Kevin M. Kleinow

Demethylation of the pesticide methoxychlor in liver and intestine from untreated, methoxychlor-treated, and 3-methylcholanthrene-treated channel catfish (Ictalurus punctatus): evidence for roles of CYP1 and CYP3A family isozymes.

Exposure to the organochlorine pesticide methoxychlor (MXC) is associated with endocrine disruption in several species through biotransformation to mono-desmethyl-MXC (OH-MXC) and bis-desmethyl-MXC (HPTE), which interact with estrogen receptors. The biotransformation of [14C]methoxychlor was examined in channel catfish (Ictalurus punctatus), a freshwater species found in the southern United States. Hepatic microsomes formed OH-MXC and HPTE, assessed by comigration with authentic standards. The Km for OH-MXC formation by control liver microsomes was 3.8 ± 1.3 μM (mean ± S.D., n = 4), and Vmax was 131 ± 53 pmol/min/mg protein. These values were similar to those of catfish pretreated with 2 mg/kg methoxychlor i.p. for 6 days (Km 3.3 ± 0.8 μM and Vmax 99 ± 17 pmol/min/mg) but less (p < 0.05) than the kinetic parameters for catfish treated with 3-methylcholanthrene (3-MC), which had Km of 6.0 ± 1.1 μM and Vmax of 246 ± 6 pmol/min/mg protein. Liver microsomes from 3-MC-treated fish produced significantly more of the secondary metabolite and more potent estrogen, HPTE. Intestinal microsomes formed OH-MXC at lower rates than liver. Methoxychlor pretreatment significantly reduced intestinal metabolite formation from 32 ± 4 to 15 ± 6 pmol/min/mg (mean ± S.D., n = 4), whereas 3-MC treatment significantly increased OH-MXC production to 72 ± 22 pmol/min/mg. Ketoconazole, clotrimazole, and α-naphthoflavone all decreased the production of OH-MXC in liver microsomes, whereas α-naphthoflavone stimulated HPTE formation, suggesting that CYP1 and CYP3 family isozymes demethylated methoxychlor. The results suggest that the formation of estrogenic metabolites from methoxychlor would be more rapid in catfish coexposed to CYP1 inducers.

Distribution and inducibility of P-glycoprotein in the catfish: immunohistochemical detection using the mammalian C-219 monoclonal.

Mounting evidence suggests that the P-glycoprotein (pgp) efflux pump may be a modulator of bioavailability and a mode of excretion for xenobiotics. Immunohistochemistry was utilized to examine the distribution and inducibility of a pgp like protein in catfish. Immunoreactivity to the MDR C-219 monoclonal antibody was noted primarily in bile canaliculi or bile preductules of the liver, discrete areas of the extratubular region of the kidney and the columnar epithelia of the intestine. Regional differences in pgp content were noted in the intestine with the distal region containing greater pgp levels than the proximal intestine. Dietary administration of vincristine, a prototypic pgp inducer and beta-naphthoflavone an Ah agonist resulted in induction of the C-219 immunoreactivity in the liver and the distal intestine. These results are consistent in location and inducibility with pgp like proteins and support a possible relationship to xenobiotic absorption and/or excretion in the catfish.

Enhanced bioaccumulation of dietary contaminants in catfish with exposure to the waterborne surfactant linear alkylbenzene sulfonate.

Fish bioaccumulate a variety of contaminants and act as an exposure portal to the human consumer. Surfactants, known pharmaceutically to alter membrane permeability, change drug bioavailability and attenuate transporter function are also found in contaminant mixtures in the aquatic environment. The overall objective of this study was to determine if the surfactant C-12 linear alkylbenzene sulfonate (LAS) at environmentally relevant concentrations, alters the disposition and enhances bioaccumulation of co-exposed dietary xenobiotics in the catfish. Included for study were the carcinogen benzo(a)pyrene (BaP), pharmaceutical, ivermectin (IVM), and P-glycoprotein (P-gp) substrate rhodamine 123 (Rho-123), each exhibiting different dispositional footprints. Rho-123 transport into bile and membrane fluidity was examined in isolated perfused livers from control and LAS exposed catfish. Mass balance residue assessments were performed on catfish following in vivo exposure for 12 days to LAS in water at 0, 100 or 300 microg/L with 6 days of (3)H-IVM or (3)H-BaP gavage treatments. LAS at 1, 5 and 20 microM in the perfused liver, significantly decreased the transport of Rho-123 (1 microM) into bile by 18.6, 38.1 and 66.7%, respectively. Fluorescence anisotropy measurements demonstrated a 29.7% increase in fluidity at the (1 microM, 348 microg/L) LAS concentration. In vivo mass balance studies indicated that waterborne LAS (100 and 300 microg/L) increased the dietary dose remaining in fish by 39% and 78% for (3)H-IVM and 50 and 157% for (3)H-BaP. LAS at environmentally relevant concentrations altered the bioavailability and disposition of dietary xenobiotics in the catfish. Co-exposure with LAS increases xenobiotic bioaccumulation, potential toxicity of mixture components to the fish and the potential for residue transfer from fish to the consumer.

