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

Semicarbazide-sensitive amine oxidase (SSAO) (E.C. 1.4.3.6) is a group of enzymes with as yet poorly understood function which is widely present in nature. The variation in methodology for determination of activity, differences in substrates used and in nomenclature have made it difficult to compare SSAO in different species and tissues. Since SSAO is implicated in the pathophysiology of diabetes mellitus and congestive heart failure, our aim was to analyse the importance and abundance of SSAO in human plasma and tissues compared to other mammals. In plasma of ten different mammals, V_max values were found to vary more than 10 000-fold, while K_M differed much less; in human plasma SSAO activity is relatively low. In some species more than one SSAO entity was present in plasma. SSAO activity was ubiquitous in tissues of human, rat and pig, but varied considerably, both between species and between tissues. In human tissues, SSAO activity is higher than in tissues from rat and pig. Relative to monoamine oxidase-B there is also wide variation in SSAO, with much higher relative activities in human than in rat and pig tissues. We conclude that in plasma, SSAO activity is highest in ruminants, while in tissues, SSAO activity is more prominently present in human than in rat and pig.

Acute effects of acephate (Ace) and methamidophos (Met) on acetylcholinesterase activity, endocrine system and amino acid concentrations were studied in rats. The rats were injected intraperitoneally with Ace (500 mg/kg) or Met (5 mg/kg) and then sacrificed at 15 or 60 min after the injection (A15 and A60 for Ace and M15 and M60 for Met). The primary aim of this study was to determine whether the mammalian toxicity of Ace is solely due to its conversion to Met or the protection of Ace against Met-inhibited AChE is also an important factor. The second aim of this study was to study the effects of Ace and Met on the endocrine system and amino acid concentrations and whether or not these effects correlate with AChE inhibition and Met accumulation. The Ace or Met injected animals did not exhibit the signs of organophosphate (OP) poisoning within 15 min after the injection, but exhibited tremors at 45 min after the injection. Blood and brain AChE activity in A15 and M15 rats exhibited 55 to 75% inhibition while the enzyme activity in A60 and M60 rats exhibited 80 to 95% inhibition. Ace was metabolized to Met in rats both in vivo and in vitro. A15 rats had significantly higher Met concentration in their liver, brain and adrenal glands compared to M15 rats, and A60 rats had significantly higher Met concentrations in their blood, liver, brain and adrenal glands compared to M60 rats. Thus, tissue Met concentrations in Ace-treated rats were significantly higher than in Met-treated rats and the inhibition of AChE activity was not consistent with the amount of metabolically formed Met, supporting the hypothesis that the Ace protection plays a role in the overall toxicity. Ace and Met both impaired circulating blood hormone and amino acid concentrations in rats. The endocrine effects of Ace and Met differed from their cholinergic effects, and were not proportional to the amount of Met present in different tissues obtained from the treatment groups. Plasma ACTH concentration was elevated in M60 rats but not in A60 rats. Thus, Ace may indirectly protect the pituitary against the toxic effects of Met. Unlike plasma ACTH levels, serum corticosterone and aldosterone levels were elevated in both A60 and M60 rats. Therefore, the effect of Met on the adrenal cortex may be mediated by the pituitary gland, while the effect of Ace may be due to direct Ace-gland interaction. The decrease in the levels of some of the serum amino acids showed an increase in the energy demands in the treatment groups.

The β₃-selective adrenergic receptor ligand BRL 37344 (BRL) was used to differentiate the presence and functional role of β-adrenergic receptor (βAR) subtypes in pig tissues. BRL did not stimulate adenylyl cyclase in membrane preparations or increase lipolysis from pig adipocytes. In contrast to some species, BRL appears to be a poor agonist for the pig βAR and is not a useful β₃AR ligand. Based on displacement of [³H]dihydroalprenolol binding, BRL exhibited a 100-fold selectivity for pig βAR subtypes in adipose and skeletal muscle membranes. The high affinity site was proposed to be the β₂AR. When used as an antagonist, BRL blockade of the high affinity site did not interfere with isoproterenol-stimulated lipolysis but did inhibit adenylyl cyclase activation. Results indicate that the high affinity βAR (β₂AR) is not linked to lipolysis, possibly due to intracellular compartmentalization. Therefore, βAR subtypes may have function-specific effects.

