Effect of photoperiod on steroid metabolism in the sea star Asterias Rubens L

• 1.1. The effect of photoperiod on steroid metabolism in Asterias rubens was studied.
• 2.2. Daylength was artificially shortened in 3 weeks from long-day (LD 18/6) to short-day (LD 6/18) conditions and its effect on the metabolism of pregnenolone and dehydroepiandrosterone was studied in homogenates of gonad and pyloric caeca tissue from male and female seastar.
• 3.3. Pregnenolone metabolism did not change during the experiment when the animals were kept continuously under the same (long-day) conditions. Pregnenolone metabolism was intensified by decreasing daylength. The production of progesterone reached its maximum at a daylength comparable to that in autumn (LD 12/12), and that of an unidentified steroid at an even shorter daylength.
• 4.4. Metabolism of dehydroepiandrosterone was influenced by photoperiod. There were indications that androstenedione production is maximal at fall conditions. This was evident for an as yet unidentified steroid.
• 5.5. Metabolism of DHEA strongly increased during the experiment in animals which were kept continuously under long-day conditions. It is discussed that this may be a reaction to crowding.

     

Progesterone and oestrone levels in the gonads and pyloric caeca of the male sea star Asterias rubens: A comparison with the corresponding levels in the female sea star

• 1.1. Levels of progesterone and oestrone were estimated by means of RIA in the gonads and pyloric caeca of male specimens of Asterias rubens throughout the reproductive cycle.
• 2.2. The patterns obtained appeared to be sex-specific on comparison with those of female specimens of A. rubens and it was concluded that progesterone and oestrone in the testes are involved in the regulation of gametogenesis.
• 3.3. The levels of progesterone in the pyloric caeca were about ten times higher than those in the testes; in the same sequence levels of oestrone were only slightly higher.
• 4.4. The onset of a new reproductive cycle, taking the abrupt decrease of the pyloric caeca-index as a marker, coincides with a strong decrease of the progesterone level (P < 0.001) and an increase of the oestrone level (P < 0.01) in the pyloric caeca.
• 5.5. It is supposed that progesterone and oestrone in the pyloric caeca are concerned with making available the materials required for gametogenesis.

     

Radioimmunological quantification of C21, C19 and C18 steroids in the gonads of Locusta migratoria migratorioides R. and F.

• 1.1. Levels of progesterone, pregnenolone, testosterone, 5α-dihydrotestosterone, estrone and estradiol were measured by radioimmunoassay in purified gonadal extracts of larval and adult male and female Locusta migratoria.
• 2.2. The average steroid contents varied between less than 1 ng to more than 160 ng/g tissue.
• 3.3. Young adults were treated with precocene or ketoconazole in an attempt to influence the steroid contents in gonads.
• 4.4. Ketoconazole treatment had no effect on the steroid contents in gonads whereas precocene treatment resulted in higher contents of androgens.

     

IDentification of a microsomal retinoic acid synthase as a microsomal cytochrome P-450-linked monooxygenase system

• 1.1. To characterize an enzyme which metabolizes retinal in liver microsomes, several properties of the enzymatic reaction from retinal to retinoic acid were investigated using rabbit liver microsomes.
• 2.2. The maximum pH of the reaction in the liver microsomes was 7.6.
• 3.3. The K_m and V_max values for all-trans, 9-cis and 13-cis-retinals were determined.
• 4.4. The reaction proceeded in the presence of NADPH and molecular oxygen.
• 5.5. The incorporation of one atom of molecular oxygen into retinal was confirmed by using oxygen-18, showing that the reaction comprised monooxygenation, not dehydrogenation.
• 6.6. The monooxygenase activity was inhibited by carbon monoxide, phenylisocyanide and antiNADPH-cytochrome P-450 reductase IgG, but not by anti-cytochrome b₅ IgG.
• 7.7. The enzymatic activity inhibited by carbon monoxide was photoreversibly restored by light of a wavelength of around 450 nm.
• 8.8. The retinal-induced spectra of liver microsomes with three isomeric retinals were type I spectra.
• 9.9. The microsomal monooxygenase activity induced by phenobarbital or ethanol were more effective than that by 3-methylcholanthrene, clotrimazole or β-naphthoflavone.
• 10.10. These results showed that the monooxygenase reaction from retinal to retinoic acid in liver microsomes is catalyzed by a cytochrome P-450-linked monooxygenase system.