Bioavailability and biotransformation of 3H-benzo[a]pyrene metabolites in in Situ intestinal preparations of uninduced and BNF-induced channel catfish

Abstract The systemic bioavailability and intestinal biotransformation of dietary [G- 3 H]-3-hydroxy-benzo [a]pyrene (3OH-BaP), benzo[a]pyrene [G- 3 H]-9-sulfate (BaP-9-SO 4 ) and 9-benzo[a]pyrenyl [G- 3 H]-β- d -glucopyranosiduronic acid (BaP-9-Glu) were evaluated using an in situ isolated perfused intestinal segment of the catfish. 3OH-BaP (2 and 20 μM), BaP-9-SO 4 (10 and 40 μM) or BaP-9-Glu (10 and 40 μM) solutions were administered via micelles into the isolated intestinal segment of non-induced and β-naphthoflavone (10 mg/kg diet) induced catfish. Following a 60 min perfusion, the efferent blood, intestinal contents and mucosa were analysed for [ 3 H] content and metabolite profiles. BNF administration did not result in any significant effect upon the transport or metabolism of BaP-9-SO 4 , BaP-9-Glu or 3OH-BaP. The appearance of radioactivity in all analysed components for all compounds followed a dose-dependent relationship which was modified by bioavailability and biotransformation. BaP-9-SO 4 and BaP-9-Glu were readily transported intact from the intestinal lumen to the systemic circulation. 3OH-BaP was extensively biotransformed to BaP-3-SO 4 and lesser amounts of BaP-3,6-dione and BaP-3-glucuronide before being absorbed into the blood. These findings support the hypothesis that preconsumptive metabolites and their intestinal biotransformation products are readily available to the systemic circulation of a consumer.

Zebrafish embryos sequester and retain petrochemical combustion products: Developmental and transcriptome consequences

Zebrafish embryos are a model for studying effects of environmental stressors on development. Incomplete combustion of the environmentally relevant volatile petrochemical, 1,3-butadiene (BD) yields butadiene soot (BDS) nanoparticles, to which polynuclear aromatic hydrocarbons (PAHs) are adsorbed. In mammalian cells these PAHs are concentrated in lipid droplets and trigger up-regulation of biotransformation, oxidative stress and inflammatory genes. The present study was designed to determine whether: (a) PAH-rich BDS elicits alterations in zebrafish embryo development; (b) BDS-exposed zebrafish embryos sequester PAHs in select tissues; and (c) developmental abnormalities are correlated with altered gene expression patterns. 1-day old zebrafish embryos were exposed for 48 h to BDS (0, 6, 30 or 60 μg/ml) sprinkled on the water surface. PAH localization was tracked by fluorescence. Developmental responses (pericardial edema, yolk sac swelling, axial malformations) were monitored by microscopy. Gene expression changes were assessed by gene microarray and qRT-PCR. Our results show that PAHs localized with endogenous lipids in the yolk sac and in hatching gland cells. PAHs were retained at least 8 days after exposures ended. Dose-dependent pericardial and yolk sac edema and axial malformations were prominent and accompanied by up-regulation of biotransformation and oxidative stress gene cascades. Thus, zebrafish embryos should be useful for predicting the potential for developmental toxicity following exposure to PAH-rich petrochemical soots, e.g., those arising from attempts at oil spill remediation by combustion.

In-vitro metabolism of benzo[a]pyrene and benzo[a]pyrene-7,8-dihydrodiol by liver and intestinal mucosa homogenates from the winter flounder (Pseudopleuronectes americanus)

Freshly prepared homogenates were used to assess the relative ability of winter flounder (Pseudopleuronectes americanus) liver and intestinal mucosal cells to metabolize the polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (BaP) and its proximate carcinogenic metabolite, BaP-7,8-dihydrodiol (7,8-Diol). Data obtained from homogenates prepared from fish previously fed β-naphthoflavone (BNF) indicated that both tissues had similar abilities to metabolize either BaP or 7,8-Diol on a per gram of protein basis. Metabolite profiles produced indicate that water-soluble metabolite formation is favored at low doses. These findings support the hypothesis that the intestine plays an important role in first-pass metabolism of dietary carcinogens in the winter flounder.

Pharmacokinetics, bioavailability, plasma protein binding and disposition of nalidixic acid in Rainbow Trout (Oncorhynchus mykiss)

1. The pharmacokinetics, disposition and bioavailability of nalidixic acid were examined in Rainbow Trout following i.v. and per os administration (5 mg/kg). 2. Nalidixic acid was biexponentially eliminated from plasma following i.v. dosing (t1/2 alpha = 0.06 h, t1/2 beta = 23.0 h). The volume of distribution (Vss) and total body clearance (Clb) were 964.7 ml/kg and 31.5 ml/kg/h, respectively. 3. In vitro plasma protein binding was specific and saturable over a range of concentrations from 0.43 microM to 20.0 mM. Binding was approx. 26% at kinetically relevant plasma concentrations. 4. Apparent oral bioavailability was determined to be > 100%, suggesting that nalidixic acid was largely bioavailable and non-linear pharmacokinetics were evoked. 5. Oral studies demonstrated the highest 14C nalidixic acid equivalent concentrations in bile, intestine and liver. Muscle contained intermediate concentrations but among all organs accounted for the greatest total amount of drug (12.2% of dose). Mass balance studies demonstrated composite values for per cent of dose administered of 23.7, 18.8, 8.5, 10.0, 7.4 and 2.3% for 1, 2, 3, 6, 9 and 15 days, respectively. 6. A glucuronic acid conjugate of nalidixic acid was identified by n.m.r. and mass spectral analysis as the single primary metabolite.

