Application of SSH and a macroarray to investigate altered gene expression in Mytilus edulis in response to exposure to benzo[a]pyrene.

The lack of genomic resources for aquatic invertebrates restricts their use as sentinel species in coastal environments. It is known that where genomic data are not available, suppression subtractive hybridisation (SSH) can generate cDNA libraries representative of pollutant-responsive gene transcription in aquatic vertebrates. To assess whether the approach was equally suited to aquatic invertebrates, altered gene expression in digestive gland of the mussel, Mytilus edulis, in response to exposure to benzo[a]pyrene (BaP) (1 mg/l) was investigated with SSH and a nylon macroarray. Screening of the subtracted libraries showed 112/250 up-regulated and 25/55 down-regulated clones were positive for differential expression and characterisation of these identified 87 with unique sequence suitable for array on a nylon membrane. The transcripts isolated were from a diverse range of genes involved in general stress, oxidative stress, cell adhesion, transcriptional and translational regulation, transport mechanisms, energy metabolism, cell metabolism, lipid metabolism, protein turnover and activation, lysosomal activity and 22 cryptic clones. Subsequent use of the clones in macroarray format to analyse expression of BaP-responsive genes (0 vs 4 day exposed) showed 0-100-fold increased levels of the forward-subtracted probes and between 0 and 0.1-fold down-regulation of the reverse-subtracted probes. Only 15% of the clones showed less than 2-fold change in expression. The gene ontology of the transcripts isolated demonstrates that BaP elicits a multitude of responses with a major feature being disruption of cellular redox status. The results indicate that the use of SSH and a macroarray is a robust method to discover novel pollutant-responsive genes in aquatic invertebrates.

Membrane topology of epoxide hydrolase.

The amino acid sequences of epoxide hydrolase from rat, rabbit and human have been subjected to hydropathy analysis and a novel model for the membrane topology of this enzyme is presented. The enzyme would appear to be retained in microsomal membranes by a single transmembrane segment located at the N-terminus and the majority (96%) of the protein is exposed at the cytosolic membrane surface. This model is significantly different from a scheme suggested by analysis of the rat enzyme alone which proposed six transmembrane domains (Porter et al. (1988) Arch. Biochem. Biophys. 248, 121-129). Experiments with rat microsomal membranes were conducted to distinguish between the two models and used proteolytic enzymes and non-permeant chemical probes. Epoxide hydrolase of intact and permeabilised membranes was resistant to digestion by a number of proteinases. However, this is likely to be related to a compact fold of the protein rather than membrane association since purified, delipidated enzyme preparations were also resistant to proteolysis. While the use of proteinases did not provide useful membrane topological information, experiments with the fluorescent probe, 3-azido-2,7-naphthalenedisulphonate strongly support the view that the majority of the protein is indeed exposed at the cytosolic surface of the membranes. The analysis illustrates the caution which must be employed in the formulation of topological models based on hydropathy plots alone and the value of considering homologous proteins.

The association of flowering time quantitative trait loci with duplicated regions and candidate loci in Brassica oleracea.

A population of 150 doubled haploid lines of rapid cycling Brassica oleracea, derived from an F1 from a var. alboglabra x var. italica cross, was scored for flowering time in two trials. Using information on 82 mapped molecular markers, spread evenly across the nine linkage groups, QTL were identified at six locations; one each on linkage groups O2 and O3 and two each on linkage groups O5 and O9. In total, these QTL explained 58 and 93% of the genetical variation in the two trials. Three of these QTL, on linkage groups O2, O3, and O9, were situated in regions showing considerable homology both with each other and with chromosome regions of B. nigra that have been shown to affect flowering time. These same regions are all homologous to a single tract of Arabidopsis chromosome 5, which contains a number of the flowering-related genes, one or more of which may be candidates for the QTL found in Brassica.

Steroid 21-hydroxylase is a major autoantigen involved in adult onset autoimmune Addison's disease.

An adrenal-specific protein reacting with autoantibodies in the sera of patients with adult onset Addison's disease has been purified from human adrenal glands. The protein, mol.wt. 55K, has the biochemical characteristics of steroid 21-hydroxylase and reacts on Western blots with rabbit antibodies to recombinant 21-hydroxylase. Absorption of the native human 55K adrenal protein with human adrenal autoantibodies prevented the subsequent reaction of the 55K protein with rabbit antibodies to 21-hydroxylase in Western blot analysis. In addition, human adrenal autoantibodies reacted with recombinant 21-hydroxylase expressed in yeast. These data indicate that the adrenal specific enzyme steroid 21-hydroxylase is a major autoantigen involved in adult onset autoimmune Addison's disease.

