Brevetoxin forms covalent DNA adducts in rat lung following intratracheal exposure

BACKGROUND: Human exposure to brevetoxins produced by the red tide organism, Karenia brevis, is an increasing public health concern. Using in vitro exposure of rat liver cells to brevetoxin B (PbTx-2), the primary toxin product of K. brevis, we previously showed that it formed C(27,28)-epoxy brevetoxin metabolites capable of covalently binding to nucleic acids, a common initiation step for carcinogenesis. OBJECTIVE: This study was undertaken to evaluate nucleic acid adduction in lung following in vitro and in vivo brevetoxin exposures. METHODS: To clarify reactions of brevetoxin epoxide with DNA, we analyzed reaction products of PbTx-6 (a C(27,28) epoxide metabolite of brevetoxin B) with nucleosides. We also analyzed adducts from nucleic acid hydrolysates of isolated rat lung cells treated with PbTx-2 or PbTx-6 in vitro and lung tissue from rats after intratracheal exposure to PbTx-2 or PbTx-6 at 45 microg toxin/kg body weight. RESULTS: Our results indicate that PbTx-2 forms DNA adducts with cytidine after treatment of isolated lung cells, and forms DNA adducts with adenosine and guanosine after intratracheal exposure. CONCLUSIONS: These results are consistent with metabolic activation of highly reactive brevetoxin intermediates that bind to nucleic acid. These findings provide a basis for monitoring exposure and assessing the hazard associated with depurination of brevetoxin-nucleotide adducts in lung tissue.     

The type B brevetoxin (PbTx-3) adversely affects development, cardiovascular function, and survival in Medaka (Oryzias latipes) embryos

Brevetoxins are produced by the red tide dinoflagellate Karenia brevis. The toxins are lipophilic polyether toxins that elicit a myriad of effects depending on the route of exposure and the target organism. Brevetoxins are therefore broadly toxic to marine and estuarine animals. By mimicking the maternal route of exposure to the oocytes in finfish, we characterized the adverse effects of the type B brevetoxin brevetoxin-3 (PbTx-3) on embryonic fish development and survival. The Japanese rice fish, medaka (Oryzias latipes), was used as the experimental model in which individual eggs were exposed via microinjection to various known concentrations of PbTx-3 dissolved in an oil vehicle. Embryos injected with doses exceeding 1.0 ng/egg displayed tachycardia, hyperkinetic twitches in the form of sustained convulsions, spinal curvature, clumping of the erythrocytes, and decreased hatching success. Furthermore, fish dosed with toxin were often unable to hatch in the classic tail-first fashion and emerged head first, which resulted in partial hatches and death. We determined that the LD(50) (dose that is lethal to 50% of the fish) for an injected dose of PbTx-3 is 4.0 ng/egg. The results of this study complement previous studies of the developmental toxicity of the type A brevetoxin brevetoxin-1 (PbTx-1), by illustrating in vivo the differing affinities of the two congeners for cardiac sodium channels. Consequently, we observed differing cardiovascular responses in the embryos, wherein embryos exposed to PbTx-3 exhibited persistent tachycardia, whereas embryos exposed to PbTx-1 displayed bradycardia, the onset of which was delayed.     

Uptake and elimination of brevetoxin in blood of striped mullet (Mugil cephalus) after aqueous exposure to Karenia brevis

There is a critical need to simply and reliably monitor brevetoxins routinely in the blood of humans and aquatic animals. We used striped mullet as laboratory test animals to better define the uptake and elimination kinetics of brevetoxin during an aqueous exposure to the brevetoxin-producing dinoflagellate Karenia brevis. Striped mullet were first exposed to sublethal densities of K. brevis (approximately 250,000 cells/L) for 1, 4, 8, 12, and 24 hr. No mortality was observed in the aquaria, and at each time point blood samples were taken and applied to blood collection cards for brevetoxin analysis using radioimmunoassay (RIA). The RIA indicated that blood levels of brevetoxin (PbTx-3) increased to values significantly different from that of the controls at all five time points during exposure (p < 0.05). Striped mullet were then exposed to a K. brevis culture with a known brevetoxin concentration of 0.5 ng/mL. Even after exposures at a low brevetoxin concentration, RIA was able to detect 2.25 +/- 0.62 ng/mL PbTx-3 equivalents in the blood of the mullet at 8 hr of exposure. When exposed to higher brevetoxin concentrations (3.5 and 5.4 ng/mL), blood brevetoxin increased to peak levels at 12 hr and then reached equilibrium after 24 hr in the continued presence of K. brevis. During this time of equilibrium, the mullet maintained brevetoxins with a blood:water coefficient of 2.2. To define the elimination of brevetoxin, striped mullet were next exposed for 8-10 hr and then transferred to fresh seawater containing no K. brevis for up to 116 hr. Blood brevetoxin levels remained elevated and decreased only by 50% 116 hr after transfer. The rate of elimination fit best to a two-phase exponential decay with a biologic half-life of 12 and 266 hr. This study, using RIA in conjunction with blood collection cards, demonstrates an effective means to monitor blood brevetoxin levels in finfish and provides a foundation to characterize biologically relevant levels of brevetoxin in other species impacted by red tide events.     

