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

The effect of anticonvulsants on the burst firing of action potentials in snail central neuron elicited by d-amphetamine was studied in the identified RP4 neuron of the African snail, Achatina fulica Ferussac. Oscillation of membrane potential and burst firing of action potentials were elicited by d-amphetamine in a concentration-dependent manner. Voltage clamped studies revealed that d-amphetamine elicited a negative slope resistance (NSR) in steady-state I–V curve between −40 and −10 mV. The burst firing of action potentials was alleviated following extracellular application of phenytoin, but was not affected after ethosuximide, carbamazepine, and valproic acid. The NSR elicited by d-amphetamine was blocked by phenytoin. However, the NSR was not altered if carbamazepine was added. These results suggest that of the four anticonvulsants tested, only phenytoin could alleviate the burst firing of action potentials elicited by d-amphetamine in snail neuron.

Atrazine (1000 ppm), endosulfan (1 ppm) or butylated hydroxyanisole (BHA) (1000 ppm) added to a semi-synthetic diet of Orthosia gothica for 2 days in the last instar did not change the soft tissue cytosolic glutathione-S-transferase (GST) activities towards 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB) and cumene hydroperoxide (CU). However, all three pesticides changed the GST subunit composition compared with the control as observed by reverse phase high performance liquid chromatography of the isozymes purified by glutathione–Sepharose affinity chromatography. The changes seem to have occurred mainly in the GST class 2 subunit. There is no obvious explanation for the changes, which may be a result of interactions between xenobiotic and GST in the cytosol as well as changes in the level of regulation of synthesis. However, the observation added to our knowledge of the processes involved when pesticides are degraded by GSTs in vivo.

Kokanee salmon (Oncorhynchus nerka kennerlyi), a landlocked subspecies of sockeye salmon, exhibited hypothalamic–pituitary–interrenal (HPI, adrenal homologue) axis activation and an increase in plasma cortisol concentration up to 639±55.9 ng/ml in association with upstream migration in the upper Colorado River even though they were not exposed to a change in salinity and lengthy migration. Kokanee salmon were collected at various stages of migration and concomitant sexual maturation. The pattern of cortisol elevation in kokanee is similar to that in ocean-run sockeye salmon (O. nerka nerka). The presence of plasma cortisol elevation in an upstream migrating, landlocked Pacific salmon suggests that stressors previously considered to cause the cortisol increase, such as long-distance migration and changes in salinity, may not be primary causes of the HPI axis activation.

Investigations were carried out to determine the role of juvenile hormone (JH) and 20-hydroxy ecdysone in the synthesis and uptake of vitellogenins, which were earlier identified, purified and characterised, in Dysdercus koenigii. The concentration(s) of vitellogenin(s) in fat body, haemolymph and that of vitellin(s) in ovary were significantly lower after chemical allatectomy at eclosion. In addition, at 70 h after emergence, chemical allatectomy reduced ovarian vitellin concentration, but vitellogenin levels remained normal in the fat body and haemolymph. The haemolymph vitellogenins were not incorporated into oocytes in such insects. Administration of JH-III at 20 h after allatectomy restored vitellogenin levels in the fat body and haemolymph, but the ovary failed to incorporate the available vitellogenins from haemolymph in such insects. However, when JH-III was administered twice, one at 20 h and then at 70 h after allatectomy, vitellogenin concentrations in fat body and haemolymph and also vitellin concentrations in ovary approached control levels. It is suggested that JH has two separate roles, one in vitellogenin synthesis and the other in uptake. 20-hydroxy ecdysone had no apparent role in either vitellogenin synthesis or uptake in D. koenigii.

This work aimed to validate the relationship between metallothioneins (MTs) and metals (Cd, Cu and Zn) in field conditions. Specimens of the marine bivalve Ruditapes decussatus (Linné, 1758) from Gargour were transferred in two sites: Gargour and Sidi Mansour, both situated along the south-eastern coast of Tunisia. The bivalves were removed from pairs of cages at day 0 (date of transplantation), day 62 and day 132. Metals (Cd, Cu and Zn) and MTs were determined in the subcellular fractions of the digestive gland. In Gargour, metal and MT levels increased significantly after 62 days of transplantation. However, they showed modest and non-significant variations in Sidi Mansour. Zn was mainly associated with the insoluble fraction, whereas Cd and Cu percentages in the soluble and the insoluble fractions were equivalent. Simple correlation analysis showed a positive and significant relationship between MTs and each metal. If all metals were taken together, multiple correlations showed that MTs were significantly correlated with Cd and Zn, with an important coefficient for Cd, but no significant relationship was observed for Cu. Gel filtration chromatography showed that in the heat stable fraction, the only cytosolic SH rich compounds have an apparent low molecular mass (about 15 kDa), which could correspond to metallothioneins. In the digestive gland of R. decussatus MTs responded to moderate increases of metal contamination, without interference with other factors, and could be a promising biochemical indicator of metal exposure.

The effects of diet and other non-anthropogenic stressors on biochemical defenses and their relationship to susceptibility have been largely ignored in wildlife populations. Lanosol is a compound found in relatively high amounts in various marine species of Rhodophyta, including Odonthalia dentata. While previous studies demonstrated that lanosol is a feeding deterrent to several marine herbivores, Cryptochiton stelleri readily feeds upon O. dentata. To examine the effects of lanosol on the profile of biochemical defenses in C. stelleri, chitons were gavaged daily with 0, 1, 2.5, 5, or 10 mg/kg of lanosol. After three days of exposure, digestive gland microsomes were probed for expression of homologous isoforms of cytochromes P450 (CYP1A, CYP3A, and CYP2) and phase II enzymatic activities. Expression of a 43 kDa CYP3A-like protein was increased by approximately 45% over control following 2.5, 5, and 10 mg/kg treatments. Estradiol hydroxylase activity tended to increase with the dose of lanosol. UDP-glucuronosyl transferase activity was highly variable but appeared to increase at the two highest treatments, while sulfotranserase activity was significantly decreased at the three highest doses. Kinetic studies of GST activity showed lanosol is a non-competitive inhibitor of both CDNB and GSH in the GST-mediated conjugation reaction. These results show that dietary exposure to the brominated-phenol, lanosol, may alter expression and activity of some phase I and II biotransformation enzymes in chitons, potentially providing a dietary advantage for the species.