A comparative study of Leishmania mexicana amastigotes and promastigotes. Enzyme activities and subcellular locations.

Leishmania mexicana mexicana amastigotes have been shown to contain greater activities than promastigotes of the enzymes that catalyse the beta-oxidation of fatty acids, but lower activities of several glycolytic enzymes, with the activity of pyruvate kinase being especially low. The results suggest the beta-oxidation of fatty acids is relatively more important to Leishmania amastigotes than promastigotes, whereas the reverse is true for glycolysis. Succinic dehydrogenase and peptidase activities were much higher in promastigotes than amastigotes. The activities of glucose-6-phosphatase, fructose-1,6-bisphosphatase, acid phosphatase and glucose-6-phosphate dehydrogenase varied less, although in each case the activity was significantly lower in the mammalian stage. A method for lysing and fractionating L. m. mexicana promastigotes has been developed. Using this procedure it has been established that many of the glycolytic and functionally related enzymes are located in cell organelles, that hexokinase is intimately connected with the particulate part of the parasite, and that the microsomal fraction of L. m. mexicana is very different in composition from the microsomes of mammalian liver cells.

Changes in hepatic xenobiotic-metabolising enzymes in mouse liver following infection with Leishmania donovani.

Infection of mice with Leishmania donovani resulted in decreased activities of several liver enzymes involved in the metabolism of xenobiotics. Microsomal membranes from infected livers contained reduced amounts of cytochromes P450 and b5 and NADPH-cytochrome P450 reductase. Several cytochrome P450 isoenzymes (P450-PB1, P450-PB3, P450-PCN and P450-UT1) and P450-mediated reactions (aminopyrine demethylase, aniline hydroxylase, benzphentamine demethylase and ethoxycoumarin deethylase) were affected similarly. The metabolism of two carcinogens (nitrosodimethylamine and 7,12-dimethylbenz[a]anthracene) by liver microsomal membrane preparations was also reduced. Leishmania infection caused an increase of cytosolic epoxide hydrolase and microsomal epoxide hydrolase and NADH-cytochrome b5 reductase were unaffected. The results suggest that Leishmania-infected animals are likely to have altered responses to exogenous toxins compared to uninfected animals.

Effects of pyrazole on nitrosodimethylamine demethylase and other microsomal xenobiotic metabolising activities.

Pyrazole administered to immature rats at one day or on four successive days prior to sacrifice increased a microsomal NDMAD with apparent Km 0.04 mM. Aniline hydroxylase activity was also increased by these treatments. Ethoxycoumarin deethylase and amino pyrine demethylase activities were not altered when animals were treated with pyrazole one day prior to sacrifice but were reduced to below control activity when animals were treated for four successive days. All microsomal mono-oxygenases were decreased when animals received a single administration of pyrazole four days prior to sacrifice and the cytochrome P-450 content of these microsomes was reduced by up to 50%. When microsomes from untreated animals or animals treated for four successive days were incubated with pyrazole in the presence of NADPH, cytochrome P-450 content decreased in a time dependent process to a limiting value. The effect was dependent on pyrazole concentration and saturable. These results suggest that pyrazole induces a cytochrome P-450 isoenzyme with high affinity for NDMA but also acts as a suicide inhibitor of the cytochrome.

Effects of metyrapone and norharmane on microsomal mono-oxygenase and epoxide hydrolase activities.

This study was undertaken to examine the possibility that metyrapone and norharmane stimulate epoxide hydrolase and inhibit mono-oxygenase activities by binding to a cytochrome P-450 component of a stable complex containing the two enzymes. The concentration of metyrapone and norharmane which inhibited mono-oxygenase activities of hepatic microsomes from untreated and diethylnitrosamine treated rats was lower than that required to stimulate epoxide hydrolase of the same microsomes. The ability of metyrapone and norharmane to stimulate epoxide hydrolase in these microsomes was not inhibited by the addition of carbon monoxide and reductant. Epoxide hydrolase activity was inhibited by detergents but the enzyme was still stimulated by metyrapone and norharmane under conditions of total membrane disaggregation. When microsomes were solubilized, epoxide hydrolase could be quantitatively recovered by immunoprecipitation. The immunoprecipitate contained no detectable cytochrome P-450 but was stimulated by metyrapone and norharmane. A purified epoxide hydrolase was stimulated by metyrapone but not by norharmane. The response of the enzyme to norharmane was not restored by the inclusion of cytochrome P-448. These findings suggest that metyrapone and norharmane act at separate sites on both cytochrome P-450 and epoxide hydrolase.