     

Purification and characterization of elastase from the pyloric caeca of rainbow trout (Oncorhynchus mykiss)

• 1.1. An elastase-like enzyme was purified from the pyloric caeca of rainbow trout by hydrophobic interaction, cation exchange and gel-filtration chromatography.
• 2.2. The approximate molecular weight of the elastase was 27 kDa and the isoelectric point was remarkably basic.
• 3.3. The pH optimum of this enzyme was 8.0, when assayed with Succinyl-Ala-Ala-Ala-p-Nitroanilide.
• 4.4. When assayed with Succinyl-Ala-Ala-Ala-p-Nitroanilide, the enzyme activity had a temperature optimum of 45°C, and the enzyme was stable up to this temperature.
• 5.5. The trout elastase exhibited a higher specific activity than porcine elastase against Succinyl-Ala-Ala-Ala-p-Nitroanilide and elastin-orcein.
• 6.6. The trout elastase was inhibited by elastatinal, PMSF, TPCK, SBTI and Bowman-Birk inhibitor.

     

Effects of inhibition of nadph: cytochrome p-450 reductase on benzo(a)pyrene metabolism in mouse liver microsomes

• 1.1. Effects of antioxidants (butylated hydroxytoluene and nor-dihydroguaiaretic acid), vitamin K-related quinones (vitamin K₁ and coenzyme Q₁₀) and inorganic copper (CuSO₄), in concentrations inhibiting NADPH: cytochrome P -450 reductase, were re-examined on benzo(a)pyrene metabolism in mouse liver uninduced microsomes.
• 2.2. It was found that all these compounds decrease production of the two-electron oxygenation products of benzo(a)pyrene (monophenoles, diols) and the amounts of glucuronides in a manner parallel to their inhibitory potency against NADPH: cytochrome P-450 reductase.
• 3.3. No correlation was found between amounts of one-electron oxidation products of benzo(a)pyrene and inhibition of NADPH: cytochrome P-450 reductase.
• 4.4. Without added UDPGA the compounds studied decreased protein associated benzo(a)pyrene metabolites in parallel to the decreased overall metabolism of this polyaromatic hydrocarbon.
• 5.5. The mode of action of the studied compounds is discussed.

     

Activities of hexokinase,phosphofructokinase and pyruvate kinase in the body wall,pyloric caeca and tube feet of Asterias vulgaris: Evidence of body wall as a major source of glycolytic activity

• 1.1. The activities of hexokinase (HK) and pyruvate kinase (PK) were significantly higher than the activity of phosphofructokinase (PFK) in the body wall, pyloric caeca and tube feet.
• 2.2. When expressed as a function of wet weight, the specific activity of HK was highest in the pyloric caeca. When expressed as a function of cytosolic protein, the specific activity of HK was highest in the body wall.
• 3.3. Specific activities PFK and PK, expressed as functions of cytosolic protein, were highest in the tube feet. These enzyme activities should reflect the high energetic requirements of the tube feet.
• 4.4. Highest total activity of PFK was found in the body wall. These results support the conclusion that the body wall is a metabolically active organ and that the energetic requirement of the body wall is a significant component of the energetic requirements of the organism.

     

Relative bioavailability and DNA adduct formation of benzo[a]pyrene and metabolites in the diet of the winter flounder

• 1.1. Radiolabeled metabolites of the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) were shown to be absorbed through the diet of the winter flounder, Pseudo pleuronectes americanus.
• 2.2. Oral bioavailability of a mixture of naturally produced metabolites was significantly less than that of the parent BaP.
• 3.3. Oral bioavailability of a pure metabolite, BaP-7,8-dihydrodiol (7,8-D) was found to be similar to that of BaP.
• 4.4. Both metabolites and BaP formed DNA adducts in liver.

