Hepatic CYP1A1 induction in rainbow trout by continuous flowthrough exposure to beta-naphthoflavone.

In order to assess the usefulness of CYP1A1 mRNA measurement as an environmental biomarker it was necessary to determine if hepatic P450 CYP1A1 mRNA induction is sustained during constant exposure to hepatic monooxygenase inducers. To accomplish this, rainbow trout (Oncorhynchus mykiss) were exposed, under flowthrough conditions, to beta-naphthoflavone (beta-NF), a known CYP1A1 inducer in fish. Trout were exposed to a beta-NF concentration of 1.0 mg beta-NF/liter, using dimethylformamide as carrier, for 1, 2, 4, and 8 days, followed by depuration in clean water for 1, 8, 14, and 35 days. In a second experiment, trout were exposed to beta-NF concentrations of 0.05, 0.10, and 0.50 mg beta-NF/liter, using dimethylformamide as carrier, for 1, 3, 7, and 14 days, followed by depuration for 7 and 28 days. At the 1.0-mg beta-NF/liter concentration, ethoxyresorufin-O-deethylase (EROD) activity was significantly decreased by 4 days of exposure when compared to controls. At beta-NF concentrations of 0.05 to 0.50 mg beta-NF/liter EROD activity was increased compared to controls but was inversely related to the beta-NF concentration. Hybridizable CYP1A1 mRNA was increased approximately 40-fold over control levels at concentrations of 0.05 to 0.50 mg beta-NF/liter for 1, 3, and 7 days of exposure. In a third experiment, trout exposed to 0.05 mg beta-NF/liter for 2, 6, 12, 24, 32, 40, and 48 hr had increased (45- to 167-fold) EROD activity by 18 and 48 hr, respectively. Immunoreactive CYP1A1 protein was increased 46-fold at 48 hr and CYP1A1 mRNA was increased 29-fold at 48 hr of continuous beta-NF exposure. This is in contrast to previous experiments using intraperitoneal injection of beta-NF in which the induced CYP1A1 mRNA decreased to near control levels by 48 hr after injection. These data indicate that both CYP1A1 catalytic activity and immunoreactive protein are decreased at high inducer concentrations while mRNA levels remain elevated and continue to increase over time during continuous exposure. In a fourth experiment trout were continuously exposed to concentrations of 0.625, 1.25, 2.5, 5.0, and 10.0 micrograms beta-NF/liter, using dimethylformamide as carrier, for 1, 3, 7, 14, and 21 days, followed by clean water depuration for 1, 3, 7, 14, and 21 days EROD activity was significantly increased in a concentration-dependent manner over control by Day 1 of exposure with concentrations of 2.5, 5.0, and 10.0 micrograms beta-NF/liter.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunochemical and immunohistochemical studies of cadmium associated proteins in Rana tadpoles.

Previous observations suggested that Rana tadpoles treated with aqueous cadmium (Cd) accumulate Cd in their liver and mesonephros. In order to study the response to Cd in these tissues we (a) exposed tadpoles in mid-limb bud stages to sublethal quantities of Cd, (b) isolated Cd-associated protein (CAP) from a liver cytosol fraction, (c) prepared a heterologous rabbit antiserum against glutaraldehyde-treated CAP (G-CAP), (d) used the rabbit anti-G-CAP antiserum in order to assess the tissue distribution of CAP in Cd-treated and untreated tadpoles, and (e) assessed species cross-reactivities of our anti-G-CAP with CAPs and metallothioneins (MTs) isolated from Cd-treated vertebrate liver cytosol fractions. We found that (a) CAP was present in higher quantities in liver cytosol obtained from Cd-treated tadpoles compared to liver cytosol obtained from untreated control tadpoles, (b) indirect immunofluorescent analysis revealed that CAP was localized in liver hepatocytes and kidney tubule epithelial cells in Cd-treated tadpoles, and (c) the anti-G-CAP crossreacted with rodent and fish CAP. These observations suggest that the developing liver and mesonephros are involved in responses to toxic metals and that our anti G-CAP antiserum may be used to gauge exposure to environmental Cd.

Effects of prolonged low-level cadmium exposure on the tadpole immune system.

