Waterborne pharmaceutical uptake and toxicity is modified by pH and dissolved organic carbon in zebrafish.

Human and veterinary pharmaceuticals have been observed in natural aquatic environments around the world, and many have been shown to impact fish health. Presently, we examined the influence of pH, dissolved organic carbon (DOC) and Na+ or Ca2+ on the bioavailability and toxicity of waterborne pharmaceuticals in larval zebrafish. Drugs included sertraline (selective serotonin reuptake inhibitor; SSRI), fluoxetine (SSRI), diclofenac (nonsteroidal anti-inflammatory drug) and ethinyl estradiol (estrogen; EE2). The 96 h-LC50s for sertraline, fluoxetine and diclofenac were influenced by pH over an environmentally relevant range (pH 5.8-8.2). Toxicity was related to the predicted concentration of non-ionized compounds, which more readily cross cell membranes than ionized compounds. For example, sertraline was 4.1-fold more toxic (as measured by 96 h-LC50s) at pH 8.2 compared to pH 5.8, while the predicted amount of non-ionized sertraline was also greater at pH 8.2 (based on previously reported pKa values). Experiments with radiolabelled drugs demonstrated that sertraline uptake was also 5.4-fold higher at pH 8.2 compared to pH 5.8. Terrigenous and autochthonous DOC samples (as low as 1 mg/L) protected against sertraline uptake and toxicity, although they were more effective at lower (environmentally relevant) drug concentrations. In contrast, the uptake of EE2, which was principally non-ionized in all water chemistries tested, was not altered by pH or DOC. There was no change in sertraline toxicity with the addition of 12 mM Na+ or 3 mM Ca2+. In conclusion, the influence of pH and DOC on drug uptake and toxicity in fish appears to be predictable based on the physicochemical properties of the drug (e.g. pKa, polar surface area). The influence of water chemistry on drug bioavailability in fish is likely relevant to all aquatic life.

The effects of chronic acetaminophen exposure on the kidney, gill and liver in rainbow trout (Oncorhynchus mykiss).

In this study, we examined if rainbow trout chronically exposed to acetaminophen (10 and 30 µgL-1) showed histological changes that coincided with functional changes in the kidney, gill and liver. Histological changes in the kidney included movement and loss of nuclei, non-uniform nuclei size, non-uniform cytoplasmic staining, and loss of tubule integrity. Histological effects were more severe at the higher concentration and coincided with concentration dependent increases in urine flow rate and increased urinary concentrations of sodium, chloride, potassium, calcium, urea, ammonia, glucose, and protein. Yet, glomerular filtration rate was not altered with acetaminophen exposure. In the gill, filament end swelling, whole filament swelling, and swelling of the lamellae were observed in exposed fish. Lamellar spacing decreased in both exposure groups, but lamellar area decreased only with 30 µgL-1 exposure. At faster swimming speeds, oxygen consumption was limited in acetaminophen exposed fish, and critical swimming speed was also decreased in both exposure groups. The liver showed decreased perisinusoidal spaces at 10 and 30 µgL-1 acetaminophen, and decreased cytoplasmic vacuolation with 30 µgL-1 acetaminophen. A decrease in liver glycogen was also observed at 30 µgL-1. There was no change in plasma concentrations of sodium, chloride, potassium, calcium, magnesium, and glucose with exposure, suggesting compensation for urinary loss. Indeed, an increase in Na+-K+-ATPase activity in the gills was found with 30 µgL-1 acetaminophen exposure. Chronic exposure of rainbow trout to the environmentally relevant pharmaceutical acetaminophen, alters both histology and function of organs responsible for ion and nutrient homeostasis.

In Vitro Biotransformation of Two Human CYP3A Probe Substrates and Their Inhibition during Early Zebrafish Development.