     

Mixed function oxidase activity in the harbour seal (Phoca vitulina) from sable is., N.S.

• 1.1. One adult male, eight pups (including two full term foetuses) and nine adult female harbour seals (Phoca vitulina) were analysed for indices of mixed function oxidase (MFO) activity.
• 2.2. MFO activity was present in liver samples, but was at or below detection limits in samples of kidney, lung and pancreas.
• 3.3. Hepatic ethoxyresorufin O-de-ethylase and benzo[a]pyrene hydroxylase activities were similar to those reported in other seals and in other mammals.
• 4.4. Cytochromes P-450 and b₅ concentrations were slightly lower than those observed in other mammals.
• 5.5. MFO activities in newborn pups and foetuses were significantly lower than those in adult females.
• 6.6. No qualitative differences in cytochrome P-450 isozyme distribution between foetal and adult samples could be discerned by electrophoresis.

     

Protein digestion and ion concentrations in rainbow trout (Salmo gairdnerii Rich.) digestive tract in sea- and fresh water

• 1.1. Rainbow trout maintained in fresh water or Actapted to sea-water for 24 hr were fed casein-based dry diet. After feeding, fish were kept in fresh water (FW) or transferred to artificial sea-water (SW) and sacrificed after 10 or 20 hr.
• 2.2. The digestive tract was separated into five parts: stomach, pyloric caeca region, middle intestine and two equal lengths of rectum.
• 3.3. The content of these parts was analysed for ions Na⁺, K⁺, Cl⁻, Mg²⁺ and for free, peptide and total amino acids.
• 4.4. In the fish stomach all ions, with the exception of Ca²⁺, indicate drinking of sea-water. In the pyloric caeca region Na⁺ appears to be efficiently absorbed in SW fish but influxed in FW fish. In the rectum of SW fish K⁺ appears to be reabsorbed but Na⁺ concentrated in faeces.
• 5.5. Free amino acid concentrations were always higher in gut lumen of SW than in FW fish in respect to time after feeding and portion of intestinal content. Free amino acids constitute at most 7.4–8.7% of total amino acids in the content of pyloric caeca region.
• 6.6. Peptide amino acids, being mostly di-, tri- and tetra-peptides, increased in stomach content from 14.7 to 28.4% of the total, from 6 to 10 hr after a meal in SW fish. Peptide amino acids constituted 80.3–89.0% of total amino acids in intestinal content of the pyloric caeca region. These peptide portions decreased in the mid-intestine (47.5–52.5%) and increased again in the rectum (73.6–76.0%).
• 7.7. It was concluded that in rainbow trout fed in both sea- or fresh water, ion concentrations do not seem to interfere with protein digestion and nutrient absorption in alimentary tract.

     

Determination of benzo(a)pyrene in isopods (Porcellio scaber Latr.) exposed to contaminated food

• 1.1. A method is presented for the determination of the polycyclic aromatic hydrocarbon benzo(a)pyrene (BaP) in the isopod Porcellio scaber, using reversed-phase HPLC with fluorescence detection.
• 2.2. This technique has a detection limit for BaP in P. scaber of approximately 3.2 ng g⁻¹ fresh weight.
• 3.3. BaP was assimilated from food by P. scaber.
• 4.4. After four weeks of ad libitum feeding on BaP-contaminated food, concentrations in the isopods were approximately 30–40 times lower than those in the food.
• 5.5. Male and female isopods did not differ in BaP concentration. Variation among males seemed to be much higher than among females.

     

Fate of ingested leucine and glucose in the sea star,Asterias rubens,during its annual reproductive cycle

• 1.1. The distribution of radiolabel from L-leucine [¹⁴C-UL] and D-glucose [¹⁴C-UL] was measured in the sea star Asterias rubens at 1, 6 and 24 hr after oral administration.
• 2.2. Incorporation of the label from both compounds was observed in pyloric caeca, coelomocytes and ovaries even after an incubation time of 1 hr.
• 3.3. Highest incorporation from both precursors was found in proteins, while substantial radioactivity was present in the amino acids, organic acids and neutral components. Lipids were hardly labelled from leucine and only slightly from glucose.
• 4.4. Radioactivity in proteins and lipids increased with increasing incubation time. No significant differences were found in the distribution patterns of radiolabel during the reproductive cycle.
• 5.5. The data obtained are discussed in terms of current knowledge on the translocation of nutrients in echinoderms.

