Ulla Tjärnlund

Wild birds of declining European species are dying from a thiamine deficiency syndrome.

Wild birds of several species are dying in large numbers from an idiopathic paralytic disease in the Baltic Sea area. Here, we demonstrate strong relationships between this disease, breeding failure, and thiamine (vitamin B1) deficiency in eggs, pulli, and full-grown individuals. Thiamine is essential for vertebrates, and its diphosphorylated form functions as a cofactor for several life sustaining enzymes, whereas the triphosphorylated form is necessary for the functioning of neuronal membranes. Paralyzed individuals were remedied by thiamine treatment. Moreover, thiamine deficiency and detrimental effects on thiamine-dependent enzymes were demonstrated in the yolk, liver, and brain. We propose that the mortality and breeding failure are part of a thiamine deficiency syndrome, which may have contributed significantly to declines in many bird populations during the last decades.

Effects of two polybrominated diphenyl ethers on rainbow trout (Oncorhynchus mykiss) exposed via food

Abstract Two polybrominated diphenyl ethers, 2, 4, 2′, 4′-tetrabromodiphenyl ether and 2, 4, 5, 2′, 4′-pentabromodiphenyl ether were added to the food given to two different groups of rainbow trout. The rainbow trout were fed for a period of 22 days. On days 6 and 22, a number of physiological and biochemical variables were investigated including condition factor, liver somatic index, spleen somatic index, hematocrit, differential count of leucocytes, haemoglobin and glucose in whole blood as well as the liver enzyme activities, glutathione reductase, catalase and ethoxyresorufin-O-deethylase. The results show that both these substances were relatively non-acutely toxic and several variables were unaffected. However, the blood variables hematocrit and blood glucose showed a slight influence from 2, 4, 5, 2′, 4′-pentabromodiphenyl ether after six days of exposure. Also the glutathione reductase activity in the liver decreased after 22 days of exposure. The 2, 4, 2′, 4′-tetrabromodiphenyl ether showed no influence on the investigated variables, with one striking exception. This structure caused a decrease of about 75% in ethoxyresorufin-O-deethylase activity in the liver after six days, and this effect was also observed after 22 days.

Biochemical biomarkers in adult female perch (Perca fluviatilis) in a chronically polluted gradient in the Stockholm recipient (Sweden)

A battery of biochemical biomarkers and the SigmaPCB concentration in adult female perch (Perca fluviatilis) verified an aquatic pollution gradient with the city of Stockholm (Sweden) as a point source of anthropogenic substances. The investigation included both an upstream gradient, 46 km westwards through Lake Mälaren, and a downstream gradient, 84 km eastwards through the Stockholm archipelago. Besides the main gradient from Stockholm, there were strong indications of pollution coming from the Baltic Sea. The results indicated a severe pollution situation in central Stockholm, with poor health status of the perch, characterised by increased specific EROD activity in the liver, increased liver EROD somatic index, decreased AChE activity in the muscle, increased amount of DNA adducts in the liver, and a high concentration of biliary 1-pyrenol. In addition, laboratory exposure to common EROD inducers elicited an abnormal response, suggestive of chronic intoxication.

Abnormalities of a gill cover bone, the operculum, in perch Perca fluviatilis from a pulp mill effluent area

Abstract Abnormalities of a gill cover bone, the operculum, were found in perch Perca fluviatilis from an area affected by pulp mill effluents in the Gulf of Bothnia, Baltic Sea, Sweden. One of the abnormalities was expressed as a shortening of the distal part of the operculum, and was termed opercular deformity. This deformity was quantified by taking two measurements of the operculum. The second abnormality was manifested as craterous formations on the operculum. Both abnormalities were studied using a sample of opercula collected in the pulp mill effluent area between the years 1982 and 1989. The maximum prevalences of the abnormalities, 34% for the craterous formation and 20% for the opercular deformity, were found in 1983 and 1984, respectively. In a reference area the prevalences were 1% and 1.4%, respectively. After 1984, the prevalences decreased together with prevalences of other fish diseases and deformities in the same area. This coincided with a reduction in concentrations of effluent. The observed high prevalence of the opercular deformity and the craterous formations were attributed to the effects of the pulp mill effluent. In addition, an analysis of the biochemical composition opercula from perch caught in another pulp mill effluent area showed a dose-dependent alteration of several constituents, indicating a relationship with exposure to the effluent. This study shows the potential of opercular abnormalities to monitor effects of pulp mill effluents, and also, it is suggested, for retrospective studies on large samples of opercula used for age determination in ecological studies of fish.