At present, the zebrafish embryo is increasingly used as an alternative animal model to screen for developmental toxicity after exposure to xenobiotics. Since zebrafish embryos depend on their own drug-metabolizing capacity, knowledge of their intrinsic biotransformation is pivotal in order to correctly interpret the outcome of teratogenicity assays. Therefore, the aim of this in vitro study was to assess the activity of cytochrome P450 (CYP)-a group of drug-metabolizing enzymes-in microsomes from whole zebrafish embryos (ZEM) of 5, 24, 48, 72, 96 and 120 h post-fertilization (hpf) by means of a mammalian CYP substrate, i.e., benzyloxy-methyl-resorufin (BOMR). The same CYP activity assays were performed in adult zebrafish liver microsomes (ZLM) to serve as a reference for the embryos. In addition, activity assays with the human CYP3A4-specific Luciferin isopropyl acetal (Luciferin-IPA) as well as inhibition studies with ketoconazole and CYP3cide were carried out to identify CYP activity in ZLM. In the present study, biotransformation of BOMR was detected at 72 and 96 hpf; however, metabolite formation was low compared with ZLM. Furthermore, Luciferin-IPA was not metabolized by the zebrafish. In conclusion, the capacity of intrinsic biotransformation in zebrafish embryos appears to be lacking during a major part of organogenesis.

Interactions of waterborne and dietborne Pb in rainbow trout, Oncorhynchus mykiss: Bioaccumulation, physiological responses, and chronic toxicity.

In Pb-contaminated environments, simultaneous exposure to both waterborne and dietborne Pb is likely to occur. This study examined the potential interactive effects of these two pathways in juvenile rainbow trout that were exposed to Pb in the water alone, in the diet alone, and in combination for 7 weeks. The highest waterborne Pb concentration tested (110µgL(-1)) was approximately equivalent to the 7-week LC20 (97µgL(-1)) measured in a separate trial, while the lowest was a concentration often measured in contaminated environments (8.5µgL(-1)). The live diet (10% daily ration on a wet mass basis) consisted of oligochaete worms (Lumbriculus variegatus) pre-exposed for 28days to the same waterborne Pb concentration, and the highest dietary dosing rate to the trout was 12.6µg Pb g fish(-1)day(-1). With waterborne exposure, whole body Pb burden increased to a greater extent in the worms than in the fish. Nonetheless, in trout waterborne exposure still resulted in 20-60-fold greater Pb accumulation compared to dietborne Pb exposure. However, combined exposure to both waterborne and dietborne Pb reduced the whole body accumulation extensively at waterborne Pb>50µgL(-1), with similar antagonistic interaction in liver and carcass (but not gill or gut) at a lower threshold of 20µgL(-1). Growth effects in trout were minimal with marginal reductions in the dietborne and combined exposures seen only at 110µgL(-1). Chronic Pb exposure reduced lipid and carbohydrates level in the worms by 50% and 80% respectively, while protein was unchanged, so growth effects in trout may have been of indirect origin. After 7 weeks, Ca(2+) homeostasis in the trout was unaffected, but there were impacts on Na(+). Blood Na(+) was reduced in waterborne and dietborne exposures, while gut Na(+)/K(+) ATPase activities were reduced in waterborne and combined exposures. This study is the first, to our knowledge to examine the interaction of waterborne and dietborne Pb exposure in fish. While physiological impacts of Pb were observed in both worms and fish, higher concentrations of dietborne Pb actually protected fish from waterborne Pb bioaccumulation and these effects. The impacts of metals on diet quality should not be neglected in future dietborne toxicity studies using live prey.

Reproductive impacts and physiological adaptations of zebrafish to elevated dietary nickel.

Nickel (Ni) concentrations in the environment can rise due to human industrial activities. The toxicity of waterborne Ni to aquatic animals has been examined in a number of previous studies; however, little is known about the impacts of elevated dietary Ni. In the present study, zebrafish were chronically fed diets containing two concentrations of Ni [3.7 (control) and 116 µg Ni/g diet]. Ni-exposed males, but not females, were significantly smaller (26%) compared to controls at 80 days. In addition, total egg production was decreased by 65% in the Ni treatment at 75-78 days of the experiment. Ni was ubiquitously distributed in control animals (similar to previous studies), and concentrations varied between tissues by 15-fold. Ni exposure resulted in modest but significant Ni accumulation in some tissues (increases were highest in brain, vertebrae and gut; 44%, 34% and 25%, respectively), an effect observed only at 80 days. The limited Ni accumulation may be due to (1) the lack of an acidified stomach in zebrafish and/or (2) the efficient upregulation of Ni transport and excretion mechanisms, as indicated by the 4.5-fold increase in waterborne (63)Ni uptake by Ni-exposed fish. Eggs from Ni-exposed adults had Ni concentrations that were 5.2-fold higher than controls. However, by 4 days post fertilization, larvae had similar Ni concentrations as controls, demonstrating a capacity for rapid Ni depuration. Larvae from Ni-exposed adults were also more resistant to waterborne Ni (35% increase in the 96-h LC50 over controls). In conclusion, elevated dietary Ni significantly affected zebrafish reproduction despite only modest tissue Ni accumulation. There were also indications of adaptation, including increased Ni uptake rates and increased Ni tolerance of offspring from Ni-exposed adults. Ni concentrations were particularly elevated in the brain with exposure; possible relations to growth and reproductive impacts require further study.