Biomonitoring brevetoxin exposure in mammals using blood collection cards

A method has been tested in laboratory mice to monitor for the presence of brevetoxins in blood after exposure. The use of blood collection cards is an adaptation of a method employed for routine diagnostic and genetic testing of newborns. Blood is collected and applied to a 0.5-inch diameter circle on a specially prepared blood collection card and allowed to dry. The blood spots are then extracted and the presence of toxin activity is first screened using a high throughput receptor binding assay. Positive samples are then examined for specific brevetoxin congeners by liquid chromatography-tandem mass spectrometry. Preliminary experiments tested the efficiency and linearity of toxin extraction from blood spiked with brevetoxin-3 (PbTx-3). Blood from treated mice was tested for the presence of brevetoxin at different times following exposure to a sublethal dose (180 microg/kg PbTx-3). Brevetoxin activity determined by receptor assay increased to 25 +/- 7.4 nM PbTx-3 equivalents within 4 hr after exposure and was still detectable in three of four animals 24 hr after exposure. Tandem mass spectrometry provided confirmation of PbTx-3, which also increased for the time points between 0.5 and 4.0 hr exposure. However, PbTx-3 was not detected at 24 hr, which suggested the formation of a biologically active metabolite. We anticipate that this approach will provide a method to biomonitor brevetoxins in living marine resources (e.g., finfish), protected species, and humans.     

Measurement of brevetoxin levels by radioimmunoassay of blood collection cards after acute, long-term, and low-dose exposure in mice

We developed a radioimmunoassay (RIA) using a sheep anti-brevetoxin antiserum to evaluate detection of brevetoxin on blood collection cards from mice treated with the brevetoxin congener PbTx-3. The RIA has high affinity for PbTx-3 [half-maximal effective concentration (EC(50)) +/- SE = 1.2 +/- 0.2 nM; n = 10] and recognizes both type 1 and type 2 brevetoxins, but not ciguatoxin. Direct comparison of the RIA with a radiolabeled [(3)H]-PbTx-3 receptor-binding assay (RBA) revealed excellent sensitivity, congener selectivity, and minimal interference from blood matrix. We first analyzed blood samples from an acute time course exposure, using a maximal nonlethal dose [180 microg/kg body weight (bw)] for 0.5, 1, 2, 4, and 24 hr. Mean blood brevetoxin levels were 36 nM at 30 min and stayed above 20 nM during the 1-4 hr time points. We next analyzed blood brevetoxin levels after longer exposure (0.5, 1, 2, 3, 4, or 7 days). Mean blood brevetoxin levels were 26.0 nM at 0.5 days, decreased to 8.2 nM at 1.0 day, and maintained a significant level (p < 0.05) of 1.3 nM at day 2. We next determined the lowest measurable dose using increasing concentrations of PbTx-3 (10-300 micro g/kg bw). Analysis of the blood samples at 60 min revealed a linear relationship between administered and internal doses (r(2) = 0.993). All doses of brevetoxin administered were detectable at 1 hr, with significant levels found for the lowest administered dose of 10 micro g/kg bw--a dose that was 10-fold lower than the lowest observable effect level. This RIA provides an optimal first-tier detection of brevetoxin from blood collection cards and, used in combination with the RBA and liquid chromatography-mass spectrometry, should provide a complete panel of methods to biomonitor brevetoxin exposure.     

