Inducible cytochrome P450 activities in renal glomerular mesangial cells: biochemical basis for antagonistic interactions among nephrocarcinogenic polycyclic aromatic hydrocarbons

BACKGROUND: Benzo(a)pyrene (BaP), anthracene (ANTH) and chrysene (CHRY) are polynuclear aromatic hydrocarbons (PAHs) implicated in renal toxicity and carcinogenesis. These PAHs elicit cell type-specific effects that help predict toxicity outcomes in vitro and in vivo. While BaP and ANTH selectively injure glomerular mesangial cells, and CHRY targets cortico-tubular epithelial cells, binary or ternary mixtures of these hydrocarbons markedly reduce the overall cytotoxic potential of individual hydrocarbons. METHODS: To study the biochemical basis of these antagonistic interactions, renal glomerular mesangial cells were challenged with BaP alone (0.03 - 30 microM) or in the presence of ANTH (3 microM) or CHRY (3 microM) for 24 hr. Total RNA and protein will be harvested for Northern analysis and measurements of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin-O-deethylase (EROD) activity, respectively, to evaluate cytochrome P450 mRNA and protein inducibility. Cellular hydrocarbon uptake and metabolic profiles of PAHs were analyzed by high performance liquid chromatography (HPLC). RESULTS: Combined hydrocarbon treatments did not influence the cellular uptake of individual hydrocarbons. ANTH or CHRY strongly repressed BaP-inducible cytochrome P450 mRNA and protein expression, and markedly inhibited oxidative BaP metabolism. CONCLUSION: These findings indicate that antagonistic interactions among nephrocarcinogenic PAHs involve altered expression of cytochrome P450s that modulate bioactivation profiles and nephrotoxic/ nephrocarcinogenic potential.     

Genome wide identification of recessive cancer genes by combinatorial mutation analysis

We devised a novel procedure to identify human cancer genes acting in a recessive manner. Our strategy was to combine the contributions of the different types of genetic alterations to loss of function: amino-acid substitutions, frame-shifts, gene deletions. We studied over 20,000 genes in 3 Gigabases of coding sequences and 700 array comparative genomic hybridizations. Recessive genes were scored according to nucleotide mismatches under positive selective pressure, frame-shifts and genomic deletions in cancer. Four different tests were combined together yielding a cancer recessive p-value for each studied gene. One hundred and fifty four candidate recessive cancer genes (p-value < 1.5 x 10(-7), FDR = 0.39) were identified. Strikingly, the prototypical cancer recessive genes TP53, PTEN and CDKN2A all ranked in the top 0.5% genes. The functions significantly affected by cancer mutations are exactly overlapping those of known cancer genes, with the critical exception for the absence of tyrosine kinases, as expected for a recessive gene-set.     

Rapid direct sequence analysis of the dystrophin gene

Mutations in the dystrophin gene result in both Duchenne and Becker muscular dystrophy (DMD and BMD), as well as X-linked dilated cardiomyopathy. Mutational analysis is complicated by the large size of the gene, which consists of 79 exons and 8 promoters spread over 2.2 million base pairs of genomic DNA. Deletions of one or more exons account for 55%-65% of cases of DMD and BMD, and a multiplex polymerase chain reaction method-currently the most widely available method of mutational analysis-detects approximately 98% of deletions. Detection of point mutations and small subexonic rearrangements has remained challenging. We report the development of a method that allows direct sequence analysis of the dystrophin gene in a rapid, accurate, and economical fashion. This same method, termed "SCAIP" (single condition amplification/internal primer) sequencing, is applicable to other genes and should allow the development of widely available assays for any number of large, multiexon genes.     

