Comparative Biochemistry and Physiology Part C: Pharmacology, Toxicology and Endocrinology最新文献

Brown trout, Salmo trutta, exposed to heavy metals (mainly Cd and Zn) for at least 2 years in the Eagle River, Colorado, were examined for liver size and activity of the growth-promoting enzyme, ornithine decarboxylase (ODC) and compared to trout living in an uncontaminated site. Liver-somatic index (LSI) was greater for trout living in the uncontaminated site with the LSI of females being significantly greater than that of males. The LSI for females at the uncontaminated site was greater than that of females at the contaminated site, but males were not different statistically. ODC activity in the livers of both males and females was lower at the contaminated site. However, males and females did not differ with respect to ODC activity. These data suggest that chronic exposure to heavy metals may have important implications for growth and reproduction and possibly survival. The activity of ODC in liver might serve as a useful biomarker when assessing chronic toxicity of metals to naturally reproducing fish populations.

The in vivo effect of menadione bisulfite adduct on both hepatic oxidative stress and heme oxygenase induction was studied. A marked increase in lipid peroxidation was observed 1 h after menadione bisulfite adduct administration. To evaluate liver antioxidant enzymatic defenses, superoxide dismutase, catalase and glutathione peroxidase activities were determined. Antioxidant enzymes significantly decreased 3 h after menadione bisulfite adduct injection. Heme oxygenase activity appeared 6 h after treatment, peaking 9 h after menadione bisulfite adduct administration. Such induction was preceded by a decrease in the intrahepatic GSH pool and an increase in hydrogen peroxide steady-state concentration, both effects taking place some hours before induction of heme oxygenase. Iron ferritin levels and ferritin content began to increase 6 h after heme oxygenase induction, and these increases were significantly higher 15 h after treatment and remained high for at least 24 h after menadione bisulfite adduct injection. Administration of bilirubin entirely prevented heme oxygenase induction as well as the decrease in hepatic GSH and the increase in lipid peroxidation when administered 2 h before menadione bisulfite adduct treatment. These results indicate that the induction of heme oxygenase by menadione bisulfite adduct may be a general response to oxidant stress, by increasing bilirubin and ferritin levels and could therefore provide a major cellular defense mechanism against oxidative damage.

We investigated roles of catecholamines in metamorphosis of the prosobranch gastropod, Crepidula fornicata. Levels of DOPA, norepinephrine (NE) and dopamine (DA) were measured by high-pressure liquid chromatography (HPLC) in competent larvae and juvenile siblings that metamorphosed in response to the natural adult-derived cue or to elevated K⁺. Competent larvae contained 1.58±0.26 (S.E.M.)×10⁻² pmol DOPA, 0.91±0.45×10⁻² pmol NE, and 0.290±0.087 pmol DA (mean values per μg total protein, n=4 batches of larvae). Levels of DA per individual were not different between larvae and juvenile siblings; levels of NE were higher in juveniles. The tyrosine hydroxylase (TH) inhibitor α-methyl-dl-m-tyrosine (α-MMT) depleted DOPA and DA to approximately half of control values without affecting levels of NE. Depletion of DOPA and DA was accompanied by inhibition of metamorphosis in response to the natural cue but not to elevated K⁺. The dopamine-β-hydroxylase inhibitor diethyldithiocarbamate (DDTC) induced high frequencies of metamorphosis at concentrations of 0.1–10 μM. In juveniles induced by 10 μM DDTC, levels of both NE and DA averaged ∼80% of those in control larvae. Catecholamines may function as endogenous regulators of metamorphosis in C. fornicata.

Neurotensin (NT), given intravenously at 10–50 pmol/kg per min to anesthetized female chickens equipped with a bile duct fistula, dose-dependently elevated hepatic bile flow and bile acid output but only when the enterohepatic circulation was maintained by returning the bile to the intestinal lumen. Infusion of NT at 10 and 50 pmol/kg per min increased the average hepatic bile acid output over a 30-min period to 138±11 and 188±13% of control, respectively. During infusion of NT, plasma levels of immunoreactive NT (iNT) increased in time from the basal level (14±1.3 pM) to reach steady state at 30 min. There was a near linear relationship between the dose of NT infused and the increment in plasma iNT. In addition, infusion of NT at 40 pmol/kg min gave a plasma level of iNT (≅88 pM) which was within the range of those observed during duodenal perfusion with lipid (54–300 pM) and near to that measured in hepatic portal blood from fed animals (52±5 pM). Perfusion of duodenum with lipid released endogenous NT and increased the rate of hepatic bile flow. When NT antagonist SR48692 was given, bile flow rate decreased to the basal level. These results suggest that intestinal NT, released by lipid, may participate in the regulation of hepatic bile acid output by a mechanism requiring an intact enterohepatic circulation.