The red tide toxin, brevetoxin, induces embryo toxicity and developmental abnormalities

Brevetoxins are lipophilic polyether toxins produced by the red tide dinoflagellate Gymnodinium breve, and their neurotoxic effects on adult animals have been documented. In this study, we characterized adverse developmental effects of brevetoxin-1 (PbTx-1) using an exposure paradigm that parallels the maternal oocyte transfer of toxin. Medaka fish (Oryzias latipes) embryos were exposed to PbTx-1 via microinjection of toxin reconstituted in a triolein oil droplet. Embryos microinjected with doses of 0.1-8.0 ng/egg (ppm) of brevetoxin-1 exhibited pronounced muscular activity (hyperkinesis) after embryonic day 4. Upon hatching, morphologic abnormalities were commonly found in embryos at the following lowest adverse effect levels: 1.0-3.0 ppm, lateral curvature of the spinal column; 3.1-3.4 ppm, herniation of brain meninges through defects in the skull; and 3.4-4.0 ppm, malpositioned eye. Hatching abnormalities were also commonly observed at brevetoxin doses of 2.0 ppm and higher with head-first, as opposed to the normal tail-first, hatching, and doses > 4.1 ng/egg produced embryos that developed but failed to hatch. Given the similarity of developmental processes found between higher and lower vertebrates, teratogenic effects of brevetoxins have the potential to occur among different phylogenetic classes. The observation of developmental abnormalities after PbTx-1 exposure identifies a new spectrum of adverse effects that may be expected to occur following exposure to G. breve red tide events.     

Distribution of brevetoxin (PbTx-3) in mouse plasma: association with high-density lipoproteins

We investigated the brevetoxin congener PbTx-3 to determine its distribution among carrier proteins, including albumin and blood lipoproteins. Using a radiolabeled brevetoxin tracer (PbTx-3), we found that 39% of the radiolabel remained associated with components in mouse plasma after > 15 kDa cutoff dialysis. Of this portion, only 6.8% was bound to serum albumin. We also examined the binding of brevetoxin to various lipoprotein fractions. Plasma, either spiked with PbTx-3 or from mice treated for 30 min with PbTx-3, was fractionated into different-sized lipoproteins by iodixanol gradient ultracentrifugation. Each fraction was then characterized and quantified by agarose gel electrophoresis and brevetoxin radioimmunoassay, respectively. In both the in vitro and in vivo experiments, the majority of brevetoxin immunoreactivity was restricted to only those gradient fractions that contained high-density lipoproteins (HDLs). Independent confirmation of brevetoxin binding to HDLs was provided by high molecular weight (100 kDa cutoff) dialysis of [3H]PbTx-3 from lipoprotein fractions as well as a scintillation proximity assay using [3H]PbTx-3 and purified human HDLs. This information on the association of brevetoxins with HDLs provides a new foundation for understanding the process by which the toxin is delivered to and removed from tissues and may permit more effective therapeutic measures to treat intoxication from brevetoxins and the related ciguatoxins.     

Response and Recovery of Brain Acetylcholinesterase Activity in the European Eel,Anguilla anguilla,Exposed to Fenitrothion

European eel (Anguilla anguilla) were exposed to sublethal fenitrothion concentrations in a continuous flow-through system for 4 days. Brain acetylcholinesterase (AChE) activity was evaluated after 2, 8, 12, 24, 32, 48, 56, 72, and 96 h pesticide exposure. Results indicated that AChE activity in eel brains decreased as the concentration of fenitrothion increased. The pesticide induced significant inhibitory effects on the AChE activity ofA. anguilla,ranging from >40% inhibition at a sublethal concentration of 0.02 ppm to >60% inhibition at a sublethal concentration of 0.04 ppm. Eel were exposed to both fenitrothion concentrations for 96 h and then allowed a period of recovery in pesticide-free water. Samples were removed at 8, 12, 24, 48, 72, 96, 144, and 192 h and eel brain AChE activity was evaluated. Following 1 week of recovery, the AChE activity of those animals previously exposed to 0.02 and 0.04 ppm fenitrothion was still different from that of the controls. So, the AChE activity of eel brains at the end of the recovery phase remained significantly depressed.     

