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An integral part of hazard and safety assessments is the estimation of a chemical’s potential to cause skin sensitization. Currently, only animal tests (OECD 406 and 429) are accepted in a regulatory context. Nonanimal test methods are being developed and formally validated. In order to gain more insight into the responses induced by eight exemplary surfactants, a battery of in vivo and in vitro tests were conducted using the same batch of chemicals. In general, the surfactants were negative in the GPMT, KeratinoSens and hCLAT assays and none formed covalent adducts with test peptides. In contrast, all but one was positive in the LLNA. Most were rated as being irritants by the EpiSkin assay with the additional endpoint, IL1-alpha. The weight of evidence based on this comprehensive testing indicates that, with one exception, they are non-sensitizing skin irritants, confirming that the LLNA tends to overestimate the sensitization potential of surfactants. As results obtained from LLNAs are considered as the gold standard for the development of new nonanimal alternative test methods, results such as these highlight the necessity to carefully evaluate the applicability domains of test methods in order to develop reliable nonanimal alternative testing strategies for sensitization testing.

The Local Lymph Node Assay (LLNA) is the preferred test for the identification of skin-sensitizing potentials of chemicals in Europe and is also the first choice method within REACH. In the formal validation, only a very few surfactant chemicals were evaluated and SDS was identified as a false positive. In this study, 10 nonionic sugar lipid surfactants were tested in an LLNA, guinea pig maximization test (GPMT) and human repeated insult patch test. Of the 10 surfactants tested in the LLNA, 5 showed stimulation indices above 3.0. Three of five positive reactions were concomitant with signs of skin irritation indicated by an increase in ear thickness. In the GPMT, all test products were classified as nonsensitizers. In human volunteers, no skin reactions suggestive of sensitization were reported. In conclusion, these results are indicative of the LLNA overestimating sensitization potentials for this category of chemicals. This may in part be due to irritant effects generated by these surfactants. Until suitable nonanimal alternative tests obtain regulatory acceptance, use of other tests, e.g. GPMTs, may in cases be justified. Results such as these need be taken into account when developing nonanimal alternative methods to ensure reliable data sets for method validation purposes.

Atlantic salmon ( Salmo salar L.), reared in sea cages for 18 months (age 36 months from hatching), were exposed to natural light (NL; control), or continuous light (LL) from January (ALJ) or March (ALM) until July. Oocyte development appeared to be arrested at the oil drop stage during winter and spring in a proportion of the females following exposure to LL. This was accompanied by a decrease in plasma oestradiol-17β and testosterone levels following the onset of LL. Somatic growth was affected by LL during winter and spring. However, the arrested sexual development in a proportion of the fish in the ALJ and ALM groups did not correlate with lower growth rate, body weight or condition factor compared with controls prior to the reduction in plasma sex steroid levels. These results suggest a photoperiodic advancement of the timing of an annual `critical period' during which sexual maturation can be arrested.

Atlantic salmon ( Salmo salar L.), reared in sea cages for 18 months (age 36 months from hatching), were exposed to natural light (NL, 61°N), or continuous additional light from January (ALJ) or March (ALM) until July. On July 13, the fish were moved to indoor raceways with brackish water (2–19‰) and ambient temperature (declining from 13.0 to 5.6°C). Fish from each treatment were subjected to either simulated natural photoperiod (SNP), continuous light (24L), or short photoperiod (8L=8 L:16D), creating a total of nine experimental groups with approx. 50 fish in each. The proportion of sexually maturing females was reduced from 91% in the NL groups, to 67% and 9% in the ALM and ALJ groups, respectively ( p ≤0.005). A similar reduction was observed among the males, from 74% in the NL groups, to 57% and 16% in the ALM and ALJ groups, respectively ( p ≤0.001). Ovulation commenced in late October in the control group (NL-SNP). Compared with control, median ovulation time was advanced by 5, 4 and 3 weeks in the ALM-8L, NL-8L and ALM-SNP groups, respectively, whereas ovulation was delayed by 1 and 6 weeks in the ALM-24L and NL-24L groups, respectively. The altered timing of ovulation among the groups was paralleled by similar shifts in the seasonal plasma oestradiol-17 β and testosterone profiles. Survival of eggs to the eyed stage was lower in the ALM-8L group (mean=64.2%) compared with the NL-SNP group (mean=92.5%), indicating a negative effect on egg quality in the most advanced group. Although abrupt changes in photoperiod can be used to control timing of ovulation in Atlantic salmon to obtain off-season eggs, the decrease in egg survival and proportion of maturing fish may set constraints on how much maturation can be advanced by use of continuous light during winter and spring. However, the effect on age at maturity may also be exploited to reduce the problem with unwanted early maturation in salmon farming.

This article considers methods for the detection of oestrogenic effects in marine fish. As a marker for oestrogenic effects, plasma vitellogenin was determined in Atlantic cod, Atlantic salmon and goldsinny wrasse using ELISA with homologous or heterologous antisera. The sensitivity of marine fish to oestrogen exposure is species-dependent and cod is a suitable species for monitoring the effects of environmental oestrogens.

Changes in metallothionein (MT) levels in liver and kidney of juvenile rainbow trout (Oncorhynchus mykiss) were followed for 8 months, as the water temperature was allowed to follow the natural temperature decrease in Tvaren, a bay in the Baltic Sea. The temperature dropped from 15 °C to 2 °C during the experimental period. The liver and kidney were analysed for MT, zinc, copper and cadmium. Both the hepatic and renal MT levels were found to increase in the control group during the first 4 months of the experiment, as the water temperature dropped to 4 °C. Once the temperature had stabilized at 2 °C there was a noticeable decrease in both hepatic and renal MT levels. One group of fish was exposed to water-borne cadmium (100 μg Cd/litre) for the duration of the experiment. Cadmium accumulated in both liver and kidney, and reached the highest levels in the kidney. Exposure of the fish to cadmium resulted in marked elevations of kidney MT levels. In the liver the MT levels did not increase above the control levels as the temperature dropped to 2 °C after 6 months. However, once the temperature stabilized at 2 °C (after 8 months) the hepatic MT levels were significantly higher in cadmium-exposed fish than in the control fish.

