Quantitative oral dosing of water soluble and lipophilic contaminants in the Japanese medaka (Oryzias latipes).

Quantitative oral dosing in fish can be challenging, particularly with water soluble contaminants, which can leach into the aquarium water prior to ingestion. We applied a method of bioencapsulation using newly hatched brine shrimp (Artemia franciscana) nauplii to study the toxicokinetics of five chlorinated and brominated halogenated acetic acids (HAAs), which are drinking water disinfection by-products. These results are compared to those obtained in a previous study using a polybrominated diphenyl ether (PBDE-47), a highly lipophilic chemical. The HAAs and PBDE-47 were bioencapsulated using freshly hatched A. franciscana nauplii after incubation in concentrated solutions of the study chemicals for 18 h. Aliquots of the brine shrimp were quantitatively removed for chemical analysis and fed to individual fish that were able to consume 400-500 nauplii in less than 5 min. At select times after feeding, fish were euthanized and the HAA or PBDE-47 content determined. The absorption of HAAs was quantitatively similar to previous studies in rodents: rapid absorption with peak body levels occurring within 1-2 h, then rapidly declining with elimination half-life of 0.3-3 h depending on HAA. PBDE-47 was more slowly absorbed with peak levels occurring by 18 h and very slowly eliminated with an elimination half-life of 281 h.

Dichloroacetate toxicokinetics and disruption of tyrosine catabolism in B6C3F1 mice: dose-response relationships and age as a modifying factor.

Dichloroacetate (DCA) is a rodent carcinogen commonly found in municipal drinking water supplies. Toxicokinetic studies have established that elimination of DCA is controlled by liver metabolism, which occurs by the cytosolic enzyme glutathione-S-transferase-zeta (GST-zeta). DCA is also a mechanism based inhibitor of GST-zeta, and a loss in GST-zeta enzyme activity occurs following repeated doses or prolonged drinking water exposures. GST-zeta is identical to an enzyme that is part of the tyrosine catabolism pathway known as maleylacetoacetate isomerase (MAAI). In this pathway, GST-zeta plays a critical role in catalyzing the isomerization of maleylacetoacetate to fumarylacetoacetate. Disruption of tyrosine catabolism has been linked to increased cancer risk in humans. We studied the elimination of i.v. doses of DCA to young (10 week) and aged (60 week) mice previously treated with DCA in their drinking water for 2 and 56 weeks, respectively. The diurnal change in blood concentrations of DCA was also monitored in mice exposed to three different drinking water concentrations of DCA (2.0, 0.5 and 0.05 g/l). Additional experiments measured the in vitro metabolism of DCA in liver homogenates prepared from treated mice given various recovery times following treatment. The MAAI activity was also measured in liver cytosol obtained from treated mice. Results indicated young mice were the most sensitive to changes in DCA elimination after drinking water treatment. The in vitro metabolism of DCA was decreased at all treatment rates. Partial restoration ( approximately 65% of controls) of DCA elimination capacity and hepatic GST-zeta activity occurred after 48 h recovery from 14 d 2.0 g/l DCA drinking water treatments. Recovery from treatments could be blocked by interruption of protein synthesis with actinomycin D. MAAI activity was reduced over 80% in liver cytosol from 10-week-old mice. However, MAAI was unaffected in 60-week-old mice. These results indicate that in young mice, inactivation and re-synthesis of GST-zeta is a highly dynamic process and that exogenous factors that deplete or reduce GST-zeta levels will decrease DCA elimination and may increase the carcinogenic potency of DCA. As mice age, the elimination capacity for DCA is less affected by reduced liver metabolism and mice appear to develop some toxicokinetic adaptation(s) to allow elimination of DCA at rates comparable to naive animals. Reduced MAAI activity alone is unlikely to be the carcinogenic mode of action for DCA and may in fact, only be important during the early stages of DCA exposure.

Stereospecific toxicokinetics of bromochloro- and chlorofluoroacetate: effect of GST-zeta depletion.