Changes in brain dopamine levels,oocyte growth and spermatogenesis in rainbow trout,Oncorhynchus mykiss,following sublethal cyanide exposure

Whole brain dopamine (DA) and norepinephrine (NE) levels were measured in sexually maturing (2 years+) male and female rainbow trout,Oncorhynchus mykiss, following posure to 0.01 mg/L hydrogen cyanide (HCN). Following a 12 day exposure period in July and August 1988, whole brain DA levels of HCN exposed fish were significantly higher (p < 0.05), relative to control fish, as measured by high performance liquid chromatography (HPLC). Brain NE levels were unaffected by HCN exposure. Whole brain DA and NE levels showed a strong correlation in control fish (r=+0.81), but not in HCN exposed fish (r=+0.28), likely due to altered DA levels in the latter group. No significant differences were found in brain DA and NE levels between males and females. Mean diameters of oocyte from ovaries of the vitellogenic females were significantly (p < 0.01) reduced from 226 to 183 m in control and HCN exposed fish respectively. Testes from males revealed significantly (p < 0.001) higher numbers of spermatogonial cysts in HCN exposed fish. Evidence is given that chronic exposure to sublethal levels of HCN significantly alters brain DA levels in both sexes of rainbow trout, reduces growth in vitellogenic oocytes of the ovary in females and interferes with the passage of spermatogonia to the spermatocyte stage in sexually maturing males. Collectively, these results suggest that sublethal HCN affects the reproductive mechanisms via the hypophyseal-gonad axis in sexually maturing rainbow trout.     

Bioaccumulation factor for 32P measured in bluegill, Lepomis macrochirus, and catfish, Ictalurus punctatus

The ratio of the bioaccumulation factors for 32P and phosphorus was determined for edible tissue in two species of freshwater fish by measuring the specific activity (32P activity per milligram phosphorus) in muscle relative to feed. The 32P tracer was added to the feed at a uniform level throughout the study. Feeding was at two levels: ad libitum and at a lower but constant intake per body weight. In the main experiment, bluegill were maintained in a large flow-through tank and sacrificed at approximately weekly intervals for 51 d of 32P accumulation and 28 d of depuration to compare the specific activity with values predicted with a calculational model. In experiments performed in smaller aquaria, the specific activity in bluegill and catfish muscle was compared at two feeding levels and two temperatures. In addition, unfed fish were exposed to 32P in water at a known specific activity to determine the extent of phosphorus uptake directly from water. The pattern of specific activity increase and decrease in fish muscle during the accumulation/depuration experiment was consistent with a one-compartment model, so that specific activity ratios at steady state could be predicted from measurements during relatively brief exposures. On this basis, the ratio of the bioaccumulation factors of 32P and phosphorus in fish feeding ad libitum was 0.081 for bluegill and 0.17 for catfish. Hence, at a mean phosphorus bioaccumulation factor of 70,000, the factors for 32P are 6,000 and 12,000, respectively. The ratios were less at lower phosphorus intakes associated with lower feeding rates; moreover, the lesser value for bluegill occurred at a much lower phosphorus intake than by catfish. The bioaccumulation factor ratio was lower by an order of magnitude at a water temperature of 11 degrees C than at 16-27 degrees C, and was lower by two orders of magnitude when phosphorus uptake was from water by unfed fish.     

Lysosomal enzyme release in the bluegill sunfish (Lepomis macrochirus Rafinesque) exposed to cadmium

Lysosomal membrane lability and enzyme activities were measured in gill and liver tissues of the bluegill sunfish (Lepomis macrochirus). Exposure to sublethal cadmium concentrations for 10 or 21 days resulted in significant increases in the lability of liver lysosomal membranes, as measured by release of N-acetyl--D-glucosaminidase (NAG) and acid phosphatase (ACP). Acid phosphatase and NAG activities in liver were decreased by 10 day exposure to cadmium. However, after 21 days of exposure, liver NAG activity was elevated. Cadmium exposure did not affect the lysosomal lability index or the total enzyme activity of lysosomes isolated from gill tissue.In vitro exposure of isolated liver lysosomes to cadmium demonstrated that the increase in membrane lability was not a direct effect of cadmium on the lysosome. Stress induced by crowding the fish had no effect on liver lysosomal membrane lability. Starved bluegill sunfish had significantly destabilized lysosomes, as measured by both NAG and ACP. However, starvation did not affect the total activity of either enzyme. Neither size nor sex had any effect on lysosomal membrane lability, which indicates that field experiments will not be confounded by these factors. The problem of metal bioconcentration in fish has created the need for diagnostic tests which can be utilized to understand the biological effects of these contaminants on fish. The lysosomal membrane lability assay currently holds promise for use as a health monitoring tool.     

