John J. Lech

Induction and characterization of hemoprotein(s) P-450 and monooxygenation in rainbow trout (Salmo gairdneri)

Hepatic microsomes obtained from control rainbow trout showed relatively low monooxygenase activities toward benzo[a]pyrene, ethoxycoumarin, ethoxyresorufin, and ethylmorphine as substrates. However, benzo[a]pyrene hydroxylation, ethoxycoumarin-O-deethylation, and ethoxyresorufin-O-deethylation were greatly induced by pretreatment of trout with β-naphthoflavone and Aroclor 1242. No increase in ethylmorphine-N-demethylation, liver/body ratio, or yield of microsomal protein were observed. Phenobarbital pretreatment of trout failed to affect any of the parameters studied. The λmax of carboxyferrocytochrome P-450 was at 449 nm in control microsomes, and although a small (40%) increase in total hemoprotein(s) P-450 was seen after β-naphthoflavone pretreatment no hypsochromic shift of the λmax was observed. No significant changes in the 430455 ratio of the EtNC ligand spectra were noted after induction. Type I and Type II binding spectra were observed in all microsomal preparations examined. The use of specific inhibitors of monooxygenation (α-naphthoflavone and metyrapone) indicated that control and induced trout hepatic hemoprotein(s) P-450 were similar to rat cytochrome P1-450. Furthermore, sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the presence of a novel hemoprotein in hepatic microsomes after pretreatment of trout with β-naphthoflavone or Aroclor 1242. This induced protein had a molecular weight of approximately 57,000.

Dose-effect relationship for induction of hepatic monooxygenase activity in rainbow trout and carp by Aroclor 1254

Abstract A range of doses of Aroclor 1254 or 3,4,3′,4′-tetrachlorobiphenyl was administered i.p. via a single injection to rainbow trout and to carp. Hepatic microsomes were prepared and examined for monooxygenase activity. Rainbow trout hepatic microsomes were assayed for ethoxycoumarin- and ethoxyresorufin-O-deethylase activities. For both deethylase activities, peak activity was observed at These results suggest that in some areas environmental exposure of fish to PCBs may be sufficient to cause induction of hepatic monooxygenase activity. It also appears that the potency of planar PCB isomers as inducers in fish is sufficiently great to account for the effect of Aroclor 1254 upon hepatic monooxygenase activity in these species.

Studies on the uptake, metabolism, and disposition of pentachlorophenol and pentachloroanisole in rainbow trout

Pentachlorophenol (PCP) and pentachloroanisole (PCA) were rapidly taken up by rainbow trout exposed to these compounds at 0.025 mg/liter in water. After exposure of trout to PCP for 24 hr, the liver, blood, fat, and muscle contained 16, 6.5, 6.0, and 1.0 μ g/g, respectively. The concentrations of PCA in the same tissues after an exposure to [ 14 C]PCA were of the same order of magnitude as was found with PCP except that fat contained as much as 80 μ g/g. Elimination rates for 14 C from the blood, muscle, fat, and liver after a similar exposure were different for PCA and PCP. The half-lives for PCP residues in the blood, liver, fat, and muscle were 6.2, 9.8, 23 and 6.9 hr, respectively, while PCA was more persistent having half-lives in these same tissues of 6.3, 6.9, 23, and 6.3 days. Thin-layer chromatographic and GC-MS analyses of the tissues of the PCP-exposed trout indicated that there was no methylation of PCP in any of the tissues studied. Bile from PCP-exposed trout contained high concentrations (250 μ g/g) of PCP, mostly as the glucuronide conjugate, but no other metabolites were detected. However, bile from PCA-exposed trout contained PCP glucuronide (10 μ g/g) as well as PCA, indicating demethylation of this compound in vivo by rainbow trout. Inclusion of 1 mg/liter of piperonyl butoxide in the PCA exposure system decreased the formation of PCP from PCA.