The chloro- and bromohaloacetates are drinking water disinfection by-products and rodent carcinogens. Chloro-bromo dihaloacetates are also mechanism-based inhibitors of glutathione S-transferase-zeta (GSTZ1-1). We studied the stereospecific toxicokinetics and in vitro metabolism of two chiral dihaloacetates in male F344 rats: (-),(+)-bromochloroacetate (BCA) and racemic chlorofluoroacetate (CFA), a non-GST-zeta-inhibiting dihaloacetate. These experiments were repeated in animals that had previously been treated with dichloroacetate (DCA) to deplete GST-zeta activity. Results indicated that the elimination half-life of (-)-BCA was 0.07 compared to 0.40 h for (+)-BCA in naive rats. A comparable difference in elimination half-life was also observed for the CFA stereoisomers (0.79 vs 0.11 h). In GST-zeta-depleted rats, stereospecific elimination of (-),(+)-BCA was absent, with both stereoisomers having an elimination half-life of approximately 0.4 h. This finding was in contrast to results for CFA, which still maintained the same relative difference in elimination rate between its stereoisomers, although overall elimination was diminished in GST-zeta-depleted rats. Results of in vitro metabolism experiments indicated (-)-BCA was affected by modulating GST-zeta activity, with the intrinsic metabolic clearance decreasing from 2.81 to 0.15 ml h(-1) mg.protein(-1) (naive, GST-zeta depleted) compared with values for (+)-BCA (0.30 and 0.31 ml h(-1) mg.protein(-1)). Incubations with 350 microM diethyldithiocarbamate preferentially decreased (+)-BCA metabolism in naive and GST-zeta-depleted cytosol. These results indicate (+)-BCA is a poor substrate for GST-zeta and its metabolism is controlled by an additional GST isoenzyme.

Toxicokinetics of bromodichloroacetate in B6C3F1 mice.

The oral and i.v. elimination kinetics were investigated for bromodichloroacetate (BDCA), a haloacetate found in drinking water. The BDCA was administered at a dose of 5, 20 and 100 mg kg-1 to B6C3F1 mice and appears to distribute to the total body water with a mean volume of distribution of 427 +/- 79 ml kg-1. It is subject to first-pass hepatic metabolism with a range of bioavailabilities of 0.28-0.73. A mean terminal half-life of 1.37 +/- 0.21 h. was calculated from the two lower doses of both i.v. and oral administration. Non-linear behavior was exhibited at doses greater than 20 mg kg-1, with a much higher than expected area under the curve (AUC), a decrease in total body clearance (CL(b)) and an increase in the terminal half-life to 2.3 h at the highest dose. The average CL(b) was 220 ml h(-1) kg-1 for the lower two doses but decreased to 156 ml h(-1) kg-1 at the high dose. The BDCA is primarily eliminated by metabolism, with only 2.4% of the parent dose being recovered in the urine at the high dose. The unbound renal clearance, as calculated from the high dose, was 15.0 ml h(-1) kg-1. The BDCA is moderately bound to plasma proteins (f(u) = 0.28) and preferentially distributes to the plasma with a blood/plasma ratio of 0.88.

Dose-response relationships and pharmacokinetics of vitellogenin in rainbow trout after intravascular administration of 17alpha-ethynylestradiol.

A commonly used endpoint in bioassays testing the estrogenicity of chemicals is the induction of the egg yolk precursor vitellogenin (VTG) in male fish. However, relatively little is known about the kinetics of induction and elimination of VTG in fish exposed to xenoestrogens. In this study, we administered graded intra-arterial doses (0.001, 0.1, 1.0 and 10.0 mg/kg) of 17alpha-ethynylestradiol (EE(2)) to male rainbow trout via a dorsal aortic cannula which allowed repetitive blood sampling from individual fish for up to 48 days after injection. The plasma concentrations of VTG was quantified using an enzyme-linked immunosorbent assay procedure and the simultaneous concentrations of EE(2) were determined by gas chromatography-mass spectrometry. The pattern of VTG induction was similar for all doses of EE(2), with a 12-h lag-time before increase from basal levels (0.006-0.008 microg/ml), then increasing sharply to maximum levels within 7-9 days (C(max)=0.05, 711, 1521 and 2547 microg/ml VTG for the 0.001, 0.1, 1.0 and 10.0 mg/kg doses, respectively). After induction by EE(2), VTG declined mono-exponentially with an elimination half-life of 42-49 h. The half-life of VTG increased to 145 h in the 10 mg/kg treated fish. The pharmacokinetics of EE(2) were distinctly nonlinear with substantial increases in the elimination half-life with increasing dose. The plasma concentration-time profiles of EE(2) were influenced by enterohepatic recirculation that caused multiple or secondary peaks in the profiles. In a separate experiment, the pharmacokinetics of purified VTG was characterized after intra-arterial injection in trout. After direct injection of VTG, plasma levels declined tri-exponentially with an apparent steady-state volume of distribution of 837 ml/kg; total body clearance was 31.1 ml/h per kg, and the elimination half-life was 43.7 h.

