies. During the establishment phase of the study, several problems were encountered and overcome in order to maintain the lungs in physiological condition. In the validation phase of the study, the lungs were removed, ventilated and perfused from 34 male Fischer 344 rats. After an equilibration period, lungs were ventilated and perfused for up to 4 h. Morphological, biochemical and functional parameters were evaluated to validate the physiological condition of the lungs. Morphological parameters included wet/dry lung weight ratios and gross and histological scoring for edema. Biochemical parameters included assays for tissue ATP and reduced glutathione content, glutathione reductase activity and glucose utilization. Functional parameters included changes in lung tidal volume, dynamic compliance and airway resistance. Results indicated that edema formation was only detected histologically, that lungs remained nearly biochemically normal for 210 min and that pulmonary function declined to about 80-90% of normal. Overall, these findings indicated that the isolated, perfused rat lung remained in acceptable physiological condition for ca. 210 min. This period of time should be adequate for conducting pulmonary metabolism studies with a variety of exogenous compounds.

Analysis of DNA adduct, S-[2-(N7-guanyl)ethyl]glutathione, by liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry.

Sensitive and specific isotope dilution liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods were developed for the detection and quantitative analysis of S-[2-(N7-guanyl)ethyl]glutathione as a DNA adduct formed upon exposure of animals to carcinogenic 1,2-dihaloethanes. Separation and analysis were performed using microbore HPLC coupled in-line to an electrospray ionization triple quadrupole mass spectrometer. S-[2-(N7-guanyl)[2H4]-ethyl] glutathione was synthesized and used as internal standard. These methods provide structural confirmation of the adduct as well as quantitative analysis with the accuracy and precision necessary to measure biologically relevant levels in small tissue sample sizes (< 1 g). The sample detection limits in in vivo tissue extracts were 100 pg and 5 pg on-column for LC/MS and LC/MS/MS methods, respectively. Selected-ion monitoring mode was used to monitor the product ions of the doubly charged molecular ion. The application of these methods was demonstrated by measuring the DNA adduct levels in rat and fish samples after exposure to 1,2-dihaloethanes. The method has application in studies of DNA adduct formation as a biological marker of exposure to carcinogens and for environmental monitoring of 1,2-dihaloethanes.

Organic and inorganic components in estuarine colloids: implications for sorption and transport of pollutants.

In colloids isolated from Chesapeake Bay and its subestuaries the concentrations of Al, Fe, and a number of trace elements were determined to vary with the clay mineral fraction that was most abundant in freshwater samples collected during the winter. The elements As, Ba, Sb, and Zn, however, increased with increasing organic content, indicating a covariance with the organic component. Organic analyses for amino acids (proteins), carbohydrates, and lipids indicate that these biopolymers comprised 4 to 22%, 20 to 60%, and less than 1%, respectively, of the colloidal organic carbon in these samples. The results are significant because amino acids and carbohydrates contain oxygen, nitrogen, and sulfur functional groups capable of reacting with trace metals and organic pollutants. The sorption properties of several neutral hydrophobic organic compounds, including PAHs and herbicides, and several aromatic amines were investigated using the estuarine colloidal material. The effects of several environmental variables on these sorption properties were determined. The results indicate that colloids have the capacity to sorb and transport relatively insoluble pollutants that otherwise might remain immobile in the environment. Colloidal organic matter in natural water systems (lakes, rivers, estuaries and the oceans, as well as groundwater) may serve as substrates for the sorption or binding of organic contaminants. Although most of the data has been developed using neutral hydrophobic organic compounds, data also exist for selected polar compound groups such as aromatic amines. The chemical behavior of these compound groups in interaction with DOM appears to have some similarity to their chemical interaction with sediments and soils. Partitioning constants are linear, except for polar compounds, and appear to be closely correlated to fundamental compound properties such as solubilities and octanol-water partition coefficients. Even though a variety of methods were used to collect the colloids for study and to determine partition coefficients, no single method appears to be superior. Ultrafiltration techniques appear to be optimal for collecting the colloids, since this method subjects the materials to less extreme chemical conditions than the preparation of humic extracts, whereas equilibrium techniques such as dialysis and head space analysis appear to yield the best results for partitioning constants. However, each of these techniques has limitations with respect to the classes of compounds that can be studied successfully.

Variations in the neurotoxic potency of trimethyltin.