In order to study temporal and spatial variation of environmental fluoride levels, we analyzed the mandibular bone fluoride content of 157 roe deer (age range: 1–11 years) from two industrialized regions (Ruhr area: n=76, sampling period 1955–1998; area W of Cologne: n=81, sampling period 1983–1998) in the federal state of North Rhine-Westphalia, Germany. Bone fluoride values (dry weight basis) ranged between 150 mg F/kg (2 year-old specimen taken in 1997) and 5724 mg F/kg (10 year-old specimen taken in 1957). In both study areas, a pronounced decline in mandibular bone fluoride concentrations occurred over the respective sampling periods. In consequence, bone fluoride content of animals (both study areas pooled) taken during the period 1990–1998 was significantly (P<0.00001) lower than that of roe deer from the period 1955–1989, while the two animal groups did not significantly differ in age. These findings are regarded as indicative of a considerable reduction of fluoride deposition into the animals’ habitats, due to effective emission control measures. Bone fluoride values for the period 1990–1998 in the roe deer from the Ruhr area significantly (P<0.005) exceeded those of the individuals from the study area W of Cologne, while the difference in age between the two groups was not significant. In both study areas, a significant (P<0.00001) positive correlation between age and mandibular bone fluoride content (Ruhr area: r_s=0.601; area W of Cologne: r_s=0.725) was found for animals taken during this period. The present study underscores the suitability of analyzing skeletal fluoride concentrations in wild roe deer in order to monitor the magnitude of environmental contamination by fluoride and thereby to assess the efficiency of measures taken to reduce fluoride emissions from industrial sources.

Metallothioneins (MTs) are cysteine-rich metal-binding proteins found in micro-organisms, plants and all invertebrate and vertebrate animals. Unicellular eukaryotes such as yeast have a copper-MT whose synthesis is induced by a copper-activated transcription factor. Most higher organisms have two major cadmium/zinc MT isoforms, whose synthesis is controlled by a zinc-activated transcription factor. The blue crab, Callinectes sapidus, has two cadmium-inducible isoforms, CdMT-I and CdMT-II, and a third isoform, CuMT-II, which is induced by copper, but not by cadmium. The cDNA sequence of the copper-specific MT, along with those of the two CdMTs, was determined utilizing 3′ and 5′ rapid amplification of cDNA ends (RACE). CuMT-II cDNA encodes a 63 amino acid protein containing 21 cysteine residues. CdMT-I and CdMT-II cDNA encode a 58 and 57 amino acid protein, respectively, each with 18 cysteines. Molecular phylogeny analysis shows that the CdMT isoforms cluster with other crustacean CdMTs, whereas the copper-specific MT is more closely related to mollusk MTs. CuMT-II shows considerable homology to a copper-specific, non-cadmium inducible, MT from the snail, Helix pomatia. The presence of copper-specific MTs in mollusks and crustaceans, both of which are dependent on hemocyanin for oxygen transport, suggests that CuMT-II is involved in copper homeostasis associated with the synthesis and degradation of hemocyanin.

Aflatoxin B₁ (AFB₁) negatively affects chicken (Gallus domesticus) growth. This effect is more severe during development. We studied the influence of age on the toxic effects of AFB₁ on plasma, renal and hepatic enzymes, under two protocols, in adult and in developing Arbor-Acres chickens. Protocol A: 100 male 4-week-old chickens (640 g), received AFB₁, 0.5, 1.0, or 2.0 μg/g of feed (daily p.o.), a fourth group received an aflatoxin-free diet. Five birds/group were slaughtered at 7, 14, 21 and 28 days of treatment. Body, hepatic and renal weights, succinate-dehydrogenase (SDH) and glutamate-dehydrogenase (GluDH) in plasma and liver were measured. Hepatic SDH and GluDH decreased (P<0.05). Protocol B: two groups of 24 male 1-week-old chickens (106 g) received either aflatoxin-free feed (n=24) or AFB₁ feed (2.0 μg/g). At days 7, 14, 21 and 28, the same parameters of Protocol A were measured. AFB₁ markedly reduced body weight gain (20–30%), plasma proteins, albumin, renal and hepatic protein content (P<0.05) and increased absolute and relative weights of the kidney (P<0.05). SDH and GluDH were reduced (P<0.05), while total renal γ-glutamyltranspeptidase (GGT) increased (P<0.05). Results suggest that serum proteins, SDH and GluDH are sensitive early indicators of this toxicity that was more severe in developing chickens. Decrease in serum albumin might be used as an early and suitable indicator of the deleterious effect of this mycotoxin in developing chickens.