Trapping and identification of the dichloroacetate radical from the reductive dehalogenation of trichloroacetate by mouse and rat liver microsomes.

A key question in the risk assessment of trichloroethylene (TRI) is the extent to which its carcinogenic effects might depend on the formation of dichloroacetate (DCA) as a metabolite. One of the metabolic pathways proposed for the formation of DCA from TRI is by the reductive dehalogenation of trichloroacetate (TCA), via a free radical intermediate. Although proof of this radical has been elusive, the detection of fully dechlorinated metabolites in the urine and the formation of lipid peroxidation by-products in microsomal incubations with TCA argue for its existence. We report here the trapping of the dichloroacetate radical with the spin-trapping agent PBN, and its identification by GC/MS. The PBN/dichloroacetate radical adduct was found to undergo an intramolecular rearrangement during its extraction into organic solvent. An internal condensation reaction between the acetate and the nitroxide radical moieties is hypothesized to form a cyclic adduct with the elimination of an OH radical. The PBN/dichloroacetate radical adduct has been identified by GC/MS in both a chemical Fenton system and in rodent microsomal incubations with TCA as substrate.

Pharmacokinetics of intravascularly administered (65)Zinc in channel catfish (Ictalurus punctatus).

Comparison was made of the pharmacokinetics of the radioisotope (65)Zinc ((65)Zn) in blood, plasma, and whole body of adult channel catfish (Ictalurus punctatus) following intravascular (iv) administration. A two-compartment model described the pharmacokinetics of (65)Zn in plasma and blood during the first 40 days following iv administration, but was unable to describe the long-term disposition of (65)Zn. Whole-body counting revealed that approximately half of the (65)Zn dose was sequestered in a slowly exchangeable pool with a half-life of 1.5 years. Greater than 99% of the circulating (65)Zn was bound to plasma proteins, whereas there was less than 1% binding to red blood cells. Synthesis of the results for channel catfish and existing data in other species indicates three phases in the pharmacokinetics of zinc. The first phase consists of initial distribution outside the vascular system to kidney, liver, and other organs (alpha phase in blood and plasma; t(1/2) of 4 to 5 h). The second phase involves distribution from organs to a slowly exchangeable zinc pool, likely consisting of bone (beta phase in blood and plasma; alpha phase in whole body; t(1/2) of 4 to 20 days). The third phase appears to involve a slow turnover of sequestered zinc (t(1/2) greater than 1 year). Blood sampling or short-term whole-body measurements will underestimate the persistence of zinc in fish, thus prolonged sampling and measurement of whole-body concentrations are necessary to characterize the pharmacokinetics of zinc.

Comparative toxicokinetics of chlorinated and brominated haloacetates in F344 rats.