Xenobiotics and xenoestrogens in fish : modulation of cytochrome P450 and carcinogenesis

As is the case with mammals, an ever-increasing number of cytochromes P450 (CYPs) are being characterized from fish. The focus of work on fish CYPs has been primarily on environmental induction of CYP1A by pollutants such as the polycyclic aromatic hydrocarbons, polychlorinated biphenyls, dioxins and dibenzofurans. This response has been the basis for a sensitive biomonitoring tool of ecosystem health for a number of years. Studies have documented a correlation between CYP1A induction, pollutant levels and tumor incidence, especially in bottom-dwelling species. The rainbow trout has been utilized as a tumor model to document the role of CYP1A modulation in the inhibition or promotion of cancer. Fish are also very responsive to the class of chemicals known as xenoestrogens. Recent evidence is presented documenting the modulation of CYPs by xenoestrogens and their potential role as modulators of the tumor response. In this paper, we summarize the current knowledge concerning the occurrence of CYPs in fish and focus on the role of CYP1A induction in environmental monitoring of various genotoxic carcinogens and in the modulation of cancer in the trout model. Finally, the important class of aquatic pollutants known as xenoestrogens have now been shown to modulate CYP levels perhaps leading to alterations in tumor response or other adverse effects.

Effect of Polycyclic Aromatic Hydrocarbons on Hepatic Microsomal Enzymes and Disposition of Methylnaphthalene in Rainbow Trout in vivo

1. The effects of 3-methylcholanthrene, 2,3-benzanthracene and beta-naphthoflavone on xenobiotic metabolism in rainbow trout were studied. 2. These three polycyclic aromatic hydrocarbons increase hepatic arylhydrocarbon (benzo[alpha]pyrene) hydroxylase activity without altering glucuronyl transferase activity. 3. All three polycyclic aromatic hydrocarbons increased hepatic microsomal cytochrome P-450 levels by approximately 50%. 4. Pretreatment of trout with 2,3-benzanthracene resulted in an increase in the metabolism and biliary excretion of 2-methylnaphthalene in vivo. 5. These studies demonstrate that the induction of mono-oxygenation by polycyclic aromatic hydrocarbons can result in significant effects upon the metabolism and excretion of xenobiotics by fish in vivo.

The effect of various types of inducing agents on hepatic microsomal monooxygenase activity in rainbow trout.

The effects of various types of inducing agents on the hepatic microsomal monooxygenase (MO) system of rainbow trout were examined and compared to the induction profiles observed following pretreatment with either 3-methylcholanthrene- or phenobarbital-type inducers. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) elevated ethoxycoumarin- and ethoxyresorufin-O-deethylase activities (ECOD, EROD) as well as the cytochrome(s) P-450 content, but had no effect on benzphetamine-N-demethylation (BeND). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of solubilized microsomes from TCDD-pretreated fish demonstrated the intensification of a protein band with a molecular weight of 57,000. Although the magnitude of induction was not as great, isosafrole pretreatment of rainbow trout resulted in an induction profile similar to that observed following TCDD administration to these animals. Neither Kepone nor mirex had a stimulatory effect on any of the measured parameters and neither caused a change in the protein profile following SDS-PAGE of solubilized microsomes. These results suggest that fish respond differently from mammals to these inducers of the hepatic microsomal MO system.

Uptake, disposition, and persistence of nonylphenol from water in rainbow trout (Oncorhynchus mykiss)

1. Nonylphenol is an environmental estrogenic compound. Little is known about its interaction with aquatic species since most of the work on oestrogenic alkylphenols has been done in vitro using cells in culture. 2. Rainbow trout (Oncorhynchus mykiss) were exposed to 14C-nonylphenol at 18 and 36 ppb in water to study its distribution, persistence, and bioaccumulation. 3. Nonylphenol, or its metabolites, were distributed through the body of rainbow trout including the edible tissues of dorsal muscle and fat. 4. Nonylphenol was rapidly taken up into most tissues and had an apparent half-life of 19-20 +/- 4 SE hours in the muscle and fat. 5. The apparent bioaccumulation factor in viscera and carcass ranged from 40 in carcass to 100 in viscera. 6. Three glucuronide metabolites were separated by thin-layer chromatography following treatment of bile with beta-glucuronidase.