Paraquat and menadione exposure of rainbow trout (Oncorhynchus mykiss)—Studies of effects on the pentose-phosphate shunt and thiamine levels in liver and kidney

Possible xenobiotic interactions with thiamine were studied in salmonid fish, by repeatedly injecting two model substances, paraquat and menadione, into juvenile rainbow trout (Oncorhynchus mykiss). These two substances were chosen because of their well-known ability to redox-cycle and cause depletion of NADPH in several biological systems. Depletion of NADPH increases metabolism through the pentose-phosphate shunt and may thereby increase the need for thiamine diphosphate by heightened transketolase activity. A special food was produced with lower thiamine content than commercial food, usually enriched with thiamine, which could mask an effect on the thiamine level. After 9 weeks of exposure, glucose-6-phosphate dehydrogenase, transketolase, glutathione reductase and ethoxyresorufin O-deethylase were analysed in liver and kidney cellular sub-fractions as well as analysis of total thiamine concentrations in liver, kidney and muscle. The results showed that paraquat caused a large increase in hepatic glutathione reductase activity and induced hepatic glucose-6-phosphate dehydrogenase activity, i.e., the rate-limiting enzyme in the oxidative part of the pentose-phosphate shunt. Despite this paraquat exposure did not affect transketolase activity and total thiamine concentration.

Hepatic activities of thiamine-dependent enzymes, glucose-6-phosphate dehydrogenase and cytochrome P4501A in Baltic salmon (Salmo salar) yolk-sac fry after thiamine treatment

Sea-run Baltic salmon (Salmo salar) populations have been affected by the M74 syndrome since 1974 causing high yolk-sac fry losses in Swedish compensatory rearing plants. M74 has been shown to be a maternally transmitted thiamine (vitamin B(1)) deficiency. The aim of this study was to investigate possible relationships between thiamine and hepatic activities of the thiamine-dependent enzymes transketolase (TK) and alpha-ketoglutarate dehydrogenase (alpha-KGDH) in addition to glucose-6-phosphate dehydrogenase (G6PDH) and cytochrome P4501A (CYP1A), measured as 7-ethoxyresorufin O-deethylase (EROD), in Baltic salmon yolk-sac fry after treatment with thiamine. Thiamine concentrations and activities of TK, alpha-KGDH and EROD were significantly lower (P<0.05) in M74 groups compared to controls (not developing M74) and family groups of thiamine injected females. In M74-developing groups the thiamine immersions reduced the mortality from 86 to 13% and restored thiamine concentrations and activities of TK, alpha-KGDH and EROD to levels slightly lower than the immersed controls. An interesting fact was that the controls showed significantly elevated (P<0.05) TK and alpha-KGDH-activities after immersions in thiamine, indicating that they also may have a stressed thiamine metabolism. The TK and alpha-KGDH-activities of unimmersed groups correlated significantly (P<0.05) with the thiamine content. We suggest that the low activities of TK and alpha-KGDH in M74 groups may be an integrative part in the pathogenesis of M74 development.

Further studies of the effects of exhaust from two-stroke outboard motors on fish

Abstract Rainbow trout were exposed to an n -hexane extract of engine exhaust condensate by intra peritoneal (i.p.) injection or by feeding with contaminated home-made cod chips. Exposure levels were calculated and chosen to reflect those conditions possibly experienced by fish in their natural habitats. Disruption of biological functions has been observed at different levels of biological organization including cellular and subcellular processes (DNA-adduct levels and enzyme activity) and physiological functions (carbohydrate metabolism). Female and male fish may be affected to a varying degree by these types of pollutants.