Induction of microsomal epoxide hydrolase by nitrosamines in rat liver. Effect on messenger ribonucleic acids.

Nitrosomethylethylamine and nitrosomethylpropylamine were found to be more potent inducers of rat liver microsomal epoxide hydrolase (styrene oxide hydrolase) than nitrosodiethylamine or nitrosodimethylamine. The time course of induction following a single administration of nitrosodimethylethylamine, nitrosomethylpropylamine or nitrosodiethylamine each showed a delay of 24 hr during which enzyme activity was unaltered. After that time activity increased and reached a maximum at between 72 and 120 hr. Increased enzyme activity following NDEA was paralleled by changes in the content of epoxide hydrolase in microsomes as measured by Western blots. Nitrosamines caused an increase of mRNA for epoxide hydrolase which was detected by probing Northern blots with a [32]-P labelled epoxide hydrolase cDNA and by in vitro translation of polyadenylated mRNA. Both methods showed a maximal increase at 72 hr after nitrosodiethylamine treatment but a significant increase was also observed at 24 hr although at this time no increase in enzyme activity was apparent.

Metabolism of 7,12-dimethylbenz[a]anthracene in hepatic microsomal membranes from rats treated with isoenzyme-selective inducers of cytochromes P450.

Previous work has shown that member(s) of the cytochrome P450IIC sub-family play significant roles in the formation of diols of 7,12-dimethylbenz[a]anthracene (DMBA) and are particularly important in formation of the proximate carcinogen (DMBA-3,4-diol). To further characterize the role of members of this subfamily in DMBA-diol formation and to assess the part played by other P450s, DMBA metabolism has been investigated in microsomes prepared from animals pre-treated with isoenzyme selective inducers. The rates of formation of DMBA-diols in membranes from phenobarbital-treated rats were very low when NADH was used as reductant and rates were not altered when NADPH and NADH were used in combination rather than using NADPH alone. This suggests that cytochrome b5 is not involved in DMBA-diol formation in these membranes. Treatment of animals with clofibrate, pyrazole and dexamethasone produced regio-selective alterations in the rates of formation of DMBA-diols at the -3,4-, -5,6- and -8,9- positions. However, none of the inducers caused increases in the rates of DMBA-diol formation of any great magnitude suggesting that the isoforms which are the major induced proteins (P450IVA1, P450IIE1 and P450IIIA1) do not play a significant role in diol formation. The content of other P450s in these membrane are also altered and these were investigated by Western blot using antibodies to P450IIC6, P450IIB1 and P450IIIA1. The results of the Western blots show that the effects of the inducing agents on DMBA-diol formation can be explained by alterations of members of the P450IIC and P450IIB subfamilies.

The role of specific cytochromes P450 in the formation of 7,12-dimethylbenz(a)anthracene-protein adducts in rat liver microsomes in vitro.

The role of specific cytochrome P450 (P450) isoforms in the formation of adducts of 7,12-dimethylbenz(a)anthracene metabolites and membrane proteins has been investigated in vitro with microsomal fractions prepared from rats pretreated with various isoenzyme selective inducers. The effects of isoenzyme selective inhibitors were also evaluated. Adduct formation was shown to be mediated by P450 catalysed reactions but was unaltered, relative to untreated animals, in membranes from pyrazole- and clofibrate-treated animals suggesting that CYP2E1 and CYP4A1 are not involved in this process. However, adduct formation was significantly increased in microsomes from Sudan III-, phenobarbital- and dexamethasone-treated rats, suggesting the involvement of the CYP1A, CYP2B and CYP3A subfamilies, respectively. These conclusions were further supported by the finding that adduct formation in these microsomes could be inhibited by the isoenzyme-selective inhibitors alpha-naphthoflavone, metyrapone and troleandomycin, respectively.

Gender-specific metabolism of benz[a]anthracene in hepatic microsomes from Long-Evans and Hooded Lister rats.