     

Mixed-function oxygenase in juvenile rainbow trout exposed to hexachlorobenzene or 3, 3′, 4, 4′-tetrachlorobiphenyl

• 1.1. 3,3',4,4'-tetrachlorobiphenyl (PCB 77), but not hexachlorobenzene, induced liver micro-somal cytochrome P-450 (Cyt P-450), ethoxycoumarin-O-deethylase (ECOD) and ethoxyresorufin-O-deethylase (EROD) in rainbow trout. Maximum induction was observed in a PCB 77 injected group of fish (1.0mg/kg, i.p. injection) 13 days after the injections being 2, 10 and 50 times the values of non-induced fish, respectively.
• 2.2. The apparent K_m value of ethoxyresorufin of this induced group of fish differed only slightly from that of non-induced fish. The apparent V_max value (EROD) was 50 times higher.
• 3.3. Freezing small pieces of liver in liquid nitrogen did not produce cytochrome P-420.
• 4.4. Fluorimetric and spectrophotometric measurements of EROD correlated.

     

Food deprivation induces differential changes in contents and microstructures of digestive tract and appendages in bluegill fish,Lepomis macrochirus

• 1.1. The digestive tract was compared with the tract appendages (caeca) in bluegill fish, Lepomis macrochirus in their response to short and long term food deprivation.
• 2.2. Fasting for 7 days resulted in 80% reduction of food content in the main tract, but only 40% reduction in appendages (caeca).
• 3.3. The intestine exhibited two different patterns of food distribution under fed and food deprived conditions.
• 4.4. The histopathological impact of starvation was more prominent on the intestine than on caeca.
• 5.5. These results suggest that digestive tract and appendages concommitantly conserve food during food scarcity, but appendages may offer advantages in retaining food longer, and in their greater resistance to starvation-induced effects.

     

Responses of mixed-function oxygenase and antioxidase enzyme system of Mytilus sp. to organic pollution

• 1.1. Mixed-function oxidase (MFO) system components (cytochrome P-450, “418-peak”, cytochrome b₅ and NADPH-cytochrome c (P-450) reductase) and inducible antioxidant enzymes (catalase, Superoxide dismutase (SOD), glutathione peroxidase (GPX) and DT-diaphorase) has been determined in digestive glands of mussels (Mylilus galloprovincialis) collected from three Mediterranean coastal locations, exhibiting an organic pollution gradient.
• 2.2. Cytochrome P-450, the “418-peak”, catalase and SOD showed a good correlation with whole body tissue PAHs and, to a lower extent, with PCBs.
• 3.3. Microsomal NADPH-dependent DT-diaphorase, but not the NADH-dependent microsomal enzyme or the cytosolic DT-diaphorases, was indicated to increase with pollution exposure.
• 4.4. The application of such measurements to environmental monitoring is discussed. Given the magnitude of differences observed, and the state of knowledge on enzyme function and mechanisms of toxicity, a multiparameter approach is considered to offer current and future potential for detecting the impact of organic pollution on bivalve molluscs.

     

Purification and characterization of carbonyl reductases from bovine liver cytosol and microsome. the cytosolic enzyme has a novel 3α/17β-hydroxysteroid dehydrogenase activity

• 1.1. Carbonyl reductase, which is distributed in both cytosolic and microsomal fractions in bovine liver, were purified to homogeneity on 12.5% sodium dodecylsulfate-polyacrylamide gel electrophoresis and shown to have molecular weights of 32 kDa and 68 kDa, respectively.
• 2.2. Both carbonyl reductases can catalyze the reduction of many carbonyl compounds including ketone, quinones and aldehyde with relatively low K_m values.
• 3.3. From the absorption spectrum result, microsomal carbonyl reductase closely resembles cytochrome P-450 reductase.
• 4.4. Cytosolic carbonyl reductase is a novel enzyme which can act on both testosterone and androsterone at low concentration.