Inhibition of acetylcholinesterase in guppies (Poecilia reticulata) by chlorpyrifos at sublethal concentrations: methodological aspects

Acetylcholinesterase activity is a potential biochemical indicator of toxic stress in fish and a sensitive parameter for testing water for the presence of organophosphates. A number of methodological aspects regarding the determination of the in vivo effect of chlorpyrifos on acetylcholinesterase in guppies have been investigated. It was found that with acetylthiocholine as a substrate, the contribution of pseudocholinesterase to the total cholinesterase activity can be neglected. Protection of acetylcholinesterase of guppies exposed to chlorpyrifos from additional, artifactual in vitro enzyme inhibition during homogenization is necessary. Very low concentrations of acetone in the exposure medium, resulting from dilution of the stock solution of chlorpyrifos in acetone, can result in large decreases in the oxygen content of this medium. This may affect the uptake rate of the toxic compound and, thereby, cholinesterase inhibition. Very low, sublethal concentrations of chlorpyrifos result in high inhibition levels of acetylcholinesterase (80-90%) in guppies within 2 weeks of continuous exposure. Recovery of the enzyme activity occurs after the exposed animals are kept in clean medium for 4 days, but the rate of recovery is considerably lower than the rate of inhibition.     

Effects of carbofuran, diuron, and nicosulfuron on acetylcholinesterase activity in goldfish (Carassius auratus)

Juvenile goldfish (Carassius auratus) were exposed to three widely used pesticides; carbofuran, diuron, and nicosulfuron. Acetylcholinesterase (AChE) activity and molecular forms of AChE were first characterized in brain and skeletal muscle of unexposed fish. Skeletal muscle had higher AChE activity than brain (306 and 215 nmol/min/mg protein, respectively). In brain, four molecular forms of AChE were found: A12, G4, G2, and G1. In the muscle, three molecular forms were found A12, A8, and G2. AChE activity was then evaluated in both tissues of fish exposed to different concentration of pesticides (5, 50, and 500 microg/L) for 6, 12, 24, and 48 h. In brain, AChE activity was significantly inhibited during all the periods of exposure in response to 50 microg/L (19-28%) and 500 microg/L (85-87%) carbofuran. Such effect was observed in the muscle only at 500 microg/L (86-92%). Carbofuran had no effect on the distribution of molecular forms. Significant inhibitions (9-12%) of brain AChE activity were also observed in response to diuron and nicosulfuron at 500 microg/L during all periods of exposure and for 50 microg/L nicosulfuron after 24 and 48 h. This study pointed out short-term effects of exposure to sublethal concentrations of the three pesticides, ranging among different chemical families, on brain and muscle AChE in goldfish.     

Acute lethality and effects of sublethal cadmium exposure on ventilation frequency and cough rate of bluegill (Lepomis macrochirus)

Several experiments were designed to determine the effects of sublethal cadmium exposure on ventilation rate and cough response of bluegill. Populations of test fish were selected to represent extremes in history of heavy metal exposures. Exposure to cadmium concentrations less than 1% of the reported median lethal threshold value produced significant increases in both ventilation frequency and cough rate. Changes in cough rates were directly proportional to the cadmium concentration. Cough response is an accurate short-term measure of chronic cadmium toxicity. There was good correlation between cadmium concentrations producing significant increases in cough rates and known MATC values. Based on acute toxicity studies and on changes in ventilation and cough rates, bluegill collected from a metal-contaminated lake did not exhibit increased tolerance to the toxic effects of cadmium.Research supported in part by funds from the U.S. Department of the Interior, Office of Water Research, Project Numbers A-036-IND and A-038-IND.     