Chloro, bromo, and mixed bromochloro haloacetates (HAs) are by-products of drinking water disinfection and are hepatocarcinogenic in rodents. We compared the toxicokinetics of a series of di-HAs, dichloro (DCA), bromochloro (BCA), dibromo (DBA) and tri-HAs: trichloro (TCA), bromodichloro (BDCA), chlorodibromo (CDBA), and tribromo (TBA) after iv and oral dosing (500 micrometer/kg) in male F344 rats. The blood concentrations of the HAs after iv injection declined in a bi-exponential manner with a short but pronounced distributive phase. The structural features that had the greatest influence on the disposition of HAs were substitution of a halogen for a hydrogen and the degree of bromine substitution. All di-HAs had blood elimination half-lives of less than 4 h (DCA > DBA, BCA) compared to the tri-HAs, which had half-lives that varied from 0.6 to 8.0 h (TCA > BDCA > CDBA > TBA). The urinary excretion of all di-HAs was low and accounted for less than 3% of the dose in contrast to the tri-HAs, where urinary excretion accounted for at least 30% of the dose. Toxicokinetic analysis indicated the steady-state apparent volume of distribution varied between 301 and 881 ml/kg among the HAs, but the variation was not statistically significant (P > 0.17). The blood concentration-time profiles for all di-HAs after oral dosing was complex and exhibited multiple peaks. This did not appear to be due to enterohepatic recirculation, as bile duct cannulated animals also displayed similar profiles. In contrast, the profiles for the tri-HAs did not exhibit multiple peaking after oral dosing and could be described using a one-compartment pharmacokinetic model. The oral bioavailability of the HAs varied between 30% (DBA) and 116% (TCA), depending on the number of halogen substituents and the degree of bromine substitution. In general, three patterns of elimination for the HAs can be broadly described: low metabolism with moderate renal clearance (TCA), high metabolism and renal clearance (BDCA, CDBA, TBA), and high metabolism, low renal clearance (DCA, BCA, DBA).

Effect of pre-treatment with dichloroacetic or trichloroacetic acid in drinking water on the pharmacokinetics of a subsequent challenge dose in B6C3F1 mice.

Dichloroacetate (DCA) and trichloroacetate (TCA) are prominent by-products of chlorination of drinking water. Both chemicals have been shown to be hepatic carcinogens in mice. Prior work has demonstrated that DCA inhibits its own metabolism in rats and humans. This study focuses on the effect of prior administration of DCA or TCA in drinking water on the pharmacokinetics of a subsequent challenge dose of DCA or TCA in male B6C3F1 mice. Mice were provided with DCA or TCA in their drinking water at 2 g/l for 14 days and then challenged with a 100 mg/kg i.v. (non-labeled) or gavage (14C-labeled) dose of DCA or TCA. The challenge dose was administered after 16 h fasting and removal of the haloacetate pre-treatment. The haloacetate blood concentration-time profile and the disposition of 14C were characterized and compared with controls. The effect of pre-treatment on the in vitro metabolism of DCA in hepatic S9 was also evaluated. Pre-treatment with DCA caused a significant increase in the blood concentration-time profiles of the challenge dose of DCA. No effect on the blood concentration-time profile of DCA was observed after pre-treatment with TCA. Pre-treatment with TCA had no effect on subsequent doses of DCA. Pre-treatment with DCA did not have a significant effect on the formation of 14CO2 from radiolabeled DCA. In vitro experiments with liver S9 from DCA-pre-treated mice demonstrated that DCA inhibits it own metabolism. These results indicate that DCA metabolism in mice is also susceptible to inhibition by prior treatment with DCA, however the impact on clearance is less marked in mice than in F344 rats. In contrast, the metabolism and pharmacokinetics of TCA is not affected by pre-treatment with either DCA or TCA.

The extent of dichloroacetate formation from trichloroethylene, chloral hydrate, trichloroacetate, and trichloroethanol in B6C3F1 mice.

Conflicting data have been published related to the formation of dichloroacetate (DCA) from trichloroethylene (TRI), chloral hydrate (CH), or trichloroacetic acid (TCA) in B6C3F1 mice. TCA is usually indicated as the primary metabolic precursor to DCA. Model simulations based on the known pharmacokinetics of TCA and DCA predicted blood concentrations of DCA that were 10- to 100-fold lower than previously published reports. Because DCA has also been shown to form as an artifact during sample processing, we reevaluated the source of the reported DCA, i.e., whether it was metabolically derived or formed as an artifact. Male B6C3F1 mice were dosed with TRI, CH, trichloroethanol (TCE), or TCA and metabolic profiles of each were determined. DCA was not detected in any of these samples above the assay LOQ of 1.9 microM of whole blood. In order to slow the clearance of DCA, mice were pretreated for 2 weeks with 2 g/liter of DCA in their drinking water. Even under this pretreatment condition, no DCA was detected from a 100 mg/kg i.v. dose of TCA. Although there is significant uncertainty in the amount of DCA that could be generated from TRI or its metabolites, our experimental data and pharmacokinetic model simulations suggest that DCA is likely formed as a short-lived intermediate metabolite. However, its rapid elimination relative to its formation from TCA prevents the accumulation of measurable amounts of DCA in the blood.