Metal and pharmaceutical mixtures: is ion loss the mechanism underlying acute toxicity and widespread additive toxicity in zebrafish?

The acute toxicities and mechanisms of action of a variety of environmental contaminants were examined using zebrafish larvae (Danio rerio; 4-8 days post fertilization). Toxic interactions were observed between metals. For example, the addition of a sublethal level of nickel (15% of the LC50, one third of the LC01) to all copper treatments decreased the copper 96 h LC50 by 58%, while sublethal copper exposure (6% of the copper LC50, 13% of the LC01) decreased the cadmium 96 h LC50 by 47%. Two predictive models were assessed, the concentration addition (CA) model, which assumes similar mechanisms of action, and the independent action (IA) model, which assumes different mechanisms of action. Quantitative comparisons indicated the CA model performed better than the IA model; the latter tended to underestimate combined toxicity to a greater extent. The effects of mixtures with nickel or ammonia were typically additive, while mixtures with copper or cadmium were typically greater than additive. Larvae exposed to cadmium, copper or nickel experienced whole body ion loss. Decreases were greatest for Na(+) followed by K(+) (as high as 19% and 9%, respectively, in 24h). Additive toxicity between copper and other pharmaceutical compounds such as fluoxetine (Prozac™), ß-naphthoflavone, estrogen and 17α-ethinylestradiol were also observed. Similar to metals, acutely toxic concentrations of fluoxetine, ß-naphthoflavone and ammonia all decreased whole body Na(+) and K(+). Overall, whole body Na(+) loss showed the greatest correlation with mortality across a variety of toxicants. We theorize that a disruption of ion homeostasis may be a common mechanism underlying the acute additive toxicity of many contaminants in fish.

Effects of copper on the acute cortisol response and associated physiology in rainbow trout.

The aim of this study was to determine the effects of chronic waterborne copper (Cu) exposure on the acute stress-induced cortisol response and associated physiological consequences in rainbow trout (Oncorhynchus mykiss). Trout were exposed to 30 µg Cu/L in moderately hard water (120 mg/L as CaCO(3)) for 40 days, following which time the acute cortisol response was examined with a series of stressors. At 40 days, a 65% increase in Cu was observed in the gill, but no accumulation was observed in the liver, brain or head kidney. Stressors such as air exposure or confinement did not elicit an increase in circulating cortisol levels for Cu-exposed fish, in contrast to controls. However, this inhibitory effect on the acute cortisol response appeared to have few implications on the ability of Cu-exposed fish to maintain ion and carbohydrate homeostasis. For example, plasma Na(+), Ca(2+) and glucose levels as well as hepatic glycogen levels were the same post-stress in control and Cu-exposed fish. Trout were also challenged with exposure to 50% seawater for 48 h, where Cu-exposed trout maintained plasma Na(+), glucose and hepatic glycogen levels. However, Cu-exposed fish experienced decreased plasma K(+) levels throughout the Cu exposure and stress tests. In conclusion, chronic Cu exposure resulted in the abolition of an acute cortisol response post-stress. There was no Cu accumulation in the hypothalamus-pituitary-interrenal axis (HPI axis) suggesting this was not a direct toxic effect of Cu on the cortisol regulatory pathway. However, the lack of an acute cortisol response in Cu-exposed fish did not impair the ability of the fish to maintain ion and carbohydrate homeostasis. This effect on cortisol may be a strategy to reduce costs during the chronic stress of Cu exposure, and not endocrine disruption as a result of toxic injury.

Metal uptake and acute toxicity in zebrafish: common mechanisms across multiple metals.