Genotoxicity in herring gulls (Larus argentatus) in Sweden and Iceland.

Adult and young herring gulls (Larus argentatus) in Sweden and Iceland were investigated with respect to DNA adducts, analysed with the nuclease-P1 version of the (32)P-postlabelling method, and micronucleated erythrocytes. Three important aims were: (1) to estimate the degree of exposure to genotoxic environmental pollutants in the Baltic Sea area and Iceland, (2) to evaluate the utility of the investigated biomarkers in birds, and (3) to investigate if there was any relationship between genotoxic effects and thiamine deficiency. The results demonstrate that both Swedish and Icelandic herring gulls are exposed to genotoxic pollution. Urban specimens have higher levels of DNA adducts than rural specimens, but background exposure to genotoxic environmental pollutants, such as PAHs, is also significant. In the herring gull the general level of DNA adducts in the liver seems to be higher than in fish. DNA adducts were most abundant in the liver, followed by the kidney, intestinal mucosa, and whole blood, in decreasing order. The frequency of micronucleated erythrocytes was probably slightly elevated in all the investigated sites, reflecting a significant background exposure. The level of DNA adducts was unrelated to the frequency of micronucleated erythrocytes, and both these variables were unrelated to symptoms of thiamine deficiency. The investigation confirmed the utility of DNA adducts, and probably also micronucleated erythrocytes, as biomarkers of genotoxicity in birds.

Physiological, biochemical and morphological studies of Baltic salmon yolk-sac fry with an experimental thiamine deficiency: relations to the M74 syndrome

Sea-run Baltic salmon (Salmo salar) populations are suffering from the M74 syndrome, a reproduction disorder affecting both broodfish and their progeny. The syndrome is usually manifested during the middle part of the yolk-sac fry stage and has been shown to be associated with a thiamine (vitamin B(1)) deficiency. Development of the disease is reversible by thiamine treatments of broodfish or progeny. This study aimed at investigating the ability of the thiamine antagonist pyrithiamine, administered by microinjections 3 days after hatch, to cause M74-like signs i.e. typical clinical symptoms, high mortality rates and histopathological changes. Furthermore, the effects of pyrithiamine on hepatic activities of the thiamine-dependent enzyme transketolase (TK), the glucose-6-phosphate dehydrogenase (G6PDH) and the cytochrome P4501A (CYP1A) were evaluated. Six family groups with differing thiamine status were sampled on three occasions during the yolk-sac fry stage. All pyrithiamine exposed groups, with the exception of the one with the highest thiamine concentration, showed M74-like symptoms and suffered from high mortality. Enzyme activities were not different in pyrithiamine groups as compared with controls. However, the TK-activities were strongly associated with the thiamine concentrations. The G6PDH-activity demonstrated small variations with the highest activities in the M74-groups. The [TK]/[G6PDH]-ratios were considerably lower in the M74-groups than in the healthy controls, indicating an imbalance between the oxidative and the non-oxidative part of the pentose-phosphate shunt due to a deficit in thiamine. The pyrithiamine-injections induced several M74-like symptoms including incoordination, lethargy, whitened liver and yolk-sac precipitates. They also caused high mortality rates, in addition to lowered glycogen levels and increased prevalence of necrotic brain cells. Moreover, the study demonstrates that the TK, G6PDH and CYP1A-activities are associated with the thiamine content.

Investigation of the Biological Effects of 2-Cycle Outboard Engines' Exhaust on Fish

Abstract A 2-cycle outboard engine was run in a tank to simulate boatload conditions. After appropriate dilution, exhaust water was led into another tank where fish were exposed. In addition, exposure from extract obtained by acetone/hexane extracted water containing exhaust was used in intraperitoneal injection of juvenile fish as well as in embryo injection experiments. The different areas of investigation were enzymatic disturbances, genotoxicological effects and reproduction disturbances. The investigation so far indicates that the biological effects of the exhaust from 2-cycle outboard engines is a serious threat to the environment. Toxicological effects have been measured in the liver, kidney and blood.