     

Selective activation of carcinogenic aromatic amines to bacterial mutagens in the marine musselMytilus gallopro vincialis

• 1.1. The postmitochondrial fraction of the marine mussel Mytilus galloprovincialis digestive gland activates selectively precarcinogenic aromatic amines, but not precarcinogenic benzo(a]pyrene, to Salmonella typhimurium TA 98 mutagens.
• 2.2. This activation potential is NADPH-dependent, is not inducible by exposure to Diesel 2 oil and a polluted environment, and is inhibited by methimazole.
• 3.3. The characteristics of this activation potential are consistent with the recent finding of the presence of FAD-containing-, and lack ofcytochrome P-450 dependent-, monooxygenase activity in Mytilus edulis.
• 4.4. The presence of such selective potential in marine invertebrate(s) may bring new insight into our understanding of the fate and the effects of carcinogens in the marine environment.

     

In vivo administration of H2 blockers,cimetidine and ranitidine,reduced the contents of the cytochrome P450IID (CYP2D) subfamily and their activities in rat liver microsomes

• 1.1. The effects of in vivo administration of H₂ blockers, cimetidine and ranitidine (0.6 mmol/kg body weight/day, for 5 days), on several P450 isozymes, the P450IID (CYP2D) subfamily, and their monooxygenase activities in rat liver microsomes were investigated.
• 2.2. In vivo administration of cimetidine and ranitidine decreased the contents of P450 isozymes and the activities of P450-linked monooxygenase systems; i.e., benzphetamine N-demethylase, aminopyrine N-demethylase, 7-ethoxycoumarine O-deethylase, debrisoquine 4-hydroxylase and bufuralol 1'-hydroxylase.
• 3.3. The inhibitory effect on the enzymatic activities of the P450IID (CYP2D)-linked monooxygenase systems was studied by Western blot analysis with serum containing antiCYP2D6 IgG, i.e., LKM1 autoantibody. The amount of P450IID (CYP2D) in liver microsomes decreased more remarkably in the group administered ranitidine or cimetidine in vivo than in controls.
• 4.4. The effects of cimetidine and ranitidine on the activities of the P450IID (CYP2D)-linked monooxygenase systems were investigated in vitro. The activities of debrisoquine 4-hydroxylase and bufuralol 1'-hydroxylase were inhibited in vitro by cimetidine or ranitidine at a higher concentration than that on in vivo administration of either H₂ blocker.
• 5.5. The kinetic parameters for cimetidine or ranitidine as to the activities of debrisoquine 4-hydroxylase and bufuralol 1'-hydroxylase in liver microsomes were determined by means of Lineweaver-Burk plots.
• 6.6. The suppressive effects of cimetidine and ranitidine on the activities of the P450IID (CYP2D)-linked monooxygenase systems in vivo were found to be due to a decrease of the content of the P450IID (CYP2D) protein.

     

Metabolism of benzo[a]pyrene and persistence of DNA adducts in the brown bullhead (Ictalurus nebulosus)

• 1.1. The in vitro metabolism of [³H]benzo[a]pyrene (BP) and [¹⁴C]benzo[a]pyrene-7,8-dihydrodiol (BP-7,8-diol) by liver of brown bullhead (Ictalurus nebulosus) was characterized, as was the formation and persistence of BP-DNA adducts in vivo.
• 2.2. Compared to rat liver microsomes, bullhead liver microsomes produced relatively larger amounts of BP-7,8-diol (predominantly the [−] enantiomer) and smaller amounts of BP-4,5-diol.
• 3.3. BP phase I metabolites were efficiently converted by freshly isolated bullhead hepatocytes to conjugates, predominantly glucuronides.
• 4.4. BP-7,8-diol was metabolized by hepatocytes 4-fold more rapidly than was BP and was converted to approximately equal amounts of glucuronides, glutathione conjugates and sulfates.
• 5.5. BP-DNA adducts formed in bullhead liver with a lag time of several days and maximum adduct formation at 25–30 days. The major adduct was anti-BPDE-deoxyguanosine.