Zebrafish larvae (Danio rerio) were used to examine the mechanisms of action and acute toxicities of metals. Larvae had similar physiological responses and sensitivities to waterborne metals as adults. While cadmium and zinc have previously been shown to reduce Ca(2+) uptake, copper and nickel also decreased Ca(2+) uptake, suggesting that the epithelial transport of all these metals is through Ca(2+) pathways. However, exposure to cadmium, copper or nickel for up to 48 h had little or no effect on total whole body Ca(2+) levels, indicating that the reduction of Ca(2+) uptake is not the acute toxic mechanism of these metals. Instead, mortalities were effectively related to whole body Na(+), which decreased up to 39% after 48 h exposures to different metals around their respective 96 h LC50s. Decreases in whole body K(+) were also observed, although they were not as pronounced or frequent as Na(+) losses. None of the metals tested inhibited Na(+) uptake in zebrafish (Na(+) uptake was in fact increased with exposure) and the observed losses of Na(+), K(+), Ca(2+) and Mg(2+) were proportional to the ionic gradients between the plasma and water, indicating diffusive ion loss with metal exposure. This study has shown that there is a common pathway for metal uptake and a common mechanism of acute toxicity across groups of metals in zebrafish. The disruption of ion uptake accompanying metal exposure does not appear to be responsible for the acute toxicity of metals, as has been previously suggested, but rather the toxicity is instead due to total ion loss (predominantly Na(+)).

Grizzly bear corticosteroid binding globulin: Cloning and serum protein expression.

Serum corticosteroid levels are routinely measured as markers of stress in wild animals. However, corticosteroid levels rise rapidly in response to the acute stress of capture and restraint for sampling, limiting its use as an indicator of chronic stress. We hypothesized that serum corticosteroid binding globulin (CBG), the primary transport protein for corticosteroids in circulation, may be a better marker of the stress status prior to capture in grizzly bears (Ursus arctos). To test this, a full-length CBG cDNA was cloned and sequenced from grizzly bear testis and polyclonal antibodies were generated for detection of this protein in bear sera. The deduced nucleotide and protein sequences were 1218 bp and 405 amino acids, respectively. Multiple sequence alignments showed that grizzly bear CBG (gbCBG) was 90% and 83% identical to the dog CBG nucleotide and amino acid sequences, respectively. The affinity purified rabbit gbCBG antiserum detected grizzly bear but not human CBG. There were no sex differences in serum total cortisol concentration, while CBG expression was significantly higher in adult females compared to males. Serum cortisol levels were significantly higher in bears captured by leg-hold snare compared to those captured by remote drug delivery from helicopter. However, serum CBG expression between these two groups did not differ significantly. Overall, serum CBG levels may be a better marker of chronic stress, especially because this protein is not modulated by the stress of capture and restraint in grizzly bears.

Adrenocorticotropic hormone suppresses gonadotropin-stimulated estradiol release from zebrafish ovarian follicles.

While stress is known to impact reproductive performance, the pathways involved are not entirely understood. Corticosteroid effects on the functioning of the hypothalamus-pituitary-gonadal axis are thought to be a key aspect of stress-mediated reproductive dysfunction. A vital component of the stress response is the pituitary secretion of adrenocorticotropic hormone (ACTH), which binds to the melanocortin 2 receptor (MC2R) in the adrenal glands and activates cortisol biosynthesis. We recently reported MC2R mRNA abundance in fish gonads leading to the hypothesis that ACTH may be directly involved in gonadal steroid modulation. Using zebrafish (Danio rerio) ovarian follicles, we tested the hypothesis that acute ACTH stimulation modulates cortisol and estradiol (E(2)) secretion. ACTH neither affected cortisol nor unstimulated E(2) release from ovarian follicles. However, ACTH suppressed human chorionic gonadotropin (hCG)-stimulated E(2) secretion in a dose-related manner, with a maximum decrease of 62% observed at 1 I.U. ACTH mL(-1). This effect of ACTH on E(2) release was not observed in the presence of either 8-bromo-cAMP or forskolin, suggesting that the mechanism(s) involved in steroid attenuation was upstream of adenylyl cyclase activation. Overall, our results suggest that a stress-induced rise in plasma ACTH levels may initiate a rapid down-regulation of acute stimulated E(2) biosynthesis in the zebrafish ovary, underscoring a novel physiological role for this pituitary peptide in modulating reproductive activity.