Ethanol effects on 2-deoxyglucose uptake into tissues obtained from LS and SS mice

The uptake of 2-deoxy-D-[¹⁴C]glucose (2-DG) has been used with some success to identify neuronal sites of drug action, and has also been useful in identifying brain regions that are differentially sensitive to ethanol in selectively bred rat lines, the alcohol tolerant (AT) and alcohol nontolerant (ANT) lines. The studies reported here utilized the 2-DG method in an attempt to ascertain whether the LS and SS mouse lines, which were selectively bred for differences in sensitivity to high dose, anesthetic effects of ethanol, do so because of disruption of specific neuronal sites. LS and SS mice were injected with saline or a 4.0-g/kg dose of ethanol 15 min before injection with 2-deoxy-D-[¹⁴C]glucose, and uptakes into blood, eight brain regions, the liver, and adrenal tissues were measured 2–60 min afterwards. Ethanol produced statistically significant decreases in 2-DG uptake into every region of the LS mouse brain, but only three brain regions showed significant decreases in uptake in the SS brain. Other SS brain regions showed a trend towards decreased uptake, but these trends were not significant. A comparison of percent decrease in 2-DG uptake across all brain regions showed that ethanol decreased 2-DG uptake approximately twice as much in LS brain regions as in SS brain regions. Since 2-DG uptake into adrenal and hepatic tissue was not affected by ethanol injection in either mouse line, it seems likely that the decreased 2-DG uptake, seen more readily in the LS brain, is due to ethanol-induced central nervous system (CNS) depression. If so, these results suggest that the anesthetic effects of ethanol arise because of widespread disruption of brain function.     

Immune response in the tilapia, Oreochromis mossambicus on exposure to tannery effluent

The objective of the study was to investigate the effect of chronic exposure to sublethal concentrations of tannery effluent (TE) on the specific immune response and nonspecific immunity in tilapia, Oreochromis mossambicus. The effluent from the tannery was collected directly from a chrome-tanning factory situated in Dindigul district, Tamil Nadu, India. Apart from chromium (88.2 ppm), the effluent contained appreciable amount of calcium carbonate and sodium sulphate. Groups of fish (45-50 g) were exposed to 0.0053, 0.053 or 0.53% [0.1%, 1% or 10% LC50] of TE for 28 days. The specific immune response of fish was assessed by antibody response to heat-killed Aeromonas hydrophila by ELISA and bacterial agglutination assay. Nonspecific immune mechanisms were assessed in terms of serum lysozyme activity, production of intracellular reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI) by peripheral blood leucocytes (PBL). The results indicate that chronic exposure of fish to 0.53% of TE, significantly suppressed antibody response, nonspecific serum lysozyme activity, and ROS and RNI production. Exposure to 0.053% (1% LC50) of TE also caused a similar suppressive effect though at a lesser degree. In conclusion, the study shows, that exposure to sublethal concentrations of TE, can lead to adverse effects on selected immune reactions in tilapia. Further, these findings may be important in terms of monitoring fish health and risk assessment during periods of fluctuating levels of pollutants in the natural and farm environments.     

Biochemical and behavioral effects of diazinon exposure in hybrid striped bass

The effects of environmental stimuli on biochemical processes may influence behavior. Environmental contaminants that alter behavior can have major impacts on populations as well as community structures by changing species' interactions. One important behavior is the ability to capture prey. We hypothesized that sublethal exposure to diazinon, an organophosphate pesticide, may lead to feeding behavior abnormalities in hybrid striped bass (Morone saxatilis x M. chrysops) through inhibition of brain acetylcholinesterase (AChE) activity. This can potentially reduce the organism's survival by affecting its ability to find and capture food. To test this hypothesis, bass were exposed to diazinon for 6 d, followed by a 6-d recovery period in clean water. Brain AChE activity and the ability of bass to capture prey fathead minnows (Pimephales promelas) were measured every third day. Exposed fish exhibited a concentration- and duration-dependent decrease in ability to capture prey. While bass in all diazinon treatment groups had significantly inhibited brain AChE activity, only the medium and high treatment groups showed a dose- and time-dependent increase in time to capture prey. Acetylcholinesterase activity also decreased in an exposure duration- and concentration-dependent manner. The AChE levels in exposed fish did not recover to control levels during the 6-d recovery period. These results suggest that sublethal exposure to AChE-inhibiting substances may decrease the ecological fitness of hybrid striped bass by reducing their ability to capture prey.     