The fatty-acid composition of lipids from ovulated eggs of wild and cultured turbot was investigated in order to estimate the nutritional requirements during embryonic and early larval development. Lipid comprised 13.8±0.5% (n=5) and 13.2±0.7% (n=7) of the egg dry weight in wild and cultured turbot, respectively. Polyunsaturated fatty acids (PUFA) of the (n-3) series accounted for 39% of total fatty acids in total lipid of both wild and cultured fish. The predominant (n-3) FUFA was docosahexaenoic acid (22:6 n-3), which also was the most abundant fatty acid in turbot eggs and comprised 24 and 23% of the total egg fatty acids in wild and cultured fish, respectively. Phospholipids, triacylglycerols and cholesterol-wax esters of turbot eggs all exhibited a specific fatty-acid profile distinctly different from that of total lipid. The general pattern of the fatty-acid distribution in lipids of eggs from wild and cultured turbot was similar, but the relative amount of 18:2(n-6) was considerably higher and 20:1(n-9) slightly higher in cultured fish. These differences were extended to all lipid classes and probably reflect the dietary intake of certain vegetable and marine fish oils. Calculations based on light microscopical studies showed that 55 to 60% of the total lipids in cultured turbot eggs are confined to the oil globule. The size of the oil globule remained constant during embryogenesis, and a reduction in size occurred first after hatching and mainly after yolk depletion. This implies that the total amount of lipids utilised during the embryonic development is considerably less than the total lipids present in ovulated turbot eggs. Comparison of the fatty-acid composition of total lipids from eggs and vitellogenin of wild turbot reveals that egg lipids contained a lower level of saturated and a higher level of monounsaturated fatty acids. Eggs also contained wax esters, which were not detected in vitellogenin, suggesting that vitellogenin is not the only source of lipids for turbot eggs.

Recently, we found that liver of squirrelfish (Holocentrus rufus) from Bermuda contains remarkably high concentrations of metallothionein (MT) and Zn. To clarify the nature and cause of the Zn accumulation, four Holocentridae species from three genera (Neoniphon sammara, Sargocentron spiniferum, Myripristis violacea, and Myripristis murdjan) were collected from a totally different area, the Great Barrier Reef, Australia. The average hepatic Zn levels in these species ranged from 67 μg g-1 (S. spiniferum) to 1000 μg g-1 (N. sammara). The intraspecies variation in the hepatic Zn level was pronounced. In both N. sammara and M. murdjan, there was a 48-fold difference between the lowest and the highest Zn concentration. Zn accumulation was closely correlated to a dramatically elevated concentration of MT in liver. In N. sammara, which possessed the highest level of MT among the investigated species, the mean hepatic MT concentration was 19 500 μg g-1. Subcellular fractionation of the liver showed that MT was responsible for the binding of up to 74% of the hepatic Zn. The concentrations of MT and Zn in gills were low, suggesting that Zn storage in fish within this family is tissue-specific. The results further indicate that the high concentrations of MT and Zn in Holocentridae liver are linked to normal physiological processes. We suggest that the Holocentridae family may be a unique model for future studies of Zn metabolism and the function of MT.

Metallothionein (MT) is a low-molecular-weight, cysteine-rich, metalbinding protein found in all vertebrates. The primary structure of the protein is evolutionary conserved, especially the location of cysteine-residues. There are nonetheless significant differences between MTs from different vertebrate classes, notably in major antigenic epitopes. Previous work has shown that MT from different fish species also differ. The objective of the present work was to characterize MT from temperate, sub-tropical and tropical fish species by their relative reactivities with two anti-fish MT antisera. One or both antisera cross-reacted with MT from all fish species investigated. Surprisingly, the relative reactivities of the two antisera with MT from species belonging to different taxonomic groups appeared to reflect differences at the level of family rather than differences between major phylogenetic lineages.

Induction of metallothionein (MT) in the hepatopancreas of the tropical, commercially-farmed shrimp (Penaeus vannamei,) was studied in the laboratory after exposure to waterborne cadmium. MT was quantified with differential pulse polarography. Two laboratory experiments were performed: a dose-response and a time-course experiment. MT was significantly induced in a dose-dependent way in response to waterborne CdCl2. In the time-course experiment a significant correlation was found both between induced MT and accumulated cadmium in the hepatopancreas, and between depuration of MT and cadmium. Final value of MT after 9 days of exposure to 1.50 mg Cd/litre was 5600 μg MT/g wet weight hepatopancreas, which is an induction of 180%.

The vitelline envelope of Atlantic halibut (Hippoglossus hippoglossus) is composed mainly of two major and two minor proteins. Estradiol-17ß induces the two major vitelline envelope proteins in halibut of both sexes. These proteins were also found in plasma of vitellogenic females. The origin of the two major vitelline envelope proteins is not restricted to the ovary, as male halibut synthesize these proteins after treatment with estradiol-17ß. Individual female halibut were followed and plasma sampled from May to March. Plasma levels of estradiol-17ß increased in October, peaked in early February, and declined until the last ovulation in March. In October, the two major vitelline envelope proteins were first detected in plasma and the amount remained elevated until early March. Prior to the last ovulation, the amount decreased. Plasma vitellogenin varied in a similar way. The results provide strong evidence that the physiological regulation of the synthesis of the major vitelline envelope proteins in teleosts is controlled by the female sex steroid estradiol-17ß.