We cloned three novel cytochrome P450 (CYP) 2D cDNAs in the Syrian hamster (Mesocricetus auratus). Each clone contained an open reading frame of 1500 nucleotides encoding a protein of 500 amino acids. The deduced amino acid sequences of these had high identities with those of the other CYP2D members, therefore, the clones were assigned as CYP2D20, CYP2D27, and CYP2D28. Northern blot analysis showed that the CYP2D27 mRNA was expressed in liver, but not in kidney, small intestine, and brain, while the CYP2D20 and CYP2D28 mRNAs were not detected in these tissues examined. The expression of CYP2D27 mRNA in liver did not show sex difference and was not induced by either 3-methylcholanthrene or phenobarbital treatment. We characterized the enzyme activities of recombinant CYP2D27 expressed in COS-7 cells. The CYP2D27 protein had the bufuralol 1′-hydroxylase and debrisoquine 4-hydroxylase activities that are specific to the CYP2D subfamily.

The plasma membrane/mitochondrial fractions of Penaeus indicus postlarvae contain Mg²⁺-dependent ATPase, Na⁺,K⁺-stimulated ATPase, Na⁺-stimulated ATPase and K⁺-stimulated ATPase. The Na⁺,K⁺-activated, Mg²⁺-dependent ATPase was investigated further in relation to different pH and temperature conditions, and at various concentrations of protein, ouabain, ATP and ions in the incubation medium. In vitro and in vivo effects of lead were studied on the enzyme activity. In vitro lead inhibited the enzyme activity in a concentration-dependent manner with an IC₅₀ value of 204.4 μM. In correlation with in vitro studies, in vivo investigations (both concentration and time dependent) of lead also indicated a gradual inhibition in enzyme activity. A maximum decrease of 85.3% was observed at LC₅₀ (7.2 ppm) of lead for concentration-dependent experiments. In time-dependent studies, the decrease was maximal (81.7%) at 30 days of sublethal exposure (1.44 ppm). In addition, the substrate- and ion-dependent kinetics of Na⁺,K⁺-ATPase was studied in relation to in vitro exposure of lead; these studies suggest a non-competitive type of inhibition.

Ethoxyquin (6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline (EQ) is a synthetic antioxidant used for preventing rancidity in animal foodstuffs. Three groups of ten fish were given a diet containing respectively 75 (control group with the commercial food), 200 and 400 ppm EQ for 16 days. The control group had a plasma osmolality and chloride concentration within the normal range of marine teleosts, but sodium concentrations of only about 110 mM, indicating the presence in the plasma of substantial amounts of another cation. Fish given food with 400 ppm EQ displayed a 70 mM increase in the plasma concentration of sodium. This indicates that EQ has disturbed the iono-regulatory mechanisms, probably by reducing the ATP production or inhibiting directly the Na/K-ATPase in the gills. The large increase in plasma sodium concentration was not accompanied by any significant increase in plasma osmolality, indicating that at least a part of the sodium added to the plasma is made osmotically inactive. In spite of the elevated plasma sodium concentration, the sodium content of erythrocytes of the 400-ppm EQ fish was reduced to half, while the content of calcium was unaffected. The transmembrane energy gradient of sodium in the EQ exposed turbot obviously increased, allowing them to use a sodium coupled antiport system to keep the cellular calcium content low when the Ca-ATPases blocked. A mechanism of this kind is also likely to be important to turbot that experience hypoxia under natural conditions. The 400-ppm group also displayed a substantial increase in liver weight, but the physiological significance of this effect is not clear. The leucocyte counts indicated the absence of obvious immunological effects.

The venom of eight individual Crotalus durissus terrificus snakes from the State of Minas Gerais, Brazil, in addition to pooled venom from Butantan Institute, were compared. Snakes were captured in distinct locations, some of them 600 km apart: Conselheiro Lafaiete, Entre Rios de Minas, Itauna, Itapecerica, Lavras, Patos de Minas, Paracatu, and Santo Antonio do Amparo. The crude venoms were tested for proteolytic, phospholipase A2, platelet aggregating, and hemagglutinating activities. The venoms were also analyzed by polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IEF). Chromatographic patterns of venom proteins on both gel-filtration and anion-exchange chromatographies were also performed. All venoms presented high phospholipase A2 and platelet-aggregating activities, but only minimal hemagglutinating or proteolytic activities were found. Gel-filtration chromatography showed a characteristic profile for most venoms where four main peaks were separated, including the typical ones where convulxin and crotoxin were identified; however, peaks with high amounts of lower molecular weight proteins were found in the venoms from the Santo Antonio do Amparo location and Butantan Institute, characterizing these venoms as crotamine positive. Anion-exchange chromatographies presented a similar protein distribution pattern, although the number of peaks (up to ten) distinguished some venom samples. Consistent with these results, polyacrylamide gels that were silver stained after venom separation by PAGE or IEF presented a similar qualitative band distribution, although a quantitative heterogeneity was detected among venoms. Our results suggest that the variability found in venom components of C. d. terrificus venoms captured in Minas Gerais State may be genetically inherited and/or environmentally induced.