Cd, Cu, Zn, Se, and Metallothioneins in Two Amphibians, Necturus maculosus (Amphibia, Caudata) and Bufo bufo (Amphibia, Anura)

The accumulation of cadmium, its affinity for metallothioneins (MTs), and its relation to copper, zinc, and selenium were investigated in the experimental mudpuppy Necturus maculosus and the common toad Bufo bufo captured in nature. Specimens of N. maculosus were exposed to waterborne Cd (85???g/L) for up to 40?days. Exposure resulted in tissue-dependent accumulation of Cd in the order kidney, gills > intestine, liver, brain > pancreas, skin, spleen, and gonads. During the 40-day exposure, concentrations increased close to 1???g/g in kidneys and gills (0.64?C0.95 and 0.52?C0.76; n?=?4), whereas the levels stayed below 0.5 in liver (0.14?C0.29; n?=?4) and other organs. Cd exposure was accompanied by an increase of Zn and Cu in kidneys and Zn in skin, while a decrease of Cu was observed in muscles and skin. Cytosol metallothioneins (MTs) were detected as Cu,Zn?Cthioneins in liver and Zn,Cu?Cthioneins in gills and kidney, with the presence of Se in all cases. After exposure, Cd binding to MTs was clearly observed in cytosol of gills as Zn,Cu,Cd?Cthionein and in pellet extract of kidneys as Zn,Cu,Cd?Cthioneins. The results indicate low Cd storage in liver with almost undetectable Cd in liver MT fractions. In field trapped Bufo bufo (spring and autumn animals), Cd levels were followed in four organs and found to be in the order kidney > liver (0.56?C5.0???g/g >0.03?C0.72???g/g; n?=?11, spring and autumn animals), with no detectable Cd in muscle and skin. At the tissue level, high positive correlations between Cd, Cu, and Se were found in liver (all r?>?0.80; ???=?0.05, n?=?5), and between Cd and Se in kidney (r?=?0.76; n?=?5) of autumn animals, possibly connected with the storage of excess elements in biologically inert forms. In the liver of spring animals, having higher tissue level of Cd than autumn ones, part of the Cd was identified as Cu,Zn,Cd?Cthioneins with traces of Se. As both species are special in having liver Cu levels higher than Zn, the observed highly preferential Cd load in kidney seems reasonable. The relatively low Cd found in liver can be attributed to its excretion through bile and its inability to displace Cu from MTs. The associations of selenium observed with Cd and/or Cu (on the tissue and cell level) point to selenium involvement in the detoxification of excessive cadmium and copper through immobilization.     

Age-dependent in situ hepatic and gill CYP1A activity in the see-through medaka (Oryzias latipes)

We used a recently introduced strain of medaka, the see-through medaka, whose internal organs can be seen through the skin, to develop an in situ toxicity assay of ethoxyresorufin-O-deethylase (EROD) activity that detected fluorescence from resorufin, a metabolite of ethoxyresorufin and thus an indicator of CYP1A activity. EROD activity in the liver and gills of 2-week post-hatch see-through medaka exposed simultaneously to various concentrations of 3-methylcholanthrene and 200 microg/L ethoxyresorufin for 24 h was proportional to the 3-methylcholanthrene dose. Activities in the liver and gills peaked at 40 microg/L of 3-methylcholanthrene and then decreased at higher doses, possibly because of 3-methylcholanthrene toxicity. At 1-week post-hatch stage, however, constant high EROD activity was observed in controls and at all 3-methylcholanthrene doses. Four-week post-hatch see-through medaka exhibited less EROD activity than 2-week post-hatch see-through medaka, and activity in the liver peaked at 100 microg/L of 3-methylcholanthrene. Adult see-through medaka were not suitable for fluorescence detection owing to their thick skin, muscle and/or tissue. In tests of oxidative activity response to ethoxyresorufin, 1-day and 1-week post-hatch see-through medaka exhibited high intrinsic EROD activity in the liver, gills, and other organs in the absence of 3-methylcholanthrene. This intrinsic activity declined with growth and explained the high constant EROD activity at 1-week post-hatch stage.     