Vitellogenin was purified from plasma of estradiol-17β-treated cod (Gadus morhua) rainbow trout (Oncorhynchus mykiss), turbot (Scophthalmus maximus), and wolffish (Anarhichas lupus) by precipitation with EDTA:Mg2+, distilled water, and high-performance ion-exchange chromatography. Vitellogenin of high purity was obtained by precipitation followed by chromatography, as evaluated by an homologous antiserum developed for each species. The instability of vitellogenin demanded consistent low temperature and the use of protease inhibitor before blood sampling. When the necessary precautions were taken, vitellogenin from rainbow trout, turbot, and wolffish eluted as one regular peak during chromatography. Cod vitellogenin eluted as two peaks and these demonstrated identical migration patterns on SDS-PAGE. The observed differences in stability between the four species suggest that isolation procedures should be modified according to the requirements for each species. Electrophoresis of plasma from treated fish revealed the presence of several smaller proteins, with a molecular mass around 50 kDa, that were considered to be vitelline envelope proteins. Other minor plasma proteins were immunoreactive to antisera, directed against vitellogenin and therefore judged to be fragments of degraded vitellogenin. © 1993 Wiley-Liss, Inc.

Minnows (Phoxinus phoxinus) were exposed in the laboratory to 3 nM Cd (Cd group), 2000 nM Zn (Zn group), or to both metals (CdZn group) in soft water for 142 d. A fourth group was kept with no metals added to the water (Low group). After 80 d, the Cd and Zn groups were statically exposed to 109Cd and 65Zn, respectively, for 24 h. The CdZn group was exposed to both tracers. Fish were sampled for 61 d after the tracer exposure. At the end of the experiment, an increased metallothionein level was detected in the kidney of fish exposed to the metal mixture (Low group: 0.14 nmol/g fish; CdZn group: 0.54 nmol/g fish). However, the fish did not acquire tolerance to the metals because mortality continued at a constant rate during the whole experiment. Metabolism of Cd and Zn was altered in fish exposed to the metal mixture compared with fish exposed to Cd or Zn alone. The main effect was a Zn-induced increase in Cd turnover rate in the fish. Nevertheless, the toxic action of metals was additive, since mortality at the end of the experiment was 24, 32, and 54% in the Zn, Cd, and CdZn groups, respectively.

The local lymph node assay (LLNA) is the assay of choice in European regulatory toxicology. As with other toxicology/sensitisation assays, it has a potential for false results, the anionic surfactant sodium lauryl sulphate (SLS) representing a classic example. In the work reported here, examples of false positives in the LLNA are compared to published "benchmarks" such as SLS. Clear false positives (e.g. oleic acid) are also contrasted with examples where data interpretation is more challenging. As the LLNA will be applicable to >30,000 chemicals under REACH, and in the light of animal welfare considerations to do no more than the absolute minimum of animal testing, results from a single LLNA often represent the only available data on sensitisation. This reinforces the need to ensure data from this assay are interpreted intelligently, using scientific analysis of results and considering the weight of evidence, before decisions are made on which substances should be classified as representing a skin sensitisation hazard. In chemical classes where the LLNA has been shown to be an inappropriate assay other standardised methods (e.g. the Buehler or Magnusson and Kligman guinea pig tests [OECD 406]) should be employed as the first choice assays.

Potential 1+ smolts of Atlantic salmon (Salmo salar) were reared under three light regimes: simulated natural photoperiod (LDN), continuous light (LD24:0), or a combination of continuous, low-intensity background light and a superimposed simulated natural photoperiod (dual photoperiod, LDD). Growth rate in freshwater was enhanced by LD24:0 and LDD, and changes associated with smoking (increased salinity tolerance, reduced condition coefficient) were advanced under LD24:0. Plasma growth hormone levels were initially high on LD24:0 and LDD whereas on LDN, plasma growth hormone levels increased gradually from February through April. Overall GH levels were negatively correlated with condition coefficient during the final stages of smoking. After 16 mo in seawater, there were no significant size differences among the groups. The incidence of sexual maturation as postsmolts was higher in the LD24:0 and LDD groups whereas the incidence of grilsing was higher in LDN. Results demonstrate the significant influence of photoperiod on growth and smoking in Atlantic salmon. An abrupt increase to continuous light in winter may be sufficient to advance important aspects of the parr–smolt transformation. Dual photoperiod may be a way to combine the increased growth rate observed under continuous light and the normal parr–smolt transformation associated with natural photoperiod.

A field study of the role of metallothionein (MT) in the binding of heavy metals in perch (Perca fluviatilis), exposed to moderate levels of copper, zinc and cadmium, was performed. Perch were sampled at four sites in Sweden in September during two consecutive years. Two sites were located in the vicinity of a brassworks and two outside the emission range. The first year, fish from the two brassworks sites and from one of the uncontaminated sites were collected. The second year, fish from the most contaminated site and from the two uncontaminated sites were caught. The levels of hepatic copper, zinc and cadmium reflected the concentrations of these metals in water and were increased in fish from the two contaminated sites. The level of cadmium in liver was relatively low. MT was induced in liver of perch caught at the most contaminated site. The hepatic MT content in individual livers correlated well to the accumulation of copper (r = 0.85, P less than 0.001) and zinc (r = 0.75, P less than 0.001). There was a low but significant correlation between the levels of MT and cadmium in the liver (r = 0.48, P less than 0.001). The relationship between MT and metals was very similar both years. Subcellular fractionation of the metals in the liver revealed that an induction of MT was followed by an increased amount of copper, zinc and cadmium bound to the protein. The relative fraction of the total hepatic copper and cadmium bound to MT was increased at the most contaminated site, whereas there was no difference in subcellular distribution of zinc between the sites. In perch from the most contaminated site, the portions of hepatic copper, zinc and cadmium found in the cytosolic fraction were 70, 57 and 81%, respectively. Seventy-one % of the copper, 29% of the zinc and 84% of the cadmium found in hepatic cytosol of fish from the same site, eluted together with MT after gel filtration chromatography. The analysis of the subcellular distribution of copper, zinc and cadmium demonstrates that MT is responsible for the binding of a large amount of the total hepatic cellular content of copper and cadmium in perch.

