Biomedical subjects

D R Buhler

Publications and source records attributed to D R Buhler.

At least 37 records · Page 2Linked to original sources

PubMed1998

Cloning, sequencing, and tissue expression of CYP3A27, a new member of the CYP3A subfamily from embryonic and adult rainbow trout livers.

Screening of lambdagt11 and lambdagt22A cDNA libraries of livers from adult females and embryos of rainbow trout (Oncorhynchus mykiss), respectively, using rabbit anti-rainbow trout cytochrome P450 LMC5 polyclonal antibodies showed that there were identical cDNAs of 1802-bp nucleotides with open reading frames coding for proteins containing 518 amino acids (59,206 Da, pI = 6.39). The cDNA was assigned CYP3A27 by the P450 Nomenclature Committee to represent the first CYP3A subfamily member reported for aquatic species. The deduced N-terminal sequence of CYP3A27 was in agreement with 8 of the first 12 confirmed amino acid residues from Edman degradation of LMC5, a P450 previously isolated from juvenile trout liver. In similarity comparisons between species by positional alignment, the deduced amino acid sequence of rainbow trout CYP3A27 was 56.4% identical with dog CYP3A12, 56.0% with monkey CYP3A8, 54.9% with human CYP3A4, 54.7% with rat CYP3A9, and 54.2% with sheep CYP3A24. Marked differences in sex, age, and tissue expression of CYP3A27 in rainbow trout were observed at the mRNA level as shown by Northern blots. The major extrahepatic expression site for CYP3A27 was upper small intestine. Females expressed considerably more CYP3A27 mRNA than male in the fish examined. Southern blot analysis of restriction enzyme-digested rainbow trout genomic DNA demonstrated that multiple CYP3A27-related genes exist in rainbow trout.

Amino Acid Sequence↗

PubMed1998

Species differences in the hepatic microsomal enzyme metabolism of the pyrrolizidine alkaloids.

Species differences in pyrrolic metabolites and senecionine (SN) N-oxide formation among eight animal species (sheep, cattle, gerbils, rabbits, hamsters, Japanese quail, chickens, rats) varying in susceptibility to pyrrolizidine alkaloid (PA) intoxication were measured in vitro by hepatic microsomal incubations. The results suggested that there is not a strong correlation between the production of pyrrolic metabolites and susceptibility of animals to PA toxicity. The rate of PA activation in hamsters, a resistant species, measured by formation of (+/-)6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP) far exceeded the rate of SN N-oxide formation (detoxification) (DHP/N-oxide = 2.29). In contrast, SN N-oxide was the major metabolite in sheep, another resistant species, with much lower production of DHP (DHP/N-oxide = 0.26). The roles of cytochrome P450s and flavin-containing monooxygenases (FMO) in bioactivation and detoxification of pyrrolizidine alkaloids (PA) were studied in vitro using sheep and hamster hepatic microsomes. Chemical and immunochemical inhibition data suggested that the conversion of SN to DHP is catalyzed mainly by cytochrome P450s (68-82%), whereas the formation of SN N-oxide is carried out largely by FMO (55-71%). There also appeared to be a high rate of glutathione-DHP conjugation in hamster (63%) and sheep (79%) liver microsomal incubation mixtures. Therefore, low rates of pyrrole metabolite production coupled with glutathione conjugation in sheep may explain the resistance of sheep to SN, whereas the high rate of GSH-DHP conjugation may be one of the factors contributing to the resistance of hamsters to intoxication by this PA.

Animals↗

PubMed1998

CYP2M1: cloning, sequencing, and expression of a new cytochrome P450 from rainbow trout liver with fatty acid (omega-6)-hydroxylation activity.

A cDNA clone was isolated from a female rainbow trout liver lambda g tau 11 library using polyclonal antibodies raised against rainbow trout cytochrome P450 LMC1. This 2149-nucleotide clone contained an open reading frame encoding a protein of 499 amino acids with a calculated M(r) of 56,850 Da. On the basis of cytochrome P450 (P450) amino acid sequence comparisons, this rainbow trout P450 was assigned by the P450 Nomenclature Committee to a new P450 subfamily designated as CYP2M1. Northern blot results suggest that the expression of CYP2M1 at the transcriptional level was generally sex, tissue, and age specific. By use of a full-length CYP2M1 cDNA probe, it was observed that this cDNA hybridized strongly to a single 2.2-kb transcript in juvenile female rainbow trout trunk kidney and in liver from juvenile and sexually mature trout from both sexes. Negligible amounts of mRNA hybridizable to CYP2M1 cDNA were found in the juvenile and sexually mature male trunk kidney. cDNA-directed expression in COS-7 cells and of recombinant baculovirus in insect cells produced a protein that was reactive with rabbit anti-trout P450 LMC1 polyclonal antibody and exhibited the unique (omega-6)-hydroxylation toward lauric acid previously observed with rainbow trout P450 LMC1.

Amino Acid Sequence↗

PubMed1998

Expression of CYP2M1, CYP2K1, and CYP3A27 in brain, blood, small intestine, and other tissues of rainbow trout.