Molecular programming of the corticosteroid stress axis during zebrafish development.

The functions of the hypothalamus-pituitary-interrenal (HPI) axis in teleosts have been studied primarily in juvenile and adult fish, whereas little is known about the molecular events leading to the onset of the stressor-induced cortisol response during development. Here we summarize a number of studies that have examined changes in the expression of genes encoding proteins critical for the functioning of the HPI axis, and the associated cortisol response in developing zebrafish embryos and larvae. The mRNA transcripts for some of these genes, including corticotropin releasing factor (CRF), proopiomelanocortin (POMC), melanocortin 2 receptor (MC2R), steroidogenic acute regulatory protein (StAR) and cytochrome P450 side chain cleavage (P450scc) have been detected during embryogenesis prior to hatch. The mRNA levels of MC2R, StAR and P450scc are up-regulated immediately prior to the dramatic rise in basal larval cortisol levels after hatch. Although all the components of the HPI axis are expressed and cortisol is synthesized at hatch, a stressor-induced cortisol response was not evident until 97 hpf. We hypothesize that this disconnect in the timing of the basal cortisol synthesis and stressor-induced cortisol synthesis is due to the delayed development of peripheral and central neural inputs relaying stressor stimuli to the hypothalamus. Overall, zebrafish appear to be an excellent model for elucidating the molecular mechanisms leading to the development of the corticoid stress axis in vertebrates.

The zebrafish stress axis: molecular fallout from the teleost-specific genome duplication event.

The teleost-specific whole genome duplication event 350 million years ago resulted in a variety of duplicated genes that exist in fish today. In this review, we examine whether molecular components involved in the functioning of the hypothalamus-pituitary-interrenal (HPI) axis are present as single or duplicate genes. Specifically, we looked at corticotropin releasing hormone (CRH), adrenocorticotropic hormone (ACTH) and the glucocorticoid receptor (GR). The focus is on zebrafish but a variety of species are covered whenever data is available through literature or genomic database searches. Duplicate CRH genes are retained in the salmoniformes and cypriniformes, and the peptide sequences are very similar or identical. Zebrafish, along with the Acanthopterygii, are the exceptions as they have a single CRH gene. Also, two copies of the proopiomelanocortin (POMC) gene, which encodes for ACTH and other peptides, have been observed in all teleosts except tilapia and sea bass. In zebrafish, ACTH is derived from only one POMC gene, since the cleavage site is mutated in the other gene. All teleosts examined to date have two GRs, including the recent discoveries of duplicate GRs in two species of cyprinids (carp and fathead minnow). Zebrafish are the only known exception with one GR gene. The loss of duplicate genes is not a general feature of the zebrafish genome, but zebrafish have lost the duplicate CRH, ACTH and GR genes in the past 33 million years, after possessing two of each for the previous 300 million years. The evolutionary pressures underlying the rapid loss of these HPI axis genes, and the implications on the development and the functioning of the evolutionarily conserved cortisol stress response in zebrafish are currently unknown.

Retinoid requirements in the reproduction of zebrafish.

This study examines whether retinoids are essential in the reproduction of zebrafish. Using RT-PCR, it was shown that the ovaries and testes express enzymes that synthesize and metabolize the hormone retinoic acid (RA) (raldh2 and cyp26a, respectively), and RA receptors (raraa, rarga, rxrba, rxrbb, rxrga but not rxrab). Three new isoforms of rxrba were also observed in a variety of tissues. In other experiments, zebrafish were exposed for 11 d to diethylaminobenzaldehyde (DEAB), an inhibitor of RA synthesis, or fed a retinoid deficient diet for 130 d in order to evaluate the functional requirements of retinoids in reproduction. DEAB altered cyp26a transcript numbers in the gonads, suggesting an impact on RA, and decreased the number of spawned eggs by 95%. The retinoid deficient diet decreased whole body retinoids (retinol and retinal) by 68% in females and 33% in males. Females fed the retinoid deficient diet also produced 73% fewer eggs that contained 78% less retinal than controls. Fertilization rates were not affected. These studies have shown that the RA receptors are expressed in zebrafish gonads, and RA is required for the spawning of eggs. Dietary retinoid content influences reproduction, while retinyl ester storage levels appear to be of little significance. Females were more susceptible to retinoid perturbation than males, likely due to the cost of retinal deposition in the eggs. Overall, these studies have shown retinoids play a fundamental role in the reproduction of zebrafish, and the lack of retinyl ester stores in controls that successfully spawned illustrates that we have only a limited understanding of the retinoid physiology and requirements of fish.