At the time of smoltification in May, smolts and sexually mature male parr were transferred to seawater (25‰ salinity) and sampled after 6 and 24 hr. Plasma levels of growth hormone (GH) were measured by radioimmunoassay. There was no difference in GH levels between smolts and mature parr in fresh water. GH levels did not change during exposure of smolts to seawater. In the mature male parr, plasma GH levels increased after 24 hr, when the levels were almost five times those of the freshwater controls. In the mature male parr, there was an increase in plasma osmolality, sodium, and magnesium after 24 hr in seawater; magnesium also increased after 6 hr. The levels of potassium and calcium did not change in either immature parr or mature male parr. The increase in plasma GH levels in the mature parr in seawater may be part of a mechanism to increase hypoosmoregulatory ability in fish not ready for seawater entry.

Induction of metallothionein (MT) and levels of cadmium, zinc and copper in liver of the subtropical fish blue-striped grunt Haemulon sciurus were studied after cadmium injection. Hepatic levels of MT, zinc and copper were also analysed in blue-striped grunt and tomtate H. aurolineatum caught at heavy-metal-polluted areas of Hamilton Harbour and Castle Harbour, Bermuda, respectively. In addition, a comparison of the hepatic content of MT, cadmium, zinc and copper was made between blue-striped grunt, tomtate and squirrelfish Holocentrus rufus, caught at unpolluted locations. Hepatic MT was assayed by a radioimmunoassay (RIA), using an antiserum raised against MT from perch Perca fluviatilis and partially purified MT from blue-striped grunt as standard. Hepatic MT was significantly (P < 0.001) induced in a dose-dependent manner in response to a single i.p. injection of CdCl2. The doses were 2200, 740, 250, 82, 27 and 0 μg Cd · kg−1 body wt, respectively. Significantly elevated hepatic MT levels (P < 0.01) were found 2 days after injection of 2200 μg Cd · kg−1 body wt and maximum content of MT in liver was reached after 6 days. MT values in fish 6 days after treatment were increased five-fold compared to the control group. Significantly increased MT levels (P < 0.05) were also found in livers of blue-striped grunt and tomtate caught at contaminated sites compared to control fish. The increased MT levels were in good accordance with the hepatic zinc and copper levels in these fish. The levels of MT, cadmium, zinc and copper in liver from blue-striped grunt and tomtate, caught at clean sites, were within the range found in other fish species, such as rainbow trout Oncorhynchus mykiss and P. fluviatilis. Squirrelfish, caught at an uncontaminated site, had exceptionally high levels of hepatic MT, zinc and copper.

An improved straight-phase HPLC method for the separation and quantification of lipid classes is described. Two binary gradient solvent systems were used, one for polar and one for neutral lipids, and detection was performed with a light-scattering detector. The developed HPLC methods were highly reproducible and allowed base-line separation of all investigated polar lipid classes (phosphatidic acid, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, phosphatidylserine, phosphatidylinositol and lysophosphatidylcholine) and neutral lipid classes (triacylglycerol, free fatty acid, diacylglycerol, cholesterol and monoacylglycerol) except of cholesterol ester and wax ester. Application of the chromatographic systems demonstrated that the methods are suitable for quantitative analysis of the major lipid classes present in lipid extracts from livers and eggs of Atlantic salmon (Salmo salar).

The vitelline envelopes of European sea bass and gilthead sea bream are both composed of mainly four proteins with the molecular masses of 90, 52, 48, 45 kDa and 75, 50, 48, 44 kDa, respectively. Each protein has an amino acid composition that is characterized by a high content of proline and glutamic acid and a low content of cysteine, similar to the whole vitelline envelope of both species. The amino acid composition suggests that each protein is distinct but related to the other vitelline envelope proteins. The use of homologous antisera shows that both species have vitelline envelope proteins that are induced by estradiol-17β. As males of both species synthesize these proteins after treatment with estradiol-17β, the origin is not restricted to the ovaries. Vitellogenin of both Eurpoean sea bass and gilthead sea bream has the apparent molecular mass of 170 kDa. © 1995 Wiley-Liss, Inc.

The plasma profiles of growth hormone (GH) in adult male and female Atlantic salmon were determined in relation to manipulation of the photoperiod and to the development and timing of sexual maturation. Fish were exposed to natural light (NL) or NL + 24L:0D additional light over the netpens from January (ALJ) or March (ALM) to July. Thereafter, these groups were brought indoors, subdivided, and subjected to simulated natural photoperiod (SNP), continuous light (24L), or short day (8L). Assay of salmon GH by RIA in monthly plasma samples revealed that GH levels were generally < 1 ng ml-1 during January to June and were only slightly affected by additional light in January or March. ALJ-24L treatment, and to a lesser extent, ALM-24L treatment, was effective in preventing sexual maturation, and GH levels of immature fish continued to be < or = 1.5 ng ml-1. On the other hand, in sexually maturing fish, GH levels increased to 2-5 ng ml-1 months prior to ovulation. Short-day photoperiod (8L) from July advanced ovulation and spermiation, whereas continuous light from July delayed these processes. The timing of the increase of GH levels was shifted in a parallel manner, indicating a functional relationship between plasma GH levels and the process of sexual maturation.