Application of different test systems and biochemical indicators for environmental monitoring of the Troitsa Bay,Sea of Japan

The DNase express test system and the SF-bioassay (sea urchin sperm fertilization bioassay) were used to assess the quality of sea and bottom sediment water sampled at 8 stations of the Troitsa Bay (Possyet Bay, Sea of Japan). The specific activities of 8 enzymes (alkaline and acid DNases, RNases, phosphatases and phosphodiesterases) were determined for the liver and gill tissues of the mussel Crenomytilus grayanus sampled at the same stations. The activities of all enzymes were higher in the liver than in the gills, and the activity of acid nucleases in both tissues was significantly higher than that of alkaline nucleases. The specific activities of some nucleases in the mussel tissues correlated to the level of seawater pollution. The activities of acid RNases and phosphodiesterases decreased in both tissues upon an increase in water pollution, up to 1.5–3 times in the gills, while the activities of alkaline and acid phosphatases and phosphodiesterases increased 1.5–4 times. Thus, the specific activities of acid RNases, phosphatases and phosphodiesterases from mussel liver can be a useful indicator of marine pollution. This approach can be used for biotesting of ocean water and for assessment of the biological condition of invertebrates adapting to various ecological and anthropogenic effects.     

Studies on the toxicity of lindane on Colisa fasciatus (part I: TLm measurements and histopathological changes in certain tissues).

1. In the present investigation static bioassay studies and histopathological changes in some tissues (gills, liver and kidney) induced by Lindane 20% E.C. in the fish Colisa fasciatus have been taken under consideration. 2. With the help of bioassay studies, the TLm were noted. The TLm values were 0.87, 0.78, 0.68 and 0.64 mg/l for the temperature range 17 degrees C to 20 degrees C and 0.60, 0.56, 0.46 and 0.41 mg/l for the temperature range 32 degrees C to 35 degrees C at 12, 24, 48 and 96 h respectively. 3. The statistical analysis of different factors such as concentrations, time and temperature range based on the observed data gave the following results: a) At both temperature ranges, survival rates at different concentrations and time are significant at 1% and 5% level. b) The difference in survival rates at different temperature range are insignificant both at 1% and 5pected survival numbers. The standard errors were also calculated for these two lines which gave the insignificant results and there was no much change in the shape of experimental and expected lines. 4. The histopathological changes in the tissues (gills, liver and kidney) shows the following results: a) In the case of the gills there was a loss of different types of cells i.e. respiratory cells and blood cells. Blood vessels were atrophied and the outer membrane of gill lamellae was ruptured. The erosion of the tips of the gill filaments were also observed. b) In the case of liver the outer membrane was ruptured. Some hepatic cells were vacuolated and some completely degenerated. The central areas were much affected. Due to the toxic effect, large splitting of tissues were found inside the liver. c) In the case of kidney the outer epithelium, the parenchymatous cells of renal tubules, cuboidal cells of uriniferous tubules were much affected. Due to the degeneration of the cells large spaces were formed inside the tissue. Much affection was observed in the central areas.     

Comparison of dietary and waterborne exposure to benzo a pyrene: bioavailability,tissue disposition and CYP1A1 induction in rainbow trout Oncorhynchus mykiss

Absorption and tissue distribution of benzo\[ a ]pyrene (BaP)-derived radioactivity were studied in juvenile rainbow trout following dietary or waterborne exposure. In order to compare the bioavailability of BaP, the fish were exposed to 1.5 mCi 3H-BaP kg-1 fish, either in the diet or in the water as a 2 days static exposure. Furthermore, tissue levels of BaP-derived radioactivity bound to macromolecules in different tissues were studied in non-induced fish, and in fish induced by additional treatment with unlabelled BaP (corresponding to 5 mg kg-1 fish) in the water. Absorption and tissue distribution of 3H BaP were studied by liquid scintillation counting and whole-body autoradiography. BaPderived radioactivity bound to macromolecules in different tissues was studied by autoradiography of solvent-extracted whole-body sections. The hepatic CYP1A induction was measured as EROD activity. Exposure to unlabelled BaP resulted in a marked induction of hepatic EROD activity in rainbow trout 2 days after the start of the exposure. Significant higher concentrations of radiolabelled compound were observed in waterborne-exposed fish, in contrast to dietary-exposed fish. High concentrations of radiolabelling were observed in the gills, liver, bile, intestines, olfactory organ, kidney and the skin of the waterborne-exposed fish. In the dietary-exposed fish, high levels of radioactivity were observed in the intestines and the bile, whereas lower concentrations were present in the liver. Only traces of radioactive compound were observed in the gills. In contrast to waterborne-exposed fish, no radioactivity was detected in the olfactory organ or skin. In autoradiograms of sections extracted with a series of polar and non-polar solvents, a large fraction of radioactivity was still present in the gills, olfactory organ, liver, kidney, skin and intestinal mucosa of the waterborne-exposed fish, indicating that reactive BaP intermediates formed by CYP1A-mediated metabolism were bound to macromolecules in these tissues.     