Expression of five constitutive forms of cytochrome P450 [(LMC1 (CYP2M1), LMC2 (CYP2K1), LMC3, LMC4, and LMC5 (CYP3A27)] in selected tissues from sexually immature 2-year old female and male rainbow trout (Oncorhynchus mykiss) were examined at the translational level by Western blot using polyclonal antibodies raised in rabbits against those purified trout hepatic P450s. Tissues examined were from brain, liver, muscle, blood, head kidney, trunk kidney, upper intestine, stomach, heart, and gonad (ovary or testis). The results showed that the liver was the major organ for expression of all the trout P450s studied. Trunk kidney was the secondary expression site except for LMC5. Selective translational expression of these P450 isoforms or similar proteins was observed for LCM1 and LMC5 in brain; for LMC2 and LMC5 in female upper intestine; and for LMC2 in blood plasma of the fish studied under the experimental and sampling conditions.

Animals↗

PubMed1998

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.

Animals↗

PubMed1998

Evaluation of chemicals as inhibitors of trout cytochrome P450s.

This study examined the ability of several inhibitors of mammalian cytochrome P450s to affect hepatic P450-mediated monooxygenase activities in microsomes from beta-naphthoflavone (BNF)-treated rainbow trout. Three monooxygenase activities, namely, lauric acid (omega-1)-hydroxylase (LA-OH), 7,12-dimethylbenz[a]anthracene hydroxylase (DMBA-OH), and progesterone 6beta-hydroxylase (PROG-OH) activities were used as functional markers for trout hepatic CYP2K1, CYP1A1, and CYP3A27, respectively. The chemicals that were examined for their inhibitory effects were reversible, quasi-irreversible, or irreversible inhibitors of mammalian P450. At 100 microM concentration, the reversible inhibitors (ketoconazole, miconazole, and clotrimazole) were most potent in inhibiting LA-OH activity. These imidazole compounds, as well as ellipticine, parathion, and alpha-naphthoflavone, were the strongest inhibitors of DMBA-OH and PROG-OH activities. In addition, isosafrole, piperonyl butoxide, gestodene, 17alpha-ethynylestradiol, 1-aminobenzotriazole, and 5,8,11,14-eicosatetraynoic acid strongly inhibited PROG-OH activity. The global inhibitors, metyrapone, chloramphenicol, and allylisopropylacetamide, had very little or no inhibitory effect on trout LA-OH and DMBA-OH activities. Triacetyloleandomycin, a CYP3A inhibitor, did not affect PROG-OH activity catalyzed by trout CYP3A27. Diethyldithiocarbamate was an activator of LA-OH activity. None of the above enzyme activities was selectively inhibited by any of the chemicals when used at a concentration of 100 microM. There was no difference in the inhibition of LA-OH activities by representative P450 inhibitors between liver microsomes from untreated trout and BNF-treated trout. The results of this study suggest that inhibition data from mammalian studies could not be directly extrapolated to trout and likely other fish species and that care must be observed when mammalian P450 inhibitors are used to determine the participation of P450s in the metabolism and toxicity of xenobiotics in nonmammalian species.

Animals↗

PubMed1998

The roles of CYP3A and CYP2B isoforms in hepatic bioactivation and detoxification of the pyrrolizidine alkaloid senecionine in sheep and hamsters.

The roles of cytochrome CYP3A and CYP2B isozymes in the bioactivation and detoxification of the pyrrolizidine alkaloid (PA) senecionine (SN) have been investigated in vitro with sheep and hamster hepatic microsomes. Our results show that the rate of SN activation measured by (+/-)-6, 7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP) formation greatly exceeded the rate of SN N-oxide formation (detoxification) in hamsters. In contrast, SN N-oxide, a detoxification product, was the major metabolite in sheep with much lower DHP production. Immunoinhibition studies with anti-sheep CYP3A and CYP2B antibodies show that members of CYP3A subfamily play the major role in the conversion of PA to pyrrolic metabolites in both species (over 90% in sheep; 68% in hamster). These enzymes also contribute 38.8 and 41. 3% of SN N-oxidation in sheep and hamsters, respectively. In contrast, CYP2B isoforms have a limited capacity toward DHP formation in both species (47% in sheep; 32% in hamster), while these enzymes catalyzed only 24.6 and 35.4% SN N-oxidation in sheep and hamster, respectively. Using triacetyloleandomycin (TAO) and gestodene, two highly selective chemical inhibitors of CYP3A isoforms, our data show that 90% of DHP formation was inhibited by either inhibitor in sheep. Gestodene appeared to be more efficient than TAO in the inhibition of DHP production in hamsters. Testosterone 6beta-hydroxylase activity, a functional marker of CYP3A, was significantly inhibited by TAO and gestodene in sheep liver microsomes and by gestodene (100 microM) in hamster liver microsomes. These results suggest that CYP3A isozymes have important roles in bioactivation and detoxification of PA in both species, whereas CYP2B subfamily members are less efficient in biotransformation of PA.

Animals↗

PubMed1998

Analysis of denatonium benzoate in Oregon consumer products by HPLC.