Role of the mucous surface coat, dietary bulk and mucosal cell turnover in the intestinal disposition of benzo(a)pyrene (BaP)

Abstract The dispositon of 3 H-benzo(a)pyrene (BaP) equivalents from the diet was examined at the lumen-mucosal cell interface of the proximal, medial and distal regions of the catfish intestine. 3 H-BaP (500 μd, 20 μg/kg) was administered via gavage in maintenance diet to two groups of catfish. One group was fed daily after dosing while the other was fasted until collection of intestinal tissues. Autoradiography of cryosectioned tissues and computer-enhanced image analysis allowed examination of BaP disposition. BaP was found to selectively localize at high concentrations in the mucous surface coat and corresponding villi of the colon. Supplemental feeding of uncontaminated diet lowered BaP concentrations in these regions. In a similar but separate experiment, 3 H-thymidine (666 μCi) was administered to two groups of catfish by i.p. injection to examine mucosal cell turnover in relation to BaP turnover. 3 H-Thymidine incorporation was greatest in the distal regions of both fasted and fed groups. Thymidine dynamics indicated that feeding was related to only marginal changes in cell turnover, which suggests that losses in intravillus BaP with feeding may not be wholly explained by intestinal cell turnover. Results from this study suggest that the mucous surface coat of the intestine is a major factor in the regional disposition of dietary carcinogens and removal of BaP from this mucin layer is related to the intake of dietary bulk.

Influence of ormetoprim on the bioavailability, distribution, and pharmacokinetics of sulfadimethoxine in rainbow trout (Oncorhynchus mykiss)

1. Half lives of distribution and elimination phases of 14C-sulfadimethoxine following i.v. dosing of sulfadimethoxine/ormetoprim (SDM/OMP, 42/8 mg/kg) were 0.4 and 16.1 hr respectively. The apparent volume of distribution was 503.9 ml/kg. 2. In vitro plasma protein binding of 14C-SDM was not altered by increasing concentrations of unlabeled OMP. Similarly, binding of 14C-OMP was not altered by SDM. 3. Peak plasma concentrations of 14C-SDM following oral administration of SDM/OMP were observed at 20 hr with an apparent bioavailability of 38%. 4. Oral dispositional studies revealed the highest concentrations of 14C-SDM in bile, intestine, liver and fat. 5. Parent SDM and N-acetylated SDM were detected in plasma from i.v. and orally dosed animals. 6. The pharmacokinetics and distribution of 14C-SDM were not influenced by OMP co-administration.

Influence of Dietary Coexposure to Benzo(a)pyrene on the Biotransformation and Distribution of 14C-Methoxychlor in the Channel Catfish (Ictalurus punctatus)

Methoxychlor (MXC) is an organochlorine pesticide whose mono- and bis-demethylated metabolites, 2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)-1,1,1-trichloroethane (OH-MXC) and 2,2-bis(4-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), respectively, are estrogenic and antiandrogenic. Studies in vitro showed that treatment of channel catfish with a polycyclic aromatic hydrocarbon increased phase I and phase II metabolism of MXC. To determine the in vivo significance, groups of four channel catfish were treated by gavage for 6 days with 2 mg/kg (14)C-MXC alone or 2 mg/kg (14)C-MXC and 2 mg/kg benzo(a)pyrene (BaP). On day 7, blood and tissue samples were taken for analysis. Hepatic ethoxyresorufin O-deethylase activity was 10-fold higher in the BaP-treated catfish, indicating CYP1A induction. More MXC-derived radioactivity remained in control (42.8 +/- 4.1%) than BaP-induced catfish (28.5 +/- 3.2%), mean percent total dose +/- SE. Bile, muscle and fat contained approximately 90% of the radioactivity remaining in control and induced catfish. Extraction and chromatographic analysis showed that liver contained MXC, OH-MXC, HPTE, and glucuronide but not sulfate conjugates of OH-MXC and HPTE. Liver mitochondria contained more MXC, OH-MXC, and HPTE than other subcellular fractions. Bile contained glucuronides of OH-MXC and HPTE, and hydrolysis of bile gave HPTE and both enantiomers of OH-MXC. The muscle, visceral fat, brain and gonads contained MXC, OH-MXC, and HPTE in varying proportions, but no conjugates. This study showed that catfish coexposed to BaP and MXC retained less MXC and metabolites in tissues than those exposed to MXC alone, suggesting that induction enhanced the elimination of MXC, and further showed that potentially toxic metabolites of MXC were present in the edible tissues.