Disposition and cellular binding of 3H-benzo(a)pyrene at subzero temperatures: studies in an aglomerular arctic teleost fish – the polar cod (Boreogadus saida)

Autoradiography at different levels of resolution was used to study the disposition of the polycyclic aromatic hydrocarbon benzo(a)pyrene (³H-BaP) in juvenile and sexually mature polar cod (Boreogadus saida). Exposure took place via the water or after intragastric administration at subzero temperatures. In water-exposed fish, high total tissue levels were found in the gills, olfactory organ, anterior kidney, liver, skin and intestinal wall. Only traces of radioactivity were present in the muscle, brain and gonads. No major differences in tissue levels or in general distribution pattern between males, females or juvenile fish were observed. The gills appeared to be the absorption site for exposure via water. After oral administration, tissue levels of ³H-BaP-derived radioactivity were negligible. Following both administration routes, levels of radioactivity were highest in the bile and intestinal contents while only traces were observed in the urine, indicating biliary excretion as the major excretory pathway in this aglomerular species. Tape-section autoradiography of fish exposed via water revealed tissue-bound residues of ³H-BaP in the olfactory organs, gills, kidney, liver, skin and intestinal mucosa. Light-microscopy autoradiography demonstrated that the bound residues in the olfactory organ, gills and anterior kidney were localized in epithelial cells, while those in liver and intestinal mucosa were evenly distributed. In conclusion, the present study shows that BaP is absorbed from the water via the gills at subzero temperatures, that tissue levels are considerably higher after water exposure than after dietary exposure, that biliary excretion is predominant and, finally, that site-specific tissue binding in the olfactory organs, gills and anterior kidney is confined to epithelial cells. Accepted: 3 January 2000     

Mitochondria-rich cells in gills and skin of an African lungfish, Protopterus annectens

We used scanning electron microscopy, the vital dye DASPEI and an antibody to the inner mitochondrial membrane to study the presence and localisation of mitochondria-rich cells in the gills and skin (opercular, dorsal and ventral) of the lungfish Protopterus annectens in its free-swimming conditions and at the beginning of aestivation. In the free-swimming period, the gills were short and thick and the pavement cells were extremely large (30-40 microns). The mitochondria-rich cells, which were distributed in the secondary and primary epithelium, occurred as two morphologically different types, i.e. elongated and oval, similar to the alpha and beta chloride cells of fresh water teleosts. In the skin, only one type of mitochondria-rich cells was found, resembling the alpha chloride cells. All the mitochondria-rich cells distributed in the gills and skin were labelled with anti Ca(2+)-ATPase serum indicating the possible uptake of Ca2+ at freshwater chloride cell level. At the start of aestivation, the skin and gills were covered by a thick layer of mucus and the epithelium of the gills was reduced. The mitochondria-rich cells were almost completely covered by the pavement cells.     

Dionchus postoncomiracidia (Monogenea: Dionchidae) from the skin of blacktip sharks, Carcharhinus limbatus (Carcharhinidae)

Postoncomiracidia of Dionchus sp. are described from specimens collected from the skin of 2 blacktip sharks Carcharhinus limbatus, captured in the northern Gulf of Mexico. The parasites resemble nonciliated oncomiracidia hatched from eggs laid by Dionchus sp. on gills of a cobia Rachycentron canadum and adults of Dionchus remorae that were collected from gills of a common sharksucker Echeneis naucrates, captured in association with a third blacktip shark. The hamuli of the postoncomiracidia were morphologically similar to those of adult D. remorae. This is the first report of dionchids from an elasmobranch and from a location other than the gills. These findings support the idea that some dionchid oncomiracidia colonize the skin of sharks or other aquatic vertebrates that sponsor remoras, prior to transferring to other remoras and maturing.     