Two methods to quantify metallothionein (MT), differential pulse polarography (DPP) and radioimmunoassay (RIA), were compared for MT analysis of liver from Zn- and Cd-injected perch ( Perca fluviatilis ). Nine perch were intraperitoneally injected, twice a week during 2 weeks with ZnSO 4 and CdCl 2 to yield a total dose of 30 mg Zn and 3 mg Cd per kilogram body weight. Two samples, 100 and 200 mg from each liver, were homogenized separately and further prepared for DPP, RIA, and atomic absorption spectroscopy. MT values obtained by DPP were in good agreement with the MT values determined by RIA ( r = 0.92). The relationship between the MT values analyzed with the two methods is described by the formula MT RIA = MT DPP × 0.99 − 0.048. Analysis of MT was not affected by sample size. MT values from individual liver samples plotted against the Cd and Zn content of the corresponding samples provided a high correlation. The correlation coefficient was 0.86 for MT values obtained by DPP and 0.92 for MT measured by RIA. It is concluded that DPP is a reliable method for analyzing MT in liver.

International audience; The effects of estradiol-17beta (E2) implants on plasma prolactin (PRL) concentrations, pituitary PRL content and pituitary PRL mRNA levels were examined in rainbow trout (Oncorhynchus mykiss). Intact immature fish treated with 1 mg estradiol-17beta did not show significant changes in both PRL mRNA levels and pituitary PRL content after 3 days of treatment. In a similar experiment, no changes were observed in plasma PRL levels followed during 7 days. Similarly, lack of estradiol-17beta effect on plasma PRL levels and on final PRL pituitary content was observed in ovariectomized female rainbow trout treated during 48 days with 25 mg estradiol-17beta and in mature male fish over a 3-week treatment period. Localization of estradiol receptor (ER) mRNAs in the pituitary was carried out by Northern blot analysis using a full-length rainbow trout estrogen receptor (rtER) cDNA as a probe. The rostral pars distalis of the pituitary which contained mostly PRL cells showed the lower amount of rtER mRNA when compared to other parts of the pituitary. Moreover, two mRNAs of different size (3.5 and 1.4 kb) were detected in different parts of the pituitary. Further hybridization experiments using probes containing part of the rtER cDNA (E domain or C and D domains) indicated that the small-sized mRNA (1.4 kb) probably encodes a truncated ER protein lacking hormone binding domain or an ER-related protein. Thus, only the 3.56 kb mRNA appeared to be involved in the regulation of pituitary function by estradiol. In situ hybridization analysis allowed a more precise localization of this rtER mRNA in the pituitary. No labeling was discernible over PRL cells whereas other parts of the pituitary were labeled. Grain counting corroborated this localization. These results indicate that (1) in vivo estradiol-17beta treatment did not modify PRL cell activity in rainbow trout, (2) PRL secretion, in this fish species, is not directly regulated by estradiol.

1. 1. The metabolism of Cu, Zn, Cd and Hg in lower vertebrates is described, using fish as a model. 2. 2. The main part of this review deals with metallothionein and the role of this protein for the storage and detoxification of these metals. 3. 3. Factors influencing the bioavailability and probable uptake routes are identified. 4. 4. The distribution of the metals within the organism is outlined. The distribution between tissues is described and the subcellular distribution discussed with reference to metallothionein.

Perches (Perca fluviatilis), kept in slightly hypotonic brackish water, were exposed to dilutions of a simulated heavy-metal-containing effluent from a sulfide ore smeltery . Biochemical and hematological effects of the effluent, as well as the metal residues in liver and muscle tissues, were investigated after 12 and 27 days of exposure. The metal analyses revealed no significant uptake of metals in liver and muscle during the experiment. In spite of this, the exposed fish showed several physiological effects. Some of these, e.g., anemia, hypocalcemia, increased muscle water content, and reduced liver size, were of a transient nature, while others, such as disturbed chloride balance and hyperglycemia, seemed to be more persistent. At the end of the experiment (29-33 days of exposure), the physiological response to stress treatment (asphyxia) and a subsequent recovery were studied in exposed and unexposed fish. This stress investigation indicates that an additional stressor may strengthen the toxic effects of the heavy-metal-containing effluent. Furthermore, the secondary stress responses were more pronounced and the ability to recover from them seemed to be impaired in exposed fish as compared to unexposed fish.

The ion balance in the flounder ( Ptatichthys flesus L.) was studied after 4 and 9 wk of exposure to sublethal cadmium levels (5–500 μg Cd/l) in brackish water. The cadmium exposure had no effect on the major blood plasma electrolytes, sodium and chloride, indicating an intact osmoregulation. In contrast, cadmium seriously affected the regulation of other ions. Potassium and calcium concentrations showed a strong and dose-dependent depression in blood plasma, whereas the plasma concentrations of inorganic phosphate and magnesium were significantly elevated. It is suggested that these ion disturbances might be associated with cadmium-induced pathological changes in ion-regulating tissues. The reduced concentrations of plasma calcium and potassium might be responsible for neuromuscular disturbances, such as hyperexcitability, spasms and tetanic body contractions, observed in some flounders exposed to the highest cadmium concentration (500 μg Cd/l). In spite of the pronounced disturbance of the calcium metabolism, the spinal columns of the cadmiumexposed flounders did not show any signs of demineralization, fractures or other deformations. This might be due to the fact that the flounder probably has an acellular bone tissue, which only to a small degree seems to be affected by the disturbed calcium balance. It is suggested that fish species with acellular bone tissue run a minor risk of suffering from skeletal damage after cadmium exposure than fish species with an active cellular bone tissue.