Development of the corticosteroid stress axis and receptor expression in zebrafish.

Using zebrafish embryos and larvae, we examined the temporal patterns of cortisol and expression of genes involved in corticosteroid synthesis and signaling. Embryonic cortisol levels decreased approximately 70% from 1.5 h postfertilization (hpf) to hatch (approximately 42 hpf) and then increased 27-fold by 146 hpf. The mRNA abundances of steroidogenic acute regulatory protein, 11beta-hydroxylase and 11beta-hydroxysteroid dehydrogenase type 2, increased severalfold after hatch and preceded the rise of cortisol levels. In contrast to other teleosts that possess two glucocorticoid receptors (GRs) and one mineralocorticoid receptor (MR), only one GR and MR were identified in zebrafish, which were cloned and sequenced. GR mRNA abundance decreased from 1.5 to 25 hpf, rebounded, and then was stable from 49 to 146 hpf. MR transcripts increased continuously from 1.5 hpf and were 52-fold higher by 97 hpf. An acute cortisol response to a stressor was not detected until 97 hpf, whereas melanocortin type 2 receptor mRNA increased between 25 and 49 hpf. Collectively, the patterns of cortisol and the expression of cortisol biosynthetic genes and melanocortin type 2 receptor suggest that the corticoid stress axis in zebrafish is fully developed only after hatch. The temporal differences in GR, MR, and 11beta-hydroxysteroid dehydrogenase type 2 gene expression lead us to propose a key role for MR signaling by maternal cortisol during embryogenesis, whereas cortisol secretion after hatch may be regulating GR expression and signaling in zebrafish.

Influence of acclimation and cross-acclimation of metals on acute Cd toxicity and Cd uptake and distribution in rainbow trout (Oncorhynchus mykiss).

The development of chronic metal toxicity models for fresh water fish is complicated by the physiological adjustments made by the animal during exposure which results in acclimation. This study examines the influence of a pre-exposure to a chronic sublethal waterborne metal on acclimation responses as well as the uptake and distribution of new metal into juvenile rainbow trout. In one series of tests, trout were exposed to either 20 or 60 microg/L Cu, or 150 microg/L Zn for a month in moderately hard water and then cross-acclimation responses to Cd were measured in 96 h LC(50) tests. Cu exposed trout showed a cross-acclimation response but Zn exposed trout did not. Using these results, a detailed examination of Cd uptake and tissue distribution in metal-acclimated trout was done. Trout were exposed to either 75 microg/L Cu or 3 microg/L Cd for 1 month to induce acclimation and subsequently, the uptake and distribution of new Cd was assessed in both Cd- and Cu-acclimated fish using (109)Cd. The pattern of accumulation of new metal was dramatically altered in acclimated fish. For example, in 3 h gill Cd binding experiments, Cd- and Cu-acclimated trout both had a higher capacity to accumulate new Cd but only Cu-acclimated fish showed a higher affinity for Cd compared to unexposed controls. Experiments measuring Cd uptake over 72 h at 3 microgCd/L showed that the Cd uptake rate was lower for Cd-acclimated fish compared to both Cu-acclimated fish and unexposed controls. The results demonstrate the phenomenon of cross-acclimation to Cd and that chronic sublethal exposure to one metal can alter the uptake and tissue distribution of another. Understanding how acclimation influences toxicity and bioaccumulation is important in the context of risk assessment. This study illustrates that knowledge of previous exposure conditions is essential, not only for the metal of concern, but also for other metals as well.

The effects of copper and benzo[a]pyrene on retinoids and reproduction in zebrafish.