Health and chemical burdens of fish species from polluted and hyper-eutrophic freshwater ecosystems in South Africa

Three aquatic ecosystems in South Africa, the Hartbeespoort, Klipvoor and Bospoort Dams, are classified as hyper-eutrophic, because of high nutrient loads and chemical pollution. Water and two fish species, Clarias gariepinus and Cyprinus carpio, were collected from these dams to assess the impact of eutrophication and chemical pollutants on their health status. Water and muscle samples were analysed for organic and inorganic chemicals. Condition factor was determined and a necropsy performed to note any macroscopic abnormalities. A histology-based fish health assessment was done on the liver, kidney, gills and gonads. A number of fish from the three dams exhibited livers with fatty change and focal discoloration, skin lesions and parasites within the visceral cavity. The prevalence and severity of histopathology in the liver resulted in higher liver index values than the index values for kidneys and gills. Aluminium, silicon and chromium were detected in the water and muscle tissue. The DDT metabolite p,p’-DDE was present in both species, as well as in fish from the reference site, Marico-Bosveld Dam. Only C. gariepinus from Hartbeespoort Dam had p,p’-DDD levels higher than 5 µg g^?1 per edible portion. Water from hyper-eutrophic dams adversely affects the health of freshwater fish.     

Carcinogenicity and mutagenicity testing, then and now.

Cancer is a dread disease worldwide. Mortality of individuals suffering from cancer is high, despite the current improved methods of precocious detection, surgery and therapy. Prevention of cancer is the recognized goal of many activities in cancer research. This aim was recognized early to involve the bioassay of environmental chemicals or mixtures. The first such study involved application of coal tar to the ear of rabbits, and later on to the skin of mice. Subsequently, laboratory rats were introduced, and hamsters were utilized as a substitute for the unwieldy tests in rabbits. Investigators also became concerned with the mechanisms of carcinogenesis, and more definitive approaches to carcinogen bioassay in laboratory animals, as possible indicators of cancer risk in humans. These tests were expensive and lengthy, and did not serve the important purpose of accurately measuring risk of cancer to humans. Once it was realized that DNA and the genetic apparatus might be a key target, rapid bioassays in bacterial and mammalian cell systems were introduced successfully. Thus, batteries of tests are now available to detect effectively human cancer risks, and provide novel approaches to determine the underlying mechanisms, as a sound basis for cancer prevention.     

爪鲵呼吸器官的胚后发育

应用石蜡切片和扫描电镜技术,对爪鲵(Onychodactylus fischeri)幼体、亚成体和成体3个不同发育阶段的皮肤、外鳃、咽等呼吸器官进行了显微观察与比较分析,旨在揭示爪鲵不同发育阶段各呼吸器官的演化规律.结果表明,爪鲵的皮肤随年龄的增长而逐渐增厚,幼体阶段其背腹皮肤厚度相差不大,亚成体及成体背部皮肤明显厚于腹部;外鳃是幼体和亚成体爪鲵呼吸器官的重要组成部分,随着发育外鳃逐渐完善,到亚成体阶段达到顶峰,随后逐步退化;咽部是爪鲵的重要呼吸器官,幼体期口腔和咽发育不完善,亚成体咽部逐渐发育,至成体时发育完善.     