Energetics of protein transport across biological membranes. a study of the thylakoid DeltapH-dependent/cpTat pathway

Among the pathways for protein translocation across biological membranes, the DeltapH-dependent/Tat system is unusual in its sole reliance upon the transmembrane pH gradient to drive protein transport. The free energy cost of protein translocation via the chloro-plast DeltapH-dependent/Tat pathway was measured by conducting in vitro transport assays with isolated thylakoids while concurrently monitoring energetic parameters. These experiments revealed a substrate-specific energetic barrier to cpTat-mediated transport as well as direct utilization of protons from the gradient, consistent with a H+/protein antiporter mechanism. The magnitude of proton flux was assayed by four independent approaches and averaged 7.9 x 10(4) protons released from the gradient per transported protein. This corresponds to a DeltaG transport of 6.9 x 10(5) kJ.mol protein translocated(-1), representing the utilization of an energetic equivalent of 10(4) molecules of ATP. At this cost, we estimate that the DeltapH-dependent/cpTat pathway utilizes approximately 3% of the total energy output of the chloroplast.     

Fluctuation of surface charge in membrane pores

Surface charge in track-etched polyethylene terephthalate (PET) membranes with narrow pores has been probed with a fluorescent cationic dye (3,3'-diethyloxacarbocyanine iodide (diO-C2-(3))) using confocal microscopy. Staining of negatively charged PET membranes with diO-C2-(3) is a useful measure of surface charge for the following reasons: 1) the dye inhibits K(+) currents through the pores and reduces their selectivity for cations; 2) it inhibits [3H]-choline+ transport and promotes 36Cl- transport across the membrane in a pH- and ionic-strength-dependent fashion; and 3) staining of pores by diO-C2-(3) is reduced by low pH and by the presence of divalent cations such as Ca2+ and Zn2+. Measurement of the time dependence of cyanine staining of pores shows fluctuations of fluorescence intensity that occur on the same time scale as do fluctuations of ionic current in such pores. These data support our earlier proposal that fluctuations in ionic current across pores in synthetic and biological membranes reflect fluctuations in the surface charge of the pore walls in addition to molecular changes in pore proteins.     

Analysis of the stereochemistry of lipoxygenase-derived hydroxypolyenoic fatty acids by means of chiral phase high-pressure liquid chromatography

A chiral phase HPLC method was developed for the simultaneous determination of the positional and optical isomers of the lipoxygenase-derived hydroxypolyenoic fatty acids. With a Bakerbond chiral phase HPLC column (dinitrobenzoyl phenylglycine as chiral phase) the positional and optical isomers of the reduced dioxygenation products (by triphenylphosphine or borohydride) of linoleic acid and arachidonic acid were separated after methylation of the carboxylic groups. No cumbersome chemical derivatization such as conversion to a diastereomer was necessary. As compared with the methods used up till now chiral phase HPLC proved to be simpler and more sensitive. About 10 pmol of hydroxy fatty acids suffice for an analysis. The chiral phase HPLC can be used for the preparative separation of the optical antipodes of the lipoxygenase products. An optical purity of more than 90% can be reached in one preparative run. The method was applied to the determination of the stereochemistry of the dioxygenation products of polyenoic fatty acids formed by the lipoxygenases from soybeans, reticulocytes, pea seeds (isoenzyme I and II), tomato fruits, by the quasilipoxygenase activity of hemoglobin, and by the methylene blue-mediated photooxidation of arachidonic acid.     

Integration and recovery processes contribute to the temporal selectivity of neurons in the midbrain of the northern leopard frog, Rana pipiens

This study examined the mechanisms underlying amplitude modulation selectivity in the anuran auditory midbrain. Single units were recorded extracellularly in the torus semicircularis of the northern leopard frog, Rana pipiens. Two physiologically distinct classes of neurons were identified, based on their response latencies and their selectivities to pulse repetition rates. Cells in one group had short response latencies (median = 31 ms) and responded best to pulse repetition rates below 40 Hz. Tuning to low amplitude modulation rates was largely determined by recovery processes and phasic response properties. Cells in the second group had much longer latencies (median=81 ms) and were generally selective for pulse repetition rates greater than 40-50 Hz. Sensitivity to higher amplitude modulation rates resulted from integration processes; these units only responded when a threshold number of pulses were presented at a minimum pulse density (amplitude modulation rate). At amplitude modulation rates above their best rate, their responses decreased, apparently due to inadequate recovery time between pulses.     