Relative and total amounts of plasma protein fractions are affected by infections, inflammation, and nutritional and physiological status, and are therefore important health indicators in free-living animals. Our objectives were: (1) to examine intercolony differences in plasma protein fractions in prefledgling gulls and terns; (2) to investigate relationships between plasma proteins and other physiological measures such as weight loss, growth, and immune function; and (3) to examine potential associations between organochlorine exposure and plasma proteins. During 1992, blood was collected from 3-week-old herring gull (Larus argentatus) chicks from six sites on Lakes Superior, Huron, Michigan, Erie, and Winnipeg and from 3-week-old Caspian tern (Sterna caspia) chicks from five sites on Lakes Huron, Michigan, and Ontario. These sites provided a wide gradient of organochlorine contamination. Plasma proteins were separated by high-resolution agarose gel electrophoresis and stained with Coomassie brilliant blue dye. Six major fractions were quantified: prealbumin, albumin, α-globulins, β₁-globulins, β₂-globulins, and γ-globulins. Total protein, prealbumin, albumin, and γ-globulin concentrations and the albumin/globulin ratio did not differ among sites. Total protein, albumin, and the albumin/globulin ratio were not decreased in birds experiencing food stress or weight loss. Intersite differences were found in α- and β-globulins. In gulls, β₂-globulins were positively associated with polychlorinated biphenyls (PCBs) and 1,1-dichloro-2,2-bis(p-chlorophenyl)ether (DDE). In terns, PCBs were negatively associated with α-globulins and positively associated with β₁-globulins. Additional research is needed to identify individual proteins and elucidate causal relationships between the particular protein concentrations and factors such as contaminants, growth, and condition.

In order to estimate the potential use of the mean wholebody acetylcholinesterase (AChE) activity from the ragworm Nereis diversicolor for the biological assessment of pollution by anticholinesterase agents in estuarine areas, we measured the effects of the main abiotic factors (i.e. temperature and salinity) on AChE activity. We report here that AChE activity tends to decrease in individuals sampled in tanks at a salinity of 30‰ as temperature increases. No tendencies in the evolution of AChE activity were observed in individuals sampled in tanks at a salinity of 15‰. In contrast, salinity seems to have a greater effect on AChE activity than temperature. At a temperature of 12°C, a salinity of 30‰ provokes a significant transient increase of AChE 2 days after the beginning of the maintenance period compared with a salinity of 15‰. The effects are short-term stress effects. We noticed only a transient increase of AChE activity between 2 days for individuals maintained in tanks at temperature of 20°C and salinity of 15 and 30‰, respectively, and 8 days for individuals maintained in tanks at salinity of 30‰ and at a temperature of 12°C after the beginning of the maintenance period, confirming the more pronounced effect of salinity over temperature.

The feasibility of a short-term ammonia toxicity test as an evaluation criterion for larval quality was assessed in three trials. In each one, Macrobrachium rosenbergii larvae originating from the same spawn were nutritionally differentiated in two groups by feeding them either a nutrient-rich (Artemia nauplii enriched for 24 h with n-3 highly unsaturated fatty acids (HUFA) and ascorbic acid (AA)) or a nutrient-poor diet (Artemia nauplii starved for 24 h). Throughout their development, larvae from both treatments were exposed during 24 h to six concentrations of total ammonia (NH₄⁺+NH₃) and a control (no ammonia added). Based on mortality rates, the median lethal concentration for 50% of the population (LC₅₀) was estimated. As expected from earlier work, larvae fed the optimal diet presented higher n-3 HUFA and AA contents as well as higher growth and metamorphosis rates. From the moment the effect of diet quality was analytically detectable in the tissues of the larvae, the ammonia test was able to distinguish both groups of larvae. Differences in ammonia tolerance were observed as early as larval stage 4 and remained evident throughout larval development. The short-term ammonia toxicity test proved to be a valuable, sensitive and reproducible criterion for the establishment of larval quality.

The antibacterial properties and cDNA sequences of two types of antibacterial peptides from the haemolymph of immunized common cutworm, Spodoptera litura larvae, were determined. Since the primary structures of peptides deduced from cDNA sequences showed significant homologies to cecropins A and B, they were named as Spodoptera cecropins A and B. Spodoptera cecropins were broadly effective against Gram-positive and negative bacteria. They also retained antibacterial activities in all conditions tested (at pH 5.6–8.0 and in the presence of 50–150 mM NaCl) that was adapted to confirm the antibacterial properties of Spodoptera cecropins. These results indicate that the change of pH and the increase of salt concentration in the media do not influence the activities of Spodoptera cecropins. For the reverse transcription (RT)-polymerase chain reaction (PCR) to obtain the complete primary sequence, the primer designed according to the conserved region of the cecropin leader sequences was used, which was determined by the comparison of the cDNA sequences of known cecropins. The results from RT-PCR presented that the partial cDNAs of Spodoptera cecropins A and B encode 57 and 58 amino acids, including the sequences of mature peptides, respectively. In addition, Northern blot analysis with ³²P-labeled PCR product coding for Spodoptera cecropin A revealed that Spodoptera cecropin genes are expressed in immunized fat body, but not in normal fat body.