Reproductive endocrine disruption in the freshwater catfish, Heteropneustes fossilis, in response to the pesticide gamma-hexachlorocyclohexane

Both male and female freshwater catfish, Heteropneustes fossilis, were exposed to safe (SC; 0.1 and 1.0 mg/L) and sublethal (SL; 10 mg/L) concentrations of an agricultural pesticide, gamma-hexachlorocyclohexane (gamma-HCH) for 4 weeks during the active pre-spawning (vitellogenic) phase of their annual reproductive cycle. On the last day of exposure, 18 h before killing, fish were treated intramuscularly (i.m.) with [I-14C]acetic acid (74 kBq per fish). After 4 weeks of exposure, we monitored the effects of gamma-HCH on gonadosomatic index (GSI); on plasma concentrations of gonadotropin (GtH), testostosterone (T), 11-ketotestosterone (11-KT), 17beta-estradiol (E2); and on hepatic incorporation of [I-14C]acetic acid into total phospholipids (TP) and the fractions thereof: phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI), and phosphatidylethanolamine (PE). In both the sexes, GSI and plasma GtH were decreased significantly in response to gamma-HCH exposure. Plasma T and 11-KT in males, and plasma T and E2 in females declined significantly in response to gamma-HCH exposure. In both the sexes, hepatic incorporation of [I-14C]acetic acid into PS and PI increased significantly, whereas incorporation into TP, PC, and PE was significantly reduced after gamma-HCH exposure. Our findings demonstrated that gamma-HCH exposure depressed GSI, plasma GtH, sex steroids, and [I-14C]acetic acid incorporation into hepatic TP, and had very selective and specific effects on various classes of TP, resulting either from the hypothalamo-hypophyseal-gonadal axis or from direct action on hepatic and steroidogenic enzymes during the pre-spawning phase, causing reproductive endocrine disruptions.     

Uptake, depuration, and biotransformation of anthracene and benzo[a]pyrene in bluegill sunfish

Bluegills (Lepomis macrochirus Raf.) were exposed to [14C]anthracene or [14C]benzo[a]pyrene (B[a]P) in water. Rates of uptake and biotransformation within the fish were followed by 14C counting and thin-layer and liquid chromatography. The initial uptake-rate coefficient for anthracene (KU = 36 hr-1) was found to be independent of exposure concentration. The presence of dissolved humics did not affect anthracene uptake but did reduce the B[a]P uptake rate significantly. Biotransformation of the anthracene was constant at 0.22 nmol/g/hr, with approximately 92% of the residue unmetabolized at 4 hr. Uptake of B[a]P was linear (KU = 49 hr-1), although biotransformation increased from 0.044 to 0.088 nmol/g/hr between 1 and 2 hr of exposure. Only 11% of the B[a]P 14C activity at 4 hr represented the parent compound. Although 6% of the anthracene was found in liver and gall bladder, 25% of the B[a]P was distributed in the two organs. Depuration rates were first order and yielded half-lives of 17 hr for anthracene and 67 hr for B[a]P. The estimated bioconcentration factors (BCF) for anthracene and B[a]P in whole fish (KU/KD) were 900 and 4900, respectively, for total 14C activity, but only 675 and 490 for parent material. These BCFs were considerably lower than those predicted from the octanol-water partition coefficients because of biotransformation.     

Oxidative stress responses in different organs of Jenynsia multidentata exposed to endosulfan

We evaluate antioxidant responses of Jenynsia multidentata experimentally exposed to sublethal concentrations of endosulfan (EDS). The main goal was to determine differences in the response between different organs to assess which one was more severely affected. Thus, we exposed females of J. multidentata to EDS during 24h, measuring the activity of GST, GR, GPx, CAT and LPO in brain, gills, liver, intestine and muscle of both exposed fish and controls. GST activity was inhibited in gills, liver, intestine and muscle of exposed fish but was induced in brain. GR and GPx activities were increased in brain and gills at 0.014 and 0.288 microg L(-1), respectively. GPx activity was inhibited in liver and muscle at all studied concentrations whereas inhibition was observed in the intestine above 0.288 microg L(-1). Exposure to 1.4 microg L(-1) EDS caused CAT inhibition and increase of LPO levels in liver. LPO was also increased in brain at almost all concentrations tested. We find that the brain was the most sensitive organ to oxidative damage. Thus, J. multidentata could be used as a suitable bioindicator of exposure to EDS measuring activities of antioxidant enzymes in brain and liver as biomarkers.