This study examines whether a link exists between toxicant exposure, retinoids and reproduction in fish. Zebrafish were fed a control diet (8.1 microg Cu/g diet, 0 microg benzo[a]pyrene/g diet) or diets containing elevated copper (100 microg, 500 microg and 1000 microg Cu/g diet) or benzo[a]pyrene (B[a]P; 30 and 150 microg B[a]P/g diet) for 260 days. Toxicant-supplemented diets did not affect growth or mortality rates. While whole body retinoid levels in control zebrafish decreased during the experiment, females exposed to Cu or B[a]P for 200 days or more experienced additional losses of retinyl esters (45-100% depleted) and retinal (45% depleted in B[a]P-fed fish). Despite the reduced retinoids, Cu and B[a]P did not effect reproduction with respect to the number of eggs spawned, fertilization rates or egg retinal content (retinal was instead increased 55-65% in eggs from B[a]P-fed fish). There were no apparent deformities observed in 36 h post fertilization embryos from any treatment. It appears that although internal retinoid stores were depleted in adults, dietary retinoids were sufficient to meet the daily requirement for retinal deposition in the eggs and retinoic acid synthesis. This study has shown that retinoid levels in female zebrafish are sensitive to Cu and B[a]P, and are a good indicator of long-term exposure. It also brings to light the resiliency of the retinoid system in fish and the importance of the diet on the toxicological response. Specifically that dietary retinoids appear to support normal reproduction in the absence of internal retinoid stores.

Dietary retinoic acid induces hindlimb and eye deformities in Xenopus laevis.

This study investigated the effects of dietary retinoic acid (RA) on frog hindlimb development. Xenopus laevis (African clawed frog) tadpoles were fed a diet supplemented with 0, 1, 10, or 100 microg of RA/g of food for 2 or 5 d at different stages of metamorphosis. Hindlimb deformities were induced in the group fed 100 microg of RA/g of diet for 5 d. Exposures beginning at mid-hindlimb bud development induced bilaterally bent tibiafibula (bony triangles), while exposures later in hindlimb development induced deformities of the feet, including fusion of the 1st and 2nd clawed digits and reduced length of the 4th and 5th digits (due to reduced, missing, or misplaced phalanges). There were also cases of extra phalanges in the 5th digit. The eye was another target of RA exposure. In one experiment, 58% of the tadpoles fed 10 microg of RA/g had a smaller or absent right eye. Additionally, 11% of the tadpoles fed 100 microg of RA/g of diet developed a smaller or absent left eye. Waterborne heavy metals (Zn or Cu) modified RA effects on the hindlimb and eye. Co-exposure to metals and RA resulted in cases of unilateral bony triangles and reduced rates of smaller eyes. There were also cases of extra hindlimb digits in Zn-exposed animals. Dietary RA exposure in tadpoles can cause some deformities that differ from waterborne RA exposures in previous studies. RA also induced deformities that resemble those in affected wild frog populations (bony triangles), although the patterns of other deformities and missing segments (phalanges and metatarsals) are not similar to those documented in the wild.

Constituents within pulp mill effluent deplete retinoid stores in white sucker and bind to rainbow trout retinoic acid receptors and retinoid X receptors.

Wild female and male white sucker (Catostomus commersoni) inhabiting an area receiving pulp mill effluent had reduced hepatic levels of retinol, didehydroretinol, retinyl esters, and didehydroretinyl esters, while vitamin E levels were unaffected. This disruption of the retinoid system led us to test methanol and dichloromethane extracts from the effluent of 11 pulp mills from across Canada for their ability to bind to rainbow trout (Oncorhynchus mykiss) retinoic acid receptors (RARs) from the gill and retinoid X receptors (RXRs) from the liver. Concentrated extracts of the final effluent from 6 of the 11 pulp mills were able to displace greater than 25% of the receptor-bound [3H]all-trans retinoic acid (RA) or [3H]9-cis RA from trout RARs and RXRs, respectively. The ability of the extracts to displace retinoic acid did not appear to be linked to the pulping or treatment processes. Moreover, extracts with the greatest activity came from thermomechanical mills, suggesting the compounds may originate from the wood furnish. In addition, extracts prepared from wood furnish (wood chips: white spruce [50%], lodgepole pine [47%], and balsam fir [3%]) from one mill were able to displace [3H]RA from the RARs and RXRs. The 4-hydroxy RA, a metabolite of RA that has been shown to be generated in greater quantities in fish exposed to P450-inducing xenobiotics, was able to displace [3H]all-trans RA from trout RARs as effectively as unlabeled all-trans RA. These results suggest that pulp mill effluent may impact the retinoid system of fish at multiple sites, either by decreasing hepatic retinoid stores or through contributing additional ligands (from the wood furnish) that can bind to RA receptors.