The normal variations in hepatic levels of metallothionein, zinc and copper were studied during an annual reproductive cycle in rainbow trout of both sexes. In female fish, the total hepatic zinc levels closely followed the estradiol-17β and the LSI levels. Hence, the zinc levels rose in September, peaked in December and dropped in January. No distinct peaks were, however, observed in the whole-liver copper content. The hepatic metallothionein levels in female fish began to increase at the onset of exogenous vitellogenesis. Maximum levels were reached after estradiol-17β and LSI levels had dropped in January. In male fish no distinct peaks in either zinc or copper levels were observed. The metallothionein levels increased somewhat during the time of spermatogenesis. It is suggested that metallothionein may regulate the hepatic zinc distribution during the annual reproductive cycle in female rainbow trout, thereby ensuring the organism of a control mechanism to keep the pool of available zinc at an appropriate level.

The functional role of calcitonin in teleost fish is in question. Data on the role of calcitonin in calcium regulation are inconsistent, and while a participation in some aspects of sexual maturation has been strongly indicated, the exact function is not known. To establish if there exists a functional relationship between 17 beta-estradiol (E2) and calcitonin in salmonid species, rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar), and coho salmon (Oncorhynchus kisutch) were injected ip with a single or repeated doses of E2. It is concluded that E2 treatment increases plasma calcitonin levels directly or indirectly, and that it is possible that E2 is responsible for the rise in calcitonin levels during late sexual maturation of female salmonids. In accord with earlier studies, no correlation was found between changes in calcitonin levels and free plasma calcium levels. It seems clear that changes in free plasma calcium levels are not the primary cause of the plasma calcitonin changes in teleost fish. It is possible that calcitonin is involved in mobilizing calcium or directing its mobilization by protection of certain calcium pools during vitellogenesis. However, the increase in calcitonin occurs so close to ovulation that a reproductive role other than a calcium regulatory one is likely. The possibility of transfer of calcitonin itself to the developing oocytes and a subsequent role in embryonic development must also be considered.

Freshwater rainbow trout,Salmo gairdneri, were injected with different doses of estradiol-17β in order to induce the synthesis of a protein, regarded as identical to vitellogenin. The plasma levels of free and protein-bound calcium, magnesium and inorganic phosphate were studied in control and estradiol-17β treated fish, using an ultrafiltration method. Estradiol-17β caused a dose-dependent increase in plasma vitellogenin levels, which strongly correlated to protein-bound levels of calcium and magnesium in plasma. Calcium and magnesium were bound to vitellogenin in a ratio of 9:1, which was considerably higer than the protein-binding ratio of these ions in normal plasma (5.2:1). The dose-dependent increase in total plasma levels of calcium, magnesium and inorganic phosphate during estradiol-17β treatment was solely due to an increase in the protein-bound fraction of these ions. It is concluded that the physiologically important plasma levels of free calcium, magnesium and inorganic phosphate are effectively regulated at normal levels during vitellogenin synthesis.

Two methods to quantify metallothionein (MT), differential pulse polarography (DPP) and radioimmunoassay (RIA), were compared for MT analysis of livers from ZnCd-injected perch ( Perca fluviatilis ). There was a strong agreement in the results obtained by the two methods. The regression line for the plot MT RIA versus MT DPP is described by the formula: MT RIA = (MT DPP × 0·99)—0·048 (r = 0·92). When the MT value of each 200 mg tissue sample was divided by the value of corresponding 100 mg tissue sample, the quotient was 1·70±0·78 for DPP and 1·86±0·34 for RIA (mean±SD). MT values, from individual liver samples, plotted against the Cd and Zn content of the corresponding samples provided a slightly lower correlation coefficient for MT values measured by DPP ( r = 0·87), than by RIA ( r = 0·92).

In a field investigation, physiological parameters were studied in whitefish (Coregonus spp.) living in lead-contaminated lakes in northern Sweden. Fish from an uncontaminated lake were used as a reference. The activity of the enzyme δ-aminolevulinic acid dehydratase in blood was inhibited up to 88% in fish from lead-contaminated lakes, as compared with fish from the reference lake, indicating the presence of lead pollution. In addition, higher blood glucose levels and lower plasma sodium content were observed in fish from lead-contaminated lakes. These results suggest that lead affects fish in the environment in a way similar to that observed in laboratory experiments.

1. 1. Changes m hypoosmoregulatory ability in smolting sea trout, Satmo trutta, were monitored by performing regular sea-water challenge tests from February to July. 2. 2. Sea-water tolerance was fully developed in April and was then maintained during spring and summer. 3. 3. A high capacity to regulate magnesium ions correlated well with the smoltification and culminated in May. 4. 4. High and stable plasma electrolyte levels were reached after 24 hr in sea-water. In order to discriminate between parr and smolt, this should be the most effective exposure time in sea-water. 5. 5. Stress of shorter duration had no influence on the outcome of the sea-water challenge test, whereas prolonged stress affected the hypoosmoregulatory capability.

An homologous radioimmunoassay for brown trout vitellogenin (VTG) was developed. Intact VTG, isolated from juvenile brown trout by selective precipitation and anion exchange chromatography was labelled with Na(125)I, with 1,3,4,6-tetrachloro-3α,6α-diphenylglycoluril (Iodogen) as the oxidizing agent. Incorporation of Na(125)I into VTG was higher than 75% and there was little degradation of the labelled protein. Labelled VTG eluted at the same position as unlabelled, purified brown trout VTG when analyzed by gel filtration on Sepharose 6B. Antisera with high titers, i.e. 1∶250 000, against brown trout VTG were raised in rabbits. The sensitivity of the assay was 5 ng VTG/ml and the standard curve was linear between 10 and 100 ng VTG/ml. Plasma from maturing female brown trout, as well as estradiol-treated and untreated juvenile brown trout diluted parallel to the standard curve, while plasma from maturing female rainbow trout and estradiol-treated arctic charr diluted non-parallel to the standard curve for brown trout VTG. Purified rainbow trout VTG and plasma from maturing female rainbow trout diluted parallel to each other, but with lower sensitivity than for brown trout VTG. Determinations of protein-bound phosphorus in the plasma of estradiol-treated juvenile brown trout correlated well with the RIA determinations of VTG. Repeated freezing and thawing of plasma samples yielded up to a hundred-fold increase in the apparent VTG level, while storage of a plasma sample for one year at -20°C did not affect the VTG level as measured by RIA.