Thermal controls of Yellowstone cutthroat trout and invasive fishes under climate change

We combine large observed data sets and dynamically downscaled climate data to explore historic and future (2050–2069) stream temperature changes over the topographically diverse Greater Yellowstone Ecosystem (elevation range = 824–4017 m). We link future stream temperatures with fish growth models to investigate how changing thermal regimes could influence the future distribution and persistence of native Yellowstone cutthroat trout (YCT) and competing invasive species. We find that stream temperatures during the recent decade (2000–2009) surpass the anomalously warm period of the 1930s. Climate simulations indicate air temperatures will warm by 1 °C to >3 °C over the Greater Yellowstone by mid‐21st century, resulting in concomitant increases in 2050–2069 peak stream temperatures and protracted periods of warming from May to September (MJJAS). Projected changes in thermal regimes during the MJJAS growing season modify the trajectories of daily growth rates at all elevations with pronounced growth during early and late summer. For high‐elevation populations, we find considerable increases in fish body mass attributable both to warming of cold‐water temperatures and to extended growing seasons. During peak July to August warming, mid‐21st century temperatures will cause periods of increased thermal stress, rendering some low‐elevation streams less suitable for YCT. The majority (80%) of sites currently inhabited by YCT, however, display minimal loss (<10%) or positive changes in total body mass by midcentury; we attribute this response to the fact that many low‐elevation populations of YCT have already been extirpated by historical changes in land use and invasions of non‐native species. Our results further suggest that benefits to YCT populations due to warmer stream temperatures at currently cold sites could be offset by the interspecific effects of corresponding growth of sympatric, non‐native species, underscoring the importance of developing climate adaptation strategies that reduce limiting factors such as non‐native species and habitat degradation.     

Immunochemical identity of peroxisomal enoyl-CoA hydratase with the peroxisome-proliferation -associated 80,000 mol wt polypeptide in rat liver

Peroxisome proliferators, which induce proliferation of hepatic peroxisomes, have been shown previously to cause a marked increase in an 80,000 mol wt polypeptide predominantly in the light mitochondrial and microsomal fractions of liver of rodents. We now present evidence to show that this hepatic peroxisome-proliferation-associated polypeptide, referred to as polypeptide PPA-80, is immunochemically identical with the multifunctional peroxisome protein displaying heat-labile enoyl-CoA hydratase activity. This conclusion is based on the following observations: (a) the purified polypeptide PPA-80 and the heat- labile enoyl-CoA hydratase from livers of rats treated with the peroxisome proliferators Wy-14,643 {[4-chloro-6(2,3-xylidino)-2-pyrimidinylthio]acetic acid} exhibit identical minimum molecular weights of approximately 80,000 on SDS polyacrylamide gel electrophoresis; (b) these two proteins are immunochemically identical on the basis of ouchterlony double diffusion, immunotitration, rocket immunoelectrophoresis, and crossed immunoelectrophoresis analysis; and (c) the immunoprecipitates formed by antibodies to polypeptide PPA-80 when dissociated on a sephadex G-200 column yield enoyl-CoA hydratase activity. Whether the polypeptide PPA-80 exhibits the activity of other enzyme(s) of the peroxisomal β-oxidation system such as fatty acyl-CoA oxidase activity or displays immunochemical identity with such enzymes remains to be determined. The availability of antibodies to polypeptide PPA-80 and enoyl-CoA hydratase facilitated immunofluorescent and immunocytochemical localization of the polypeptide PPA- 80 and enoyl-CoA hydratase in the rat liver. The indirect immunofluorescent studies with these antibodies provided direct visual evidence for the marked induction of polypeptide PPA-80 and enoyl-CoA hydratase in the livers of rats treated with Wy-14,643. The present studies also provide immunocytochemical evidence for the localization of polypeptide PPA- 80 and the heat-labile enoyl-CoA hydratase in the peroxisome, but not in the mitochondria, of hepatic parenchymal cells. These studies, therefore, provide morphological evidence for the existence of fatty acyl-CoA oxidizing system in peroxisomes. An increase of polypeptide PPA-80 on SDS polyacrylamide gel electrophoretic analysis of the subcellular fractions of liver of rodents treated with lipid-lowering drugs should serve as a reliable and sensitive indicator of enhanced peroxisomal β- oxidation system.