Pharmacokinetics and metabolism of dichloroacetate in the F344 rat after prior administration in drinking water.

The effect of prior administration of dichloroacetate (DCA) in drinking water on the pharmacokinetics of DCA in male F344 rats was studied. Rats were provided with DCA in their drinking water at 0.2 and 2.0 g/liter for 14 days and then challenged with iv bolus iv or gavage doses of [14C1,2]DCA, 16 hr after pretreatment withdrawal. The blood concentration-time profiles of DCA and the disposition of 14C was characterized and compared with controls. The effect of pretreatment on the in vitro metabolism of DCA in hepatic cytosol was also evaluated. Pretreatment caused a significant increase in the blood concentration and AUC0-->infinity of DCA (433.3 versus 2406 microg ml-1 hr). Pharmacokinetic analysis indicated that pretreatment significantly decreased total body clearance (267.4 versus 42.7 ml hr-1 kg-1), which was largely due to decreased metabolism since only modest differences in the urinary clearance of DCA were observed. Pretreatment significantly decreased the formation of 14CO2 after both iv and oral doses of [14C]DCA. The decrease in CO2 formation was also observed after pretreatment with DCA at 0.2 g/liter. Pretreatment also increased the urinary elimination of DCA and several metabolites, particularly glycolate. The in vitro experiments demonstrated that DCA pretreatment inhibited the conversion of DCA to glyoxylate, oxalate, and glycolate in hepatic cytosol. These results indicate that DCA has an auto-inhibitory effect on its metabolism and that pharmacokinetic studies using single doses in naïve rats will underestimate the concentration of DCA at the target tissue during chronic or repeated exposures.

Quantification of benzocaine and its metabolites in channel catfish tissues and fluids by HPLC.

Methods for extraction and gradient HPLC quantification were developed for benzocaine (BZ) and three of its metabolites to be used in conjunction with a reverse isotope technique. The metabolites were p-aminobenzoic acid (PABA), acetyl-p-aminobenzoic acid (AcPABA) and acetylbenzocaine (AcBZ). The matrixes studied were white muscle, red muscle, skin, liver, trunk kidney, head kidney, plasma and the bile of channel catfish. Analytes were validated for each of the compounds at 25 and 100 nmol per sample in the various tissues and fluids. The intraday variability (R.S.D.) was less than 13% in all tissues and fluids except for BZ in the liver. Recoveries varied from matrix to matrix for each analyte. The highest recoveries were obtained from plasma which ranged from 82.8-99.8% depending on the concentration. The average recovery of the compounds from tissues was between 50 and 78%, except for liver where the recovery of PABA and BZ was below 30%. Detection was by UV absorbance at 286 nm and the linear range was 2.5-15 nmol 100 ml-1 for all analytes. The method was selective; no interference peaks coeluted with the analytes.

Toxicokinetics and disposition of inorganic mercury and cadmium in channel catfish after intravascular administration.

To better understand the distribution and elimination of inorganic mercury (Hg) and cadmium (Cd) in fishes, channel catfish (Ictalurus punctatus) were administered either 6.4 micrograms/kg 203Hg as HgCl2 or 4.0 micrograms/kg 109Cd as CdCl2 via a dorsal aortic cannula. Blood, plasma, and red blood cells (RBCs) were serially sampled up to 156 (Hg) or 335 (Cd) days. The fraction of the injected dose remaining in the animal (Xf) was also determined at selected times by whole animal counting. The blood concentration and Xf-time profiles were simultaneously fitted to a three-compartment toxicokinetic model. The plasma concentration-time profile was also separately fitted to the same three-compartment model for comparison of parameter estimates. Toxicokinetic analysis of the blood concentration and Xf-time profile provided the following values: steady-state volume of distribution = 13.8 +/- 2.8 ml/g (Hg), 41.4 +/- 0.3 ml/g (Cd); total body clearance = 0.021 +/- 0.0006 ml/day/g (Hg), 0.0031 +/- 0.0008 ml/day/g (Cd); biological half-life (t1/2, beta) = 722 +/- 309 days (Hg), 9627 +/- 2206 days (Cd). Estimates of the t1/2 beta were up to 94 times longer if determined by simultaneous fitting of the blood concentration and Xf-time profiles. A time-dependent accumulation of Hg and Cd by RBCs was observed with maximum RBC concentrations of Hg and Cd occurring at 7 and 12 days after injection. After injection, the tissues with the highest accumulation of Hg were the liver, trunk and head kidney, muscle, and skin, but the amount of Hg in the liver gradually increased over 156 days. Most of the Cd was accumulated by the liver and trunk kidney within 24 hr, with little change occurring after 335 days. This study demonstrates the usefulness of intravascular injection and simultaneous analysis of blood and whole body amount data in determining the elimination of metals from fishes.