The effects on hematology, blood plasma ion balance and carbohydrate metabolism were studied in rainbow trout, Salmo gairdneri, kept in brackish water, and after 18 and 30 wk of exposure to sublethal levels of cadmium (10 and 100 μg Cd/liter), after 25 and 57 wk of subsequent recovery in clean water. Cadmium exposure caused anemia, hypocalcemia and hypermagnesemia. These effects have previously been observed in fish during shorter periods of exposure, but disappeared in the present study within 25 wk of recovery. This suggests that it is the presence of free cadmium ions during the exposure that is responsible for most of the hematological and biochemical effects. Contrary to these findings, the hyperglycemia, that was present during the exposure, persisted throughout the recovery period. Further evidence for a disturbed carbohydrate metabolism after cessation of the exposure was indicated by a dose-dependent decrease in muscle glycogen content after 25 wk of recovery, and by a dose-dependent increase in liver glycogen content after 57 wk of recovery. These effects on the carbohydrate metabolism could be mediated via the selective uptake of cadmium ions in the pancreas and an inhibitory effect on the insulin secretion, thus resulting in an abnormal endocrine control of the metabolism. Analyses of cadmium revealed a dose-dependent accumulation of cadmium in the muscle, that had reached equilibrium after 18 wk of exposure, while a continuing and dose-dependent accumulation was found in the liver throughout the exposure. The levels attained in the liver were about 100 times higher than in the muscle. The slow elimination from liver and kidney suggest a biological half-life for cadmium of more than 1 yr in these tissues. A close positive correlation was found between cadmium levels in liver and in kidney (r = 0.90) in fish after 57 wk of recovery.

The partial development and evaluation of a radioimmunoassay for horse metallothionein (MT) with low amounts of antigen is described. Factors that affect the yield of the conjugation reaction between horse MT and bovine IgG are discussed. Dot-blot has been used as a simple, rapid and inexpensive test for antibody screening.

Rainbow trout ( Salmo gairdneri ), kept in slightly hypotonic brackish water, were exposed to inorganic lead in three different concentrations (10, 75, and 300 μg Pb/liter) for 30 days and then allowed to recover in lead-free brackish water for 49 days. Analyses of residue levels after exposure revealed a rapid dose-dependent accumulation of lead in the liver, while this process was slower in muscle tissue. After recovery, the lead levels dropped markedly in the liver, but were only changed to a minor degree in the muscle. Analyses of the biochemical blood composition showed that lead caused disturbances in the ion regulation, indicated by dose-dependent increases in blood plasma potassium concentrations after both exposure and recovery, and by a dose-dependent reduction in blood plasma chloride concentrations after recovery. Furthermore, an impaired glucose homeostasis was suggested by a significant reduction in blood glucose levels in fish after exposure to inorganic lead.

Effects of cadmium exposure on plasma levels of calcitonin and free and protein-bound calcium were studied in vitellogenic female rainbow trout kept in brackish water (7%(.)). Fish were exposed to 100 μg cadmium litre−1 for four weeks. Exposure of female rainbow trout in the stage of vitellogenesis, with increased total plasma levels of calcium, resulted in a complex hypocalcemic response. Thus, hypocalcemia was found to be due to three different processes: (1) a decrease in the free plasma calcium, and a reduction in protein-bound calcium; due both to (2) decreased plasma levels of vitellogenin; and (3) a reduced binding of calcium to vitellogenin. These findings support the concept of an interference of cadmium with ionregulating tissues as a mechanism for hypocalcemia in rainbow trout. A direct effect on the vitellogenin-binding of calcium was also observed and reproductive function in the females was affected by decreased plasma levels of vitellogenin. In spite of the marked changes of plasma calcium in exposed fish, no significant effects on plasma calcitonin were observed, indicating a lack of a direct relationship between plasma calcium and calcitonin levels in rainbow trout.

The secondary or physiological stress responses occurring immediately after capture were measured in a wild fish population of perch (Perca fluviatilis) and during a recovery period of eight days. Capture induced blood hyperglycemia and hyperlactemia, a depletion of liver and muscle glycogen, a reduced number of circulating lymphocytes, and an increased number of circulating neutrophilic granulocytes. Further, the activity of the enzyme ALA-D in blood, the plasma levels of magnesium and inorganic phosphate, and the muscle water content were increased after capture, while the liver size appeared to be reduced. No consistent effects were observed in hematocrit values, blood hemoglobin content, red blood cell count, plasma sodium, chloride and total protein content, or in liver water content. Parameters affected by capture were found to recover and stabilize within two to four days after capture. The experimental set-up involving a careful handling of fish captured with gill nets and including a subsequent recovery in fish chests was found to be a useful approach for the assessment of the physiology of wild fish populations.

A sensitive radioimmunoassay (RIA) for metallothionein (MT) from perch (Perca fluviatilis) has been developed. The method described is a double antibody RIA with rabbit anti-perch-MT as first antibody, goat anti-rabbit-immunoglobulin G as second antibody and perch MT conjugated to [125I] Bolton-Hunter reagent as tracer. The antiserum cross-reacts with MT from four fish species tested in addition to perch, but has low avidity for horse kidney MT. At a dilution of 1:2000, the MT antibodies were able to bind 36% of the tracer when no cold ligand was present. The sensitivity of the assay was 15 pg perch MT/tube and the practical working range was found to be 0·15–250 ng perch MT/tube. The RIA allows determination of MT in plasma or lysed blood cells at concentrations as low as 3·0 ng/ml and in tissues at levels over 9·0 ng/g (wet weight). Intra- and interassay coefficients of variation were 6 and 10% respectively.