Daily changes in parameters of energy metabolism in liver, white muscle, and gills of rainbow trout: dependence on feeding

We assessed the daily patterns of parameters involved in energy metabolism in liver, white muscle, and gills of rainbow trout. Where daily rhythms were found, we analyzed the potential influence of feeding. Immature rainbow trout were randomly distributed in 3 groups: fish fed for 7 days, fish fasted for 7 days, and fish fasted for 7 days and refed for 4 days. On sampling day, fish of fed and refed groups were fed at 11.00 h, and all fish were sampled from each treatment group using the following time schedule: 14.00, 18.00, 21.00, 00.00, 04.00, 07.00, 10.00 and 14.00 h. The results obtained from metabolic parameters can be grouped into four different categories, such as i) those displaying no daily changes in any group assessed in liver (acetoacetate and lactate levels), white muscle (protein levels, and low Km (glucose) hexokinase (HK) and HK-IV activities) and gills (protein levels), ii) those displaying no 24 h changes in fed fish but in refed or fasted fish in liver (glucose, glycogen, amino acid and protein levels, and HK-IV activity), white muscle (glycogen and amino acid levels) and gills (glucose levels), iii) those displaying 24 h changes that were apparently dependent on feeding since they disappear in fasted fish in liver (Low Km (glucose) HK, lactate dehydrogenase (LDH-O), glucose 6-phosphatase (G6Pase), fructose 1,6-bisphosphatase (FBPase) , alpha-glycerophosphate dehydrogenase (G3PDH), glutamate dehydrogenase (GDH) and aspartate aminotransferase (Asp-AT) activities), white muscle (glucose levels, and pyruvate kinase (PK), LDH-O, G3PDH and Asp-AT activities) and gills (glycogen and lactate levels, and Low Km (glucose) HK, HK-IV, LDH-O and Asp-AT activities), and iv) those parameters displaying 24 h changes apparently not dependent on feeding in liver (lactate levels and PK activity) and gills (amino acid levels, and PK and GDH activities). In general, most 24 h changes observed were dependent on feeding and can be also related to daily changes in activity.     

Unit responses in the vagal lobe of the carp brain to stimulation of chemoreceptors of the skin,mouth, and gills

Experiments on curarized carp showed that neurons of the vagal lobe of the medulla respond to stimulation not only of the mouth and gills, but also of chemoreceptors of the skin of the head. The vagal lobe was shown to contain considerably fewer neurons responding selectively to stimulation of the skin than neurons responding to stimulation of the mouth or gills. Differences were found between responses of neurons to a natural food stimulus (extract from a fish food product) and to solutions of hydrochloric acid and common salt. The different roles of the vagal and facial lobes in the processing of impulsation from skin chemoreceptors is demonstrated.A. A. Zhdanov Leningrad State University. Translated from Neirofiziologiya, Vol. 12, No. 4, pp. 397–404, July–August, 1980.     

CO2‐metabolism in early tetrapods revisited: inferences from osteological correlates of gills,skin and lung ventilation in the fossil record

One of the most important physiological changes during the conquest of land by vertebrates was the increasing reliance on lung breathing, with the concomitant decrease in importance of gill breathing. The main problem involved here was to cope with the excessive accumulation of CO₂ in the body and to avoid respiratory acidosis. In the past, several often mutually contradicting hypotheses of CO₂‐elimination via skin, lungs and gills in early tetrapods have been proposed, based on theoretical physiological considerations and comparison with extant air‐breathing fishes and amphibians. This study proposes a revised scenario of CO₂‐elimination in early tetrapods based on fossil evidence, that is recently identified osteological correlates of gills, skin structure and mode of lung ventilation. In stem tetrapods of the Devonian and Carboniferous, O₂‐uptake via the lungs by buccal pumping was decoupled from CO₂‐release via internal gills, and the rather gas‐impermeable skin played a minor role in gaseous exchange. The two main lineages of crown‐group tetrapods, the amphibian and amniote lineage, used different strategies of CO₂‐elimination. As in stem tetrapods, O₂‐uptake and CO₂‐release remained always largely decoupled in temnospondyls, which ventilated their lungs via buccal pumping and relied mainly on their internal gills for CO₂‐release. Temnospondyls were not able to reduce their internal gills before their skin became more gas permeable and their body size was reduced, to shift from internal gills to the skin as the major site of CO₂‐elimination, a pattern that is retained in most lissamphibians. In contrast, internal gills were lost very early in stem amniote evolution. This was associated with the evolution of the more effective aspiration pump that allowed the elimination of the bulk of CO₂ via the lungs, leading to a coupled O₂‐uptake and CO₂‐loss in stem amniotes and later in amniotes.     