The cytochrome P450 isoforms responsible for the regio-selective metabolism of benz[a]anthracene (BA) are poorly defined but as with other polycyclic aromatic hydrocarbons (PAHs) may include members of the CYP2C sub-family. Since the expression of some of these is regulated in a gender-specific manner and may be altered by age, rat strain or by phenobarbital treatment, the effects of these variables on metabolism of BA to diols was investigated. These studies used hepatic, microsomal membranes from immature and adult Long-Evans rats and adult Hooded Lister rats. BA-diols were resolved by normal phase HPLC into three discrete peaks identified as benz[a]anthracene-5,6-diol (BA-5,6-diol), benz[a]anthracene-10, 11-diol (BA-10,11-diol) and a mixture of benz[a]anthracene-3,4- and -8,9-diols (BA-3,4-diol and BA-8,9-diol and termed Peak(3/8)). Significant gender-related differences were found in the rates of diol formation in adults of both the Long-Evans and Hooded Lister rat strains. Formation of BA-10,11-diol and to a lesser extent the components of Peak(3/8) were greater in the male compared to female animals by factors of at least 14 and two, respectively. An age-dependent effect is also observed in the Long-Evans rat since these differences are still apparent in prepubertal animals but to a lesser extent (gender ratio male:female BA-10,11-diol 9X; Peak(3/8) 1.4X). In contrast BA-5,6-diol was formed at similar rates by membranes from female and male rats whether mature (Long-Evans and Hooded Lister) or immature (Long-Evans). Phenobarbital treatment of the adult Long-Evans rats resulted in a moderate increase in the formation of each diol other than at the 10,11-position and the induction was not gender specific. The rate of formation of BA-10, 11-diol was decreased in phenobarbital-treated male rats suggesting modulation of a male specific isoform. Measurement of microsomal epoxide hydrolase revealed no gender or age differences and suggests that this enzyme is not rate limiting in BA-diol formation and thus is not responsible for the differences in BA-diol formation observed. The results suggest that CYP2C11 along with a male-specific isoenzyme not regulated by age are important in the formation of BA-10,11-diol and a component(s) of Peak(3/8) in males. CYPs 2B2 and/or 2C6 appear to be involved in formation of BA-5,6-diol in male and female. Identification of the CYPs involved in the regio-selective metabolism of BA may lead to an explanation of the lower carcinogenic potency of this PAH compared to dimethylbenz[a]anthracene and this study provides novel clues concerning the identities of the CYPs, which are important.

The contribution of specific cytochromes P-450 in the metabolism of 7,12-dimethylbenz[a]anthracene in rat and human liver microsomal membranes.

The role of specific cytochrome P-450 isoenzymes in the regio-selective metabolism of 7,12-dimethylbenz[a]anthracene (DMBA) has been studied in microsomal membranes from rat and human liver. An antibody inhibition study using membranes from phenobarbital-treated rats demonstrates that a member(s) of the CYP2C family accounts for up to 90% of the formation of the proximate carcinogen, DMBA-3,4-diol, and makes significant contributions to the formation of DMBA-5,6-diol and DMBA-8,9-diol. In these membranes the formation of DMBA-5,6-diol can be entirely accounted by the combined activity of members of the CYP2C and CYP2B families. The metabolism of DMBA has been investigated in human using microsomes from 10 individuals and the metabolites formed by these membranes were found to be mainly hydroxymethyl- and -diol products. The rates of formation of each metabolite show considerable interindividual variation and there was no correlation between these rates for any pairing of metabolites. The CYP content in these membranes of specific members of families 1, 2, 3 and 4 did correlate with the rates of formation of individual metabolites. Surprisingly there was no correlation between the content of CYP2C and formation of DMBA-3,4-diol but an antibody to rat CYP2C6 partially inhibited the formation of this metabolite. The results indicate that in human both inducible sub-families of CYPs, particularly of the PB-type, and constitutively expressed CYPs may be important in DMBA metabolism and that each metabolite may be produced by the combined activity of several CYP isoforms.

Metabolism of benz[alpha]anthracene by human bone marrow in vitro.

The metabolism of polycyclic aromatic hydrocarbons by bone marrow, mononuclear cells from normal donors and leukaemia patients in remission has been investigated. When benz[alpha]anthracene (BA) was included with marrow under cell culture conditions, it was converted to materials which were resolved into three peaks by normal phase HPLC, and which had the chromatographic characteristics of BA-dihydrodiols. Formation of hydroxymethyl-or dihydrodiol-derivatives of 7, 12-dimethylbenz[alpha]anthracene were not detected under the same conditions. The BA-metabolites were identified as BA-5,6-dihydrodiol, BA-10,11-dihydrodiol and BA-8,9-dihydrodiol. The identification was based upon chromatographic properties of the metabolites during normal and reverse phase chromatography and on UV spectral and fluorometric characterization. It was not possible to detect the formation of BA-3,4-dihydrodiol since this dihydrodiol co-elutes with BA-8,9-dihydrodiol and BA-10,11-dihydrodiol during normal phase and reverse phase chromatography, respectively. the UV spectra of BA-3,4-dihydrodiol does not have features which enable it to be readily identified in the presence of these other compounds. Formation of the dihydrodiol-metabolites was dependent on cell number and temperature. Two general cytochrome P450 inhibitors, carbon monoxide and piperonyl butoxide, blocked the formation of metabolites but the cyclooxygenase inhibitor, indomethacin had no effect. Large variations were observed in the capacity of marrow from different individuals to form benz[alpha]anthracene-dihydrodiols but, in each sample where dihydrodiols were formed, the relative amount of each metabolite was BA-8,9-dihydrodiol >> BA-5,6-dihydrodiol > BA-10,11-dihydrodiol. Factors which may contribute to this variation, including disease status, genetic and environmental agents, are considered.