Uninjected (Group I) and sheep erythrocyte (SRBC)-injected (Group II) Rana tadpoles were exposed to varying sublethal concentrations of cadmium (Cd) for 6 weeks. In order to assess possible effects on the tadpole immune system we determined pre-B, B mu, and plasma cell (PC mu) frequencies in liver and mesonephros of Group I larvae, and hemagglutinating antibody (HA) titers of Group II animals. Group I and Group II control animals were cultivated in water with no added Cd (0 ppm), while treatments were set at 0.1, 0.2, 0.4 and 0.8 ppm Cd. We found that (a) Cd appeared to stimulate a significant increase in the frequency of B mu cells in animals treated at 0.4 and 0.8 ppm, (b) certain treated Group II larvae contained significantly increased amounts of HA in their serum, while their serum protein concentrations were not significantly different, and (c) there was a dose-related increase in tissue Cd levels in treated Group II larvae. Our observations suggest that chronic low-level exposure to Cd may (a) result in a slight increases in the frequency of B mu cells in unimmunized animals, (b) increase immune responsiveness of immunized larvae, and (c) increase liver and mesonephros accumulations of Cd. Preliminary studies indicated that cytosolic Cd is associated with a protein which appears to be similar to mammalian metallothionein.

Bioavailability, disposition and pharmacokinetics of 14C-ormetoprim in rainbow trout (Salmo gairdneri).

1. Uptake, bioavailability, tissue disposition, and elimination of 14C were examined in rainbow trout following intravascular and per os dosing of 14C-ormetoprim (8 mg/kg). 2. Plasma clearance was rapid following a single i.v. dose (t1/2 alpha = 0.54 h, t1/2 beta = 17.5 h) with an apparent volume of distribution (Vss) of 4.85 l/kg. An increase in both t1/2 alpha (0.67 h) and t1/2 beta (36.7 h) was seen following multiple i.v. doses (steady state). 3. Oral dispositional studies revealed the highest concentration of OMP in bile, kidney and liver, indicating significant renal and hepatic extraction. Significant OMP residues were seen in skin (0.90 p.p.m.) and muscle (0.15 p.p.m.) at 38 days. 4. Peak absorption time of orally administered OMP was 12 h with an apparent bioavailability of 87%. 5. Intravascularly administered OMP was 33% and 31% protein-bound at 1 and 6 h respectively, and binding was non-specific and non-saturable by Scatchard analysis.

The effect of piperonyl butoxide on hepatic cytochrome P-450-dependent monooxygenase activities in rainbow trout (Salmo gairdneri).

Although piperonyl butoxide (PBO) is commonly used both in vivo and in vitro as an inhibitor of cytochrome P-450-dependent monooxygenase (MO) activity in a wide variety of species, the effect of PBO on the hepatic MO of fishes has never been characterized. The MO activity in hepatic microsomes from rainbow trout exposed to either 1 or 2 ppm PBO for 24 hr in a static system was induced to a similar level in both treatment groups. Conversely, when PBO was administered in a flow-through system to trout, the hepatic microsomes of treated animals contained MO activities that were induced in a dose-dependent manner. Furthermore, total cytochrome P-450 was significantly increased in the livers of trout treated in a flow-through system with 1 ppm or more of PBO. The in vitro inhibition kinetics of PBO toward the 7-ethoxycoumarin-O-deethylase (ECOD) and 7-ethoxyresorufin-O-deethylase (EROD) activities of hepatic microsomes from trout treated with beta-naphthoflavone (BNF) (100 mg/kg, ip) or PBO (4 ppm by flow-through) and untreated trout were compared with Dixon plots. With respect to ECOD activity, the slopes of Dixon plots from control, BNF- and PBO-treated animals were similar. However, the slopes of Dixon plots of EROD inhibition by PBO in microsomes from BNF- and PBO-treated trout were significantly different from each other. Treatment of trout with PBO in a flow-through system resulted in an increase in ECOD and EROD activity in hepatic microsomes while simultaneously decreasing their activity toward [14C]rotenone oxidation. These data suggest that the cytochrome P-450 isozyme composition in hepatic microsomes from PBO-treated rainbow trout may be qualitatively different from that of BNF-treated trout. Also, the activity of hepatic microsomes from PBO-treated trout toward a specific substrate may be either inhibited or induced.