In the present experiment, metabolically active primary cultures of rainbow trout hepatocytes were utilized to study the inducibility of metallothionein after cortisol treatment. Glucocorticoid induction of metallothionein has previously only been demonstrated in mammalian systems. We now demonstrate the inducibility of rainbow trout metallothionein by cortisol treatment of primary hepatocytes. A 90% elevation above control levels was achieved within 8 days of treatment. Zinc treatment was performed to evaluate the system, and 100 microns zinc in the culture medium resulted in a 350% increase of the metallothionein levels. We conclude that primary culture of rainbow trout hepatocytes constitutes an efficient system for studies of MT induction kinetics.

The subcellular distribution of cadmium, copper and zinc in the liver, and the binding of these heavy metals to metallothionein (MT) were studied in a wild population of perch ( Perca fluviatilis ). The fish were caught in two areas of the cadmium-contaminated river Eman in the southeast of Sweden. The livers were analyzed for zinc, copper and cadmium and the subcellular distribution of the heavy metals in mitochondrial, microsomal and cytosolic fractions was determined. The cytosols were chromatographed on Sephadex G-75 columns to determine the partition of heavy metals between high molecular weight and MT fractions. Metallothionein was determined, in tissue extracts, by differential pulse polarography. The elevated hepatic cadmium levels found in perch from the contaminated region of the river did not significantly alter the distribution of zinc and copper in the liver. With increasing amounts of cadmium present in the liver cytosol, the distribution of cadmium was altered. The increased cadmium content correlated with an increased MT level in the liver ( r =0.84). The relationship between cadmium and MT is described by the formula [MT] = 7.2 + 0.53 [Cd]. Chromatography of the liver revealed that all of the applied cadmium was bound to MT. The variations in zinc and copper showed low correlation to the MT content of the liver ( r =0.51 and r =−0.15, respectively).

The use of high-performance anion-exchange chromatography on a Mono Q column for isolation of a glycolipophosphoprotein, vitellogenin, from turbot plasma has been evaluated. The method is an effective, rapid one-step procedure, which gives a pure preparation of vitellogenin as assessed by electrophoresis, [32P]orthophosphate incorporation and amino acid composition.

Hepatic levels of Cu, Zn and metallothionein (MT) in perch, caught in a Cu/Zn gradient from a brassworks, reflected the water concentration of Cu (1·0–9·4 ppb) and Zn (0·56–59 ppb). Significant correlations were found between hepatic Cu and MT levels ( r = 0·72), and between Zn and MT levels ( r = 0·69). There was an increase of the amount of Cu and Zn in the cytosolic fraction of the liver with increased hepatic levels of the metals. When liver samples. from perch caught at the most contaminated location, were run on a gel filtration column (Sephadex G-75) 78% of the cytosolic Cu and 24% of the Zn in the cytosol eluted together with MT.

1. 1. Juvenile rainbow trout received weekly injections of estradiol-17β in two different experiments (exp. I and exp. II). In exp. I, relatively low doses (0.05 and 0.5 mg/kg) were administered and sampling performed 4, 7 and 14 days after the first injection. In exp. II, higher doses (1.0 and 10 mg/kg) were administered and sampling was performed after 7 and 14 days. 2. 2. In exp. I, estradiol generally caused a depletion of liver glycogen, no change in the amount of liver phospholipids, protein, DNA or liver water content, while dose-dependent increases in the amount of RNA and triglycerides were observed. Only a very slight hypertrophy of the liver was noted. 3. 3. Higher doses of estradiol (exp. II) increased the liver size by a factor two and decreased the amount of liver DNA per weight unit. The effects of estradiol were otherwise similar to those observed in exp. I. 4. 4. When the total amount of each component was calculated in exp. II, i.e. on whole liver basis, the amount of triglycerides, phospholipids, protein and RNA were all drastically increased, while the liver content of DNA was not changed. 5. 5. The presented data indicate that estradiol affects the carbohydrate metabolism by reducing the liver glycogen content, increases the ability of the liver to synthesize new proteins, as shown by increases in RNA and protein, and that estradiol has a lipogenic effect, which is more marked for triglycerides than for phospholipids.

Rainbow trout were exposed to 200 μg cadmium/l in the water during four months at 6-10°C. The liver, kidney and gills were analyzed for cadmium, copper, zinc, metallothionein and metallothionein mRNA. Cadmium accumulated in all three organs and reached the highest concentration in the kidney. The tissue zine and copper concentrations showed no major alterations during the experiment. The cytosolic distribution of cadmium, copper and zinc was followed during four months of exposure by Sephadex G-75 chromatography. It was found that cadmium was predominantly associated with proteins of an apparent molecular vieght of 10,000 daltons. These proteins were further identified as metallothioneins after fast protein liquid chromatography on a Mono-Q column. The metallothionein concentration was significantly higher in liver of exposed fish than in control fish after only one month. The kidneys reached significantly elevated levels of metallothionein in the exposed group after three months. In the gills, elevated metallothionein concentrations were observed after four months of exposure. After four months of exposure, the metallothionein mRNA content of liver and kidney was analyzed using a rainbow trout anti-sense RNA probe. Elevated MT mRNA levels were observed in both kidney and liver. These results demonstrate thatde novo synthesis of metallothionein is induced by cadmium in rainbow trout after exposure to the metalvia water.