The effect of gender, sexual maturation and xenobiotic treatment on the formation of hydroxymethyl metabolites from 7, 12-dimethylbenz[a]anthracene in rat liver microsomes.

The effects of age, gender, strain, phenobarbital (PB) treatment and pituitary influence on the regioselective metabolism of 7, 12-dimethylbenz[a]anthracene to hydroxmethyl metabolites were investigated. Studies used hepatic microsomal membranes from immature and mature Long Evans (LE) rats and adult Hooded Lister (HL) animals. Hydroxymethyl metabolites were resolved by both normal and reverse phase HPLC with on-line diode array detection. The CYP isoform(s) responsible for oxidation at the 12 methyl position exhibited no gender or developmental regulation and the rate of formation was not altered following hypophysectomy. PB-treatment of adult rats caused a significant increase in the rate of formation of both male and female animals (29 and 41-fold, respectively) suggesting a major contribution from a PB-inducible isoform, such as CYP2B. The rate of formation of 7OHMe12MBA exhibited no gender dependency in immature animals but was 2-fold greater than that observed for 12OHMe7MBA suggesting that steric hindrance resulting from the adjacent 1,2 benzyl ring favours substrate oxidation at the 7-methyl position. Male predominant formation of 7OHMe12MBA was apparent following sexual maturation of the LE rats and was significantly reduced upon hypophysectomy suggesting the involvement of a male-specific GH dependent isoform e.g. CYP2C11.

Construction and characterization of a forward subtracted library of blue mussels Mytilus edulis for the identification of gene transcription signatures and biomarkers of styrene exposure.

Transcriptional profiling can elucidate adaptive/toxicity pathways participating in achieving homeostasis or leading to pathogenesis in marine biota exposed to chemical substances. With the aim of analyzing transcriptional responses in the mussel Mytilus edulis exposed to the corrosive and putatively carcinogenic hydrocarbon styrene (3-5 ppm, 3days), a forward subtracted (SSH) cDNA library was produced. Female mussels were selected and digestive gland mRNA was isolated. A library with 1440 clones was produced and a total of 287 clones were sequenced, 53% being identified through BlastN analysis against Mytibase and DeepSeaVent databases. Those genes included GO terms such as 'response to drugs', 'immune defense' and 'cell proliferation'. Furthermore, sequences related to chitin and beta-1-3-glucan metabolism were also up-regulated by styrene. Many of the obtained sequences could not be annotated constituting new mussel sequences. In conclusion, this SSH study reveals novel sequences useful to generate molecular biomarkers of styrene exposure in mussels.

Microsomal epoxide hydrolase of rat liver. Purification and characterization of enzyme fractions with different chromatographic characteristics.

Microsomal epoxide hydrolase was purified from rat liver, and different fractions of the purified enzyme, which varied in their contents of phospholipid, were obtained by ion-exchange chromatography. One fraction (A), which did not bind to CM-cellulose, had a high phospholipid content, and a second fraction (B), which was eluted from CM-cellulose at high ionic strength, had a low phospholipid content. Removal of most of the phospholipid from fraction A altered its chromatographic behaviour. When the delipidated material was re-applied to CM-cellulose, most of the enzyme bound to the cation-exchanger. The specific activities of all the fractions described (with styrene epoxide [(1,2-epoxyethyl)benzene] as substrate) were altered by adding the non-ionic detergent Lubrol PX or phospholipid. Lubrol PX inhibited enzyme activity, and phospholipid reversed this inhibition. The various enzyme fractions isolated appeared to be different forms of the same protein, as judged by their minimum Mr values and immunochemical properties. These results indicate that different fractions of epoxide hydrolase isolated by ion-exchange chromatography probably are not different isoenzyme forms.