Regulation of mitochondrial functions in vivo by catecholamines was examined indirectly by depleting the catecholamines stores by reserpine treatments of the experimental animals. Reserpine treatment resulted in decreased respiratory activity in liver and brain mitochondria with the two NAD⁺-linked substrates: glutamate and pyruvate+malate with succinate ATP synthesis rate decreased in liver mitochondria only. With ascorbate+TMPD system, the ADP/O ratio and ADP phosphorylation rate decreased in brain mitochondria. For the heart mitochondria, state 3 respiration rates decreased for all substrates. In the liver mitochondria basal ATPase activity decreased by 51%, but in the presence of Mg²⁺ and/or DNP increased significantly. In the brain and heart mitochondria ATPase activities were unchanged. The energy of activation in high temperature range increased liver mitochondrial ATPase while in brain mitochondria reserpine treatment resulted in abolishment in phase transition. Total phospholipid (TPL) content of the brain mitochondria increased by 22%. For the heart mitochondria TPL content decreased by 19% and CHL content decreased by 34%. Tissue specific differential effects were observed for the mitochondrial phospholipid composition. Liver mitochondrial membranes were more fluidized in the reserpine-treated group. The epinephrine and norepinephrine contents in the adrenals decreased by 68 and 77% after reserpine treatment.

Insulin-like growth factor-I (IGF-I) is an important stimulator of collagen biosynthesis and prolidase activity in connective tissue cells. The disturbances in skin collagen metabolism (reflected by significant decrease in skin collagen content, collagen biosynthesis and prolidase activity) in fasted rats were accompanied by decrease in serum IGF-I level. Fasted rat serum was found to contain about 58% of IGF-I (101.6±15.4 ng/ml) as compared to control rat serum (175.7±19.8 ng/ml), while the skin of control and fasted rats contained similar concentrations of IGF-I (about 77 ng/g tissue). The insulin-like growth factor binding proteins (IGFBPs) of sera and tissue extracts (known to regulate IGF-I activity) were analysed by ligand blotting. In the serum of control rats one IGFBP band of about 46 kDa (corresponding to the acid-dissociated IGFBP-3) was detected. In the serum of fasted rats the 46 kDa IGFBP was not observed, however, an other IGFBP of about 30 kDa (corresponding to low molecular weight IGFBPs, e.g. IGFBP-1 or IGFBP-2) was found. The intensity of IGF-I binding to the 30 kDa IGFBP was much higher than that of IGFBP-3, found in control rat serum. Control and fasted rat skin contained similar IGFBPs, however their IGF-I binding abilities were much lower, compared to their serum counterparts. It was found that 46 kDa and 30 kDa proteins, observed in ligand blotting represent IGFBP-3 and IGFBP-1 or IGFBP-2, respectively as demonstrated by western immunoblot analysis. An increase in IGF-binding to 30 kDa IGFBP-1 and/or IGFBP-2 (known as an inhibitors of IGF-dependent functions) in the skin of fasted rats may explain the mechanism of reduced collagen biosynthesis and deposition in tissues during fasting.

The objectives of the present study were to determine the effect of supplementary vitamin-E (200, 400 and 600 mg/kg feed) on lipid peroxidation (LPX) and antioxidant defence system in gills and hepatopancreas of the freshwater prawn, Macrobrachium rosenbergii. Results indicated that vitamin-E inhibited LPX in the hepatopancreas in a comparatively lower dose than gills. Superoxide dismutase (SOD) activity was decreased significantly in gills in response to all the three supplemented diet, but in hepatopancreas decrease was observed only in response to higher doses of vitamin-E (400 and 600 mg/kg feed). Catalase (CAT) activity was reduced significantly only in gills but not in hepatopancreas. While glutathione peroxidase (GPX) activity was significantly elevated in the hepatopancreas by vitamin-E, its activity remains unaltered in gills. On the contrary, glutathione reductase (GR) activity was decreased in gills but that of hepatopancreas was constant. Glutathione (GSH) content of both gills and hepatopancreas was substantially elevated in the vitamin-E supplemented prawns. Although the ascorbic acid (ASA) content of gills was unchanged by vitamin-E, its level elevated significantly in hepatopancreas. Thus the findings of the present investigation suggest that dietary vitamin-E is capable of reducing LPX level and can modulate antioxidant defence system in gills and hepatopancreas, nevertheless, the response is highly tissue specific. It is further observed that highest dose of vitamin-E (600 mg/kg feed) could not render much additional protection in both the tissues.