The organometallic compound, trimethyltin (TMT), is used as a selective denervation tool to validate morphological, biochemical and functional approaches to the detection and characterization of neurotoxicity. Variations in nervous system response TMT have been reported and may complicate the use of this compound as a research tool. We examined the contribution of sample-to-sample differences to variations in TMT-induced neurotoxicity. Seven samples of TMT obtained from three commercial sources were evaluated for neurotoxic potency in the rat. Hippocampus weight, histology and assays of the astrocyte protein, glial fibrillary acidic protein (GFAP), were used as indices of neurotoxicity. A single administration (8.0 mg/kg, IV) of different samples of TMT resulted in markedly different degrees of neurotoxicity as assessed by hippocampus weight and GFAP assays. Subsequent analysis of each sample for trace metal and speciated organotin content revealed that sample-to-sample differences in neurotoxic potency could be attributed to the presence of several impurities. Indeed, in several samples, sodium was present at levels high enough to affect neurotoxic potency simply by diluting the TMT content. A number of samples also showed contamination with the nonneurotoxic organotin, dimethyltin. The data indicate that different sources of TMT produce quantitatively different degrees of neurotoxicity, differences that may be attributed to sample-to-sample variations in TMT content.

Assessment of the mutagenicity of fractions from s-triazine-treated Zea mays.

A water-soluble extract from maize plants exposed to 3 s-triazine herbicides (atrazine, simazine and cyanazine) has been shown to be mutagenic in strain TA100 of Salmonella. No mutagenic activity was observed in any control plant extracts using either water or a variety of organic solvents. Gel permeation studies of the extracts suggest that the mutagen(s) are small molecules (less than 1000 MW). HPLC fractionation suggests that the mutagens formed from each of the 3 herbicides are similar in polarity and water solubility, eluting in a 50/50 water:methanol fraction. Approximately 89% of 14C-labeled HPLC chromatographable metabolites of atrazine were also associated with this fraction, suggesting a close chemical link between a labeled but unidentified metabolite and the mutagenic activity.

Microbially mediated leaching of low-sulfur coal in experimental coal columns.

The leaching of a low-sulfur bituminous coal was investigated with experimental coal columns subjected to simulated rainfall events. Leachates from the columns became dominated by iron-oxidizing bacteria as evidenced by specific enrichment cultures and measurements of CO(2) assimilation. Heterotrophic microorganisms were also present in the coal leachates, but their numbers and activity decreased with decreasing pH. This pattern could be reversed by increasing the pH of the coal with lime. Organosulfur-utilizing bacteria made up a substantial portion of the heterotrophic community. Measurements of microbial activity in coal cores indicated that although much of the microbial community remained associated with coal particles, the relative abundance of heterotrophs and autotrophs in leachate seemed to reflect that in coal cores. When bacterial growth was delayed by autoclaving coal samples, acid production and leaching of iron and sulfur were also delayed. Rapid leaching of materials from coal thus appears to be strongly dependent on the presence of the natural bacterial microflora.

Anaerobic microbial methylation of inorganic tin in estuarine sediment slurries.

Estuarine sediment slurries and microorganisms were examined for the ability to methylate inorganic tin. Under controlled redox conditions, tin was methylated only in oxygen-free sediment slurries. Monomethyltin usually comprised greater than 90% of the alkyltin products formed, although dimethyltin was also produced. Autoclaved anoxic sediments did not produce organotins. Several bacterial cultures, most notably sulfate-reducing bacteria isolated from anoxic estuarine sediments, formed monoand dimethyltin from inorganic tin in the absence of sediment. The results suggest that inorganic tin methylation in estuarine environments is an anaerobic process catalyzed primarily by sulfate-reducing microorganisms.

A methodology for speciation of methyltins in mammalian tissues.

Exposure to methyltin compounds results in morphologically detectable damage to several mammalian organ systems. Although reliable dose-response relationships have been described, a fast method for quantitating levels of various methyltin species in target organs has been unavailable. It has been possible to measure organotins as total tin using atomic absorption spectrometry, but speciation of methyltins has proved difficult. We present a rapid method for quantitative analysis and speciation of methyltins, direct from mammalian tissues. Methyltin compounds (monomethyltin trichloride, dimethyltin dichloride, trimethyltin chloride, and tetramethyltin) are purged from freshly homogenized mouse kidney and brain tissues using NaBH4. The volatile organotin hydrides produced are cryogenically trapped on the head of a gas chromatographic column (at -40 degrees C) and eluted using a linear temperature program (15 degrees C/min to 190 degrees C). The compounds are detected using selected ion monitoring in a Hewlett Packard 5985-B mass spectrometer. Quantitation is achieved by integration of the areas of the chromatographic peaks. Linear response is obtained over the range of 1 ng to 30 micrograms for each compound. Recoveries of methyltins spiked into tissues are greater than 85%.

Methylation of tin by estuarine microorganisms.

Mixed inoculums of microorganisms from Chesapeake Bay sediments transformed inorganic tin (SnCl(4) . 5H(2)O) to organotin compounds. Dimethyltin and trimethyltin species were identified as products by gas chromatography-mass spectrometry. Methylated tin species were not observed in sterile controls or in poisoned controls. Thus, estuarine microorganisms have the potential for transforming tin to toxic organotins and for mobilizing tin in the ecosystem.