Toxicokinetics of parathion and paraoxon in rainbow trout after intravascular administration and water exposure.

Fish are less sensitive than mammals to organophosphate insecticide toxicity. The species differences have been mainly investigated by biochemical studies of AChE and organophosphate interaction. To examine whether species differences in the toxicokinetics of the organophosphate insecticides were also involved in their differential toxicity, rainbow trout were fitted with a dorsal aorta cannula and administered parathion and its active metabolite paraoxon intraarterially (ia) and via water exposure. Serial blood samples were removed and the plasma concentrations of parathion and paraoxon were determined by capillary GC with EC detection. Plasma protein binding was determined by equilibrium dialysis and ultrafiltration. After ia injection the plasma concentration-time profiles of parathion and paraoxon were multiexponential, with a terminal t1/2 of 56.1 and 0.528 hr. The steady-state volumes of distribution and total body clearances (CLb) for parathion and paraoxon were 1370 and 1080 ml kg-1 and 21.4 and 3020 ml hr-1 kg-1; the plasma unbound fractions were 1.23 and 52.5%. The large difference in CLb between parathion and paraoxon appeared to result primarily from differences in plasma protein binding. Parathion had greater persistence in trout than rat, suggesting that sensitivity difference were unrelated to toxicokinetic differences.

Body size and the toxicokinetics of trifluralin in rainbow trout.

Rainbow trout (Oncorhynchus mykiss) ranging from 0.2 to 3395 g were exposed to trifluralin (TF) in water at concentrations of 0.6-2.0 micrograms/liter. Trout of all body sizes rapidly accumulated TF from the water. The uptake clearance (P, ml hr-1g-1) of TF from the water decreased as body weight (BW, g) increased. This decrease followed the allometric equation P (ml/hr) = 182.BW0.66. Other kinetic parameters affected by body size were the steady-state volume of distribution which had a BW exponent value of 1.07 and the biological half-life which increased in larger fish. The relatively larger volume of distribution in larger fish reflected an increased capacity for TF in peripheral compartment-associated tissues. Metabolic elimination and the bioconcentration factor of TF did not change systematically with changes in body size. Variation in total body lipid content could not adequately explain the increase in peripheral storage capacity for TF; the decreased plasma protein binding that was observed in larger trout may also have been involved.

Presystemic branchial metabolism limits di-2-ethylhexyl phthalate accumulation in fish.

Despite the high lipophilicity of di-2-ethylhexyl phthalate (DEHP), fish do not extensively accumulate this ubiquitous environmental contaminant. Experiments with rainbow trout (Salmo gairdneri) fitted with an indwelling cannula showed that the majority of [14C]DEHP did not reach the systemic circulation of the fish, but was present in the exposure water as metabolites. Pharmacokinetic analysis, using a compartmental model that included the gill as a separate metabolic compartment, indicated that DEHP was extensively metabolized as it diffused from water to blood. Isolated perfused gill arches of trout metabolized DEHP in the exposure bath to monoethylhexyl phthalate, demonstrating the ability of the gill to prevent DEHP entry into the fish. The relationship between metabolic clearance and tissue perfusion further suggests that metabolism in the gill can play an important role in determining the accumulation and toxicity of organic chemical pollutants in fish.