X-cells in the gills of an Antarctic teleost, Pagothenia borchgrevinki

X-cell tumours have been described previously from teleost fish of the Northern Hemisphere, in which they occurred as lesions of the skin, pseudobranchs or gills. The present study describes X-cell tumours from the gills of an Antarctic teleost, Pagothenia borchgrevinki , thus extending the range to the Antarctic and also the Southern Hemisphere. Gills of affected fish were distinctly swollen and white in appearance, indicating that the gills were not functional as gas exchange organs. The affected gill tissue contained large numbers of X-cells, large spherical cells with a distinct extracellular coat and many densely staining membrane-bound granules. The possible origin of the cells is discussed.     

Routes of entry of Piscirickettsia salmonis in rainbow trout Oncorhynchus mykiss.

Since 1989, Piscirickettsia salmonis, the causal agent of piscirickettsiosis, has killed millions of farmed salmonids each year in southern Chile. The portal of entry for the pathogen was investigated by use of selected experimental infections in juvenile rainbow trout (12 g). The methods used were intraperitoneal injection, subcutaneous injection, patch contact on skin, patch contact on gills, intestinal intubation and gastric intubation. Cumulative mortalities at Day 33 post-inoculation were 98, 100, 52, 24, 24, and 2%, respectively. It was shown that intact skin and gills could be penetrated by P. salmonis. The high mortality obtained in subcutaneously injected fish indicated that skin injuries could facilitate the invasion of this pathogen. Results suggested that the main entry sites are through the skin and gills and that the oral route may not be the normal method by which P. salmonis initiates infection of salmonids.     

Routes of heat transfer in two teleosts,Ictalurus punctatus andLepomis macrochirus

Synopsis The proportion of heat transferred via the gills (Pg) was estimated for two morphologically divergent freshwater teleosts,Ictalurus punctatus (channel catfish) andLepomis macrochirus (bluegill). Methodology involved monitoring a fish's core temperature while its gills and skin surface were perfused with water of different temperatures. In both species, between 20 and 40 percent of heat was transferred via the gills. Pg depended on direction of the gill-skin temperature differential, suggesting that both species altered distribution of blood flow in response to polarity of the temperature differential. Pg of channel catfish was correlated positively with body mass; whereas, Pg and mass were uncorrelated in bluegill.     

蟹栖异阿脑虫寄生大菱鲆及其组织病理学研究

论文首次报道了蟹栖异阿脑虫(Mesanophrys carcini)在养殖大菱鲆体内的寄生情况以及所引起宿主的组织病理学变化。观察结果表明,感染的苗期大菱鲆的大部分器官组织包括皮肤、鳍、鳃、眼、脑、心脏、肝脏、肾脏、脾脏、胰腺和消化道等以及器官组织间隙中都发现了寄生的蟹栖异阿脑虫,属于全身性感染;而对于成鱼,蟹栖异阿脑虫主要寄生于皮肤、鳍和鳃等器官组织;严重感染者,蟹栖异阿脑虫可侵入其脑、肝脏、肾脏和心脏等器官组织,但数量不多。蟹栖异阿脑虫在大菱鲆体内的寄生能够引起病鱼各器官组织发生不同程度的组织病理学改变。该病在幼鱼和成鱼身上所表现出较一致的病理学特征为:鳃小叶呼吸上皮细胞增生;脑膜炎和脑组织液化性坏死;肾脏和脾脏造血组织坏死;肝脏中央静脉和窦状隙扩张;肌纤维发生凝固性坏死和眼的脉络膜充血、水肿;单核巨噬细胞浸润。研究认为,蟹栖异阿脑虫寄生引起的鳃损害使病鱼呼吸困难,窒息是病鱼发生死亡的主要原因,而虫体寄生导致的各器官组织变性,坏死,使得病鱼器官功能衰竭可加速鱼的死亡。另外,文中还探讨了蟹栖异阿脑虫入侵大菱鲆的可能途径,认为破损的皮肤和鳃是入侵的主要途径。研究结果告诉我们,在大菱鲆养殖过程中防止鱼体擦伤,保持池底清洁对于预防疾病的发生非常重要;对该病的治疗应采取外部杀虫和内部口服药物相结合的方式。