The transfer of 2,4,5,2′,4′,5′-hexachlorobiphenyl to fetuses and nursing offspring: I. Disposition in pregnant and lactating mice and accumulation in young

Abstract The disposition of 2,4,5,2′,4′,5′-hexachlorobi[ 14 C]phenyl (6-CB) in pregnant and lactating mice and its transfer to fetuses and nursing offspring were examined by administration of 6-CB to virgin female mice 2 weeks prior to mating. No differences were observed in tissue concentrations of [ 14 C]6-CB in pregnant animals when compared to virgin controls. However, on the day of birth, liver, adipose tissue, and kidney 6-CB contents in pregnant mice were higher than those in virgins sacrificed concurrently. Transplacental passage of 6-CB was minimal. Lactating mothers eliminated essentially their entire body burden of the PCB by 20 days postpartum and 6-CB was, in turn, accumulated by nursing offspring. No measurable elimination of 6-CB was observed from virgin controls during this time. Thus, although little passage of 6-CB occurred across the placenta once it had been sequestered into storage depots, it was readily transferred to suckling offspring through the lactating mammary gland.

Estrogenic effects of nonylphenol on pS2, ER and MUC1 gene expression in human breast cancer cells-MCF-7

Many chemicals have recently been discovered to have estrogenic activity, including the surfactant intermediate nonylphenol (NP). It has been well documented that estrogen is a facilitator of human breast cancer development under certain conditions, and environmental estrogens such as NP are currently under intense investigation. Using the expression of pS2 (a trefoil peptide expressed in breast cancer cells), MUC1 (a member of the mucin family) and ER (the human estrogen receptor) genes as estrogen-responsive reporter genes, the effects of estradiol and NP on human breast cancer cells-MCF-7 were studied. In the time course study, the mRNA expressions were detected after NP (10 microM) or estradiol (E2, 0.1 microM) treatments using the RT-PCR technique. The results indicated: (1) NP and E2 induced pS2 mRNA expression after a 2-h exposure and (2) NP induction produced the highest level of MUC1 mRNA after 2 h, which was reduced to only 42% of control at 48 h. E2 treatment resulted in a gradual increase in MUC1 expression over the course of the exposure. The highest level of MUC1 mRNA was at 48 h. This indicates that NP may stimulate MUC1 expression by a different mechanism than E2. (3) NP affected ER expression in the same manner as MUC1. In contrast, E2 stimulated ER expression in a similar manner as pS2; the highest level was at 2 h and expression remained elevated through the 48-h point. NP is an estrogenic compound that alters pS2, MUC1 and ER gene expression in MCF-7 cells. NP may affect MUC1 expression through a different mechanism than E2. The link between aberrant MUC1, PS2 and ER expression and the development of breast cancer also needs to be elucidated through further investigation.

Isolation and identification of a polar metabolite of tetrachlorobiphenyl from bile of rainbow trout exposed to14C-tetrachlorobiphenyl

The presence of polychlorinated biphenyls (PCBs) in a wide variety of fish has been reported by VEITH and LEE (1971) and STALLING and MAYER (1972). Laboratory studies have demonstrated the uptake of dietary PCB by rainbow trout (LIEB, et al., 1974) and lake trout (SCHOETTGER, 1973), and aqueous PCB by goldfish (HATTULA and KARLOG, 1973), spot and pinfish (HANSEN, et al., 1971) and yellow perch and rainbow trout (MELANCON, 1974). Once absorbed by fish the PCBs do not appear to be readily eliminated. Although over 50% of the PCB accumulated by spot and pinfish during preexposume to 1 ppb PCB was released during 8 weeks following exposure (HANSEN, et al., 1971; HANSEN, eta!., 1974) this extent of PCB elimination is not typical of fresh water fish. HATTULA and KARLOG (1973) reported that goldfish released almost 80% of accumulated PCBs during a i0-week washout period, but the amount of fat tissue in these fish showed an unexpected comparable decrease such that PCB levels in fat tissue remained constant at about 3000 ppm. LIEB, et al. (1974) examined the elimination of previously accumulated dietary PCBs by rainbow trout utilizing a PCB-f-ree diet (16 weeks) or fast (8 weeks). As the trout receiving the PCB-free diet grew, the PCB level decreased, but the total amount of PCB per fish remained constant. The fasted fish lost weight and the PCB level increased but the total amount of PCB per fish remained constant. Another study of the elimination of previously accumulated dietary PCB was reported by SCHOETTGER (1973). In this case, lake trout showed slightly reduced PCB concentrations, but it was not reported if this was simply a growth effect.