Gonadal steroids in the salmonid brain, acting through cellular receptors, may be responsible for the modulation of neuronal activity and organization of reproductive behaviors. We report our findings on the use of [³H]17β-estradiol (E₂) to identify intracellular estrogen receptors (ERs) in the hypothalamus of juvenile rainbow trout, Oncorhynchus mykiss. Specific binding (B_SP) of [³H]E₂ was tissue dependent between 0.5 and 2.25 hypothalamus equivalents for cytosol and nuclear extract preparations, respectively. B_SP in cytosol fractions increased with time and reached maximum levels (4.18 nM) at 2.5 h incubation; by contrast, B_SP in nuclear extract increased with time to achieve maximum levels (3.9 nM) by 2 h incubation. The association rate constants (k₊₁) for cytosol and nuclear extract preparations were 1.10±0.02×10⁸ M⁻¹ min⁻¹ and 1.27±0.04×10⁸ M⁻¹ min⁻¹, respectively. Equilibrium bound B_SP dissociated from cytosol preparations with a half life (t_1/2) of 42 min and a dissociation rate constant (k₋₁) of 1.01±0.03 min⁻¹. B_SP dissociated from nuclear extract preparations with a t_1/2=45 min and k₋₁=0.92±0.01 min⁻¹. B_SP was saturable in both extract preparations with a calculated equilibrium dissociation constant (K_d) of 1.46±0.1 nM (cytosol) and 2.37±0.2 nM (nuclear), and a maximum number of binding sites (B_MAX) of 50.85±3.2 fmol mg⁻¹ protein and 61.74±2.65 fmol mg⁻¹ protein, respectively. In both preparations, B_SP was differentially displaced by structurally similar compounds with a rank order of potency of E₂>estrone>estriol>17α-ethynyl estradiol>testosterone≫progesterone=tamoxifen≫cortisol>dexamethasone≫>β-sitosterol. These properties of specifically bound [³H]E₂ suggest the presence of an ER in the hypothalamus of juvenile rainbow trout comparable with ERs identified in salmonid liver.

We measured rates of oxidative metabolism of two tetrachlorobiphenyl (TCB) congeners by hepatic microsomes of two marine mammal species, beluga whale and pilot whale, as related to content of selected cytochrome P450 (CYP) forms. Beluga liver microsomes oxidized 3,3′,4,4′-TCB at rates averaging 21 and 5 pmol/min per mg for males and females, respectively, while pilot whale samples oxidized this congener at 0.3 pmol/min per mg or less. However, rates of 3,3′,4,4′-TCB metabolism correlated with immunodetected CYP1A1 protein content in liver microsomes of both species. The CYPIA inhibitor α-naphthoflavone inhibited 3,3′,4,4′-TCB metabolism by 40% in beluga, supporting a role for a cetacean CYP1A as a catalyst of this activity. Major metabolites of 3,3′,4,4′-TCB generated by beluga liver microsomes were 4-OH-3,3′,4′,5-TCB and 5-OH-3,3′,4,4′-TCB (98% of total), similar to metabolites formed by other species CYP1A1, and suggesting a 4,5-epoxide-TCB intermediate. Liver microsomes of both species metabolized 2,2′,5,5′-TCB at rates of 0.2–1.5 pmol/min per mg. Both species also expressed microsomal proteins cross-reactive with antibodies raised against some mammalian CYP2Bs (rabbit; dog), but not others (rat; scup). Whether CYP2B homologues occur and function in cetaceans is uncertain. This study demonstrates that PCBs are metabolized to aqueous-soluble products by cetacean liver enzymes, and that in beluga, rates of metabolism of 3,3′,4,4′-TCB are substantially greater than those of 2,2′,5,5′-TCB. These directly measured rates generally support the view that PCB metabolism plays a role in shaping the distribution patterns of PCB residues found in cetacean tissue.

The aryl hydrocarbon receptor (AhR) was cloned from the chick embryo and its function and developmental expression characterized. Chicken AhR cDNA coded for 858 amino acid protein and 396 bp of 3′ UTR. The basic helix–loop–helix domain exhibited 87–100% amino acid identity to avian, mammalian, and amphibian AhR, and 69–74% to piscine AhR. The PAS (Per-ARNT-Sim) region was slightly less well conserved with (a) 97% identity to other avian sequences, (b) 81–86% to amphibian and mammalian AhR, and (c) 64–69% with piscine AhR. The carboxy terminus diverged the most among species with less than 53% amino acid identity between chicken and any available mammalian and piscine AhR sequences. The chicken AhR mRNA and protein were 6.1 kb and 103 kDa, respectively. Chicken AhR dimerized with human AhR nuclear translocator and bound the mammalian dioxin-response element in a ligand-dependent manner. AhR protein was detected in neural ganglia; smooth, cardiac, and skeletal muscle; and epithelium involved in epithelial-to-mesenchymal transformations, such as pituitary, gastrointestinal tract, limb apical-ectodermal ridge, and kidney collecting ducts. AhR mRNA was detected in all tissues expressing protein, except myocardium. Cytochrome P4501A4 mRNA was highly induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in a subset of tissues expressing AhR, including small intestine, liver, kidney, blood vessels, and outflow tract myocardium. In conclusion, the AhR sequence and function is highly conserved between birds and mammals, and although many tissues express AhR during chick embryo development, only a subset are responsive to TCDD induction of CYP1A4.