Gun Åkerman

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.

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.

A reliable and improved methodology to expose fish in the early embryonic stage

Abstract A method has been developed to inject different substances into the yolk of fish eggs before gastrulation. Newly fertilized eggs are placed in holes in the surface of an agarose gel and the injections are made with fine glass micropipettes. The injection volume is controlled by pressure technique. The embryos are kept in the gel until hatching, where they can easily be studied under a stereo-microscope during different developmental stages, with minimized handling and therefore without disturbances. Three different species have been tested with a wide range of egg diameters. Mortality due to injection has always been lower than 1%. Due to the low mortality, different biological effects can be monitored with high accuracy and sensitivity. Therefore this methodology can be useful when investigating disturbances in reproduction as well as toxic effects at young life stages.

MEASURING BIOCONCENTRATION FACTORS IN FISH USING EXPOSURE TO MULTIPLE CHEMICALS AND INTERNAL BENCHMARKING TO CORRECT FOR GROWTH DILUTION

Modern chemical legislation requires measuring the bioconcentration factor (BCF) of large numbers of chemicals in fish. The BCF must be corrected for growth dilution, because fish growth rates vary between laboratories. Two hypotheses were tested: (1) that BCFs of multiple chemicals can be measured simultaneously in one experiment, and (2) that internal benchmarking using a conservative test substance in the chemical mixture can be used to correct for growth dilution. Bioconcentration experiments were conducted following major elements of the OECD 305 guideline. Fish were simultaneously exposed to 11 chemicals selected to cover a range of BCFs and susceptibility to biotransformation. A method was developed to calculate the growth-corrected elimination rate constant from the concentration ratio of the analyte and a benchmarking chemical for which growth dilution dominated other elimination mechanisms. This method was applied to the experimental data using hexachlorobenzene as the benchmarking chemical. The growth dilution correction lowered the apparent elimination rate constants by between 5% and a factor of four for eight chemicals, while for two chemicals the growth-corrected elimination rate constant was not significantly different from zero. The benchmarking method reduced the uncertainty in the elimination rate constant compared to the existing method for growth dilution correction. The BCFs from exposing fish to 10 chemicals at once were consistent with BCF values from single-chemical exposures from the literature, supporting hypothesis 1.

A flow-through passive dosing system for continuously supplying aqueous solutions of hydrophobic chemicals to bioconcentration and aquatic toxicity tests

A continuous supply of water with defined stable concentrations of hydrophobic chemicals is a requirement in a range of laboratory tests such as the OECD 305 protocol for determining the bioconcentration factor in fish. Satisfying this requirement continues to be a challenge, particularly for hydrophobic chemicals. Here we present a novel solution based on equilibrium passive dosing. It employs a commercially available unit consisting of ~16000 polydimethylsiloxane (PDMS) tubes connected to two manifolds. The chemicals are loaded into the unit by repeatedly perfusing it with a methanol solution of the substances that is progressively diluted with water. Thereafter the unit is perfused with water and the chemicals partition from the unit into the water. The system was tested with nine chemicals with logK(OW) ranging from 4.1 to 6.3. The aqueous concentrations generated were shown to be largely independent of the water flow rate, and the unit to unit reproducibility was within a factor of ~2. In continuous flow experiments the aqueous concentrations of most of the study chemicals remained constant over 8d. A model was assembled that allows prediction of the operating characteristics of the system from the logK(OW) or PDMS/water partition coefficient of the chemical. The system is a simple, safe, predictable and flexible tool that generates stable aqueous concentrations of hydrophobic chemicals.

Studies with oxythiamine to mimic reproduction disorders among fish early life stages

Abstract Newly fertilised eggs of sea trout (Salmo trutta) were injected with the thiamine antagonist oxythiamine or with oxythiamine in combination with 3,3′,4,4′-tetrachlorobiphenyl (PCB #77). Rainbow trout (Oncorhynchus mykiss) eggs were injected with PCB #77. A decrease in liver transketolase activity was used as a biomarker to indicate a physiological deficiency in thiamine. Exposure to oxythiamine caused deformities in the newly hatched larvae, as well as high mortality during the larval stages. Exposure to oxythiamine in combination with PCB #77 caused a synergistic higher frequency of disorders. Analysis of liver transketolase activity in the larvae injected with PCB #77 indicated that PCB #77 did not decrease the half-life of thiamine in the offspring. The data obtained indicate that oxythiamine increased the sensitivity to PCB #77 in the offspring, however through mechanisms other than those causing thiamine deficiency in feral fish.