Denatonium benzoate (DB) is being added to a variety of consumer products as an aversive agent or denaturant. In 1995, Oregon was the first state to mandate the addition of DB to antifreeze and windshield washer fluids regardless of whether the product was marketed in a bottle with a child-proof cap. We have modified published high pressure liquid chromatography (HPLC) methods to measure the concentration of DB and validated it for use in Oregon consumer products. Analysis of standards showed a linear curve in the range of 1.25 to 50 ppm. This makes it useful for most analytical applications as DB usually is added in the 10 to 30 ppm range according to DB's manufacturer. The peak analyzed was collected and verified as DB by mass spectrometry. The method was applied to a variety of commercially-available products with particular emphasis on antifreeze and windshield washer fluids which are required by Oregon law to contain an aversive agent. Products analyzed were purchased in either 1994 or 1996 and showed a wide variation in concentration of DB ranging from below the limit of detection in many of the windshield washer products to above 30 ppm in many of the antifreeze formulations. There was no significant change in levels of DB after storage of antifreeze or windshield washer fluids at room temperature for six months.

Chromatography, High Pressure Liquid↗

PubMed1998

Rainbow trout cytochrome P450s: purification, molecular aspects, metabolic activity, induction and role in environmental monitoring.

Cytochromes P450 (P450s or CYPs) constitute a superfamily of heme-thiolate proteins that play important roles in oxidative metabolism of endogenous and exogenous compounds. This review provides some limited history but addresses mainly the research progress on the cytochrome P450s in rainbow trout (Oncorhynchus mykiss), their purification, structures at the primary level, role in metabolism, responses to chemicals and environmental pollutants, application to biomonitoring and the effect of various factors on their expression or activities. Information obtained to date suggests that the rainbow trout P450 systems are as complex as those seen in mammals. Fourteen P450s have been purified from liver or trunk kidney to relatively high specific content. cDNAs belonging to seven different P450 families have been documented from trout liver, kidney and ovary. Two CYP1A genes, nine cDNAs containing open reading frames, and a cDNA fragment were entered into GenBank. Among them, CYP2K1, CYP2K3, CYP2K4, CYP2M1, CYP3A27 and CYP4T1 are the most recently described forms. CYP2K1, CYP2M1 and CYP4T1 represent newly identified P450 subfamilies first described in the rainbow trout. In many cases, the cloned rainbow trout P450s have subsequently been expressed in heterologous expressions systems such as COS-7 cells, yeast and baculovirus infected insect cells. Some of the overexpressed P450 isoforms have been partially characterized. Potential future research directions are discussed.

Animals↗

PubMed1998

Induction of lauric acid hydroxylase activity in catfish and bluegill by peroxisome proliferating agents.

In mammals, sensitivity to peroxisome proliferation by peroxisome proliferating agents (PPAs) appears to be correlated with inducibility of lauric acid hydroxylase activity. Bluegill and catfish have been shown to respond to PPAs by induction of lauric acid hydroxylase immunoreactive proteins (Haasch, 1996). In this investigation, induction of lauric acid hydroxylase activity was confirmed by HPLC and mass spectral analysis of specific hydroxylation products and possible species-specific differences in metabolism were investigated. Male bluegill, channel catfish and rat, were administered the model PPAs, clofibrate (200 mg kg-1, i.p.), ciprofibrate (100 mg kg-1, i.p.), or olive oil as vehicle control (both sexes of catfish), 48 h prior to hepatic, trunk kidney (catfish only) or kidney (rat) microsome preparation. In general, total metabolism of lauric acid was similar in all species, but female catfish metabolize lauric acid to a greater extent than males. Ciprofibrate treatment produced significant induction of omega- and omega-6 hydroxylation in male catfish kidney. In male bluegill liver, omega-, omega-4 and omega-5 hydroxylations were significantly induced by clofibrate treatment. The data indicate that induction of lauric acid hydroxylase cytochrome(s) P450 occurs in PPA-exposed fish which may be a consideration for environmentally-exposed responsive species.

Animals↗

PubMed1997

In vitro metabolism of 7,12-dimethylbenz[a]anthracene by rainbow trout liver microsomes and trout P450 isoforms.

Liver microsomes from juvenile trout metabolized DMBA to unknown highly polar metabolites (X) and to DMBA-t-5,6-diol, DMBA-t-8,9-diol, 7-OHM-12-MBA, 7M-12-OHMBA, 2-OH-DMBA, 4-OH-DMBA, and trace amounts of DMBA-t-3,4-diol. Treatment of trout with beta-naphthoflavone (BNF) and isosafrole (ISF) increased the formation of these products except for the hydroxymethyl derivatives of DMBA. The production of DMBA-t-3,4-diol, 2-OH-DMBA, and 4-OH-DMBA was much greater in BNF-induced liver microsomes than that in ISF-induced liver microsomes. In contrast, the yield of DMBA-t-8,9-diol and 7-OHM-12-MBA was greater in ISF-induced microsomes than that in BNF-induced microsomes. Trout CYP1A1 (P450 LM4b) purified from BNF-treated trout catalyzed the formation of the same metabolites generated by BNF-induced microsomes in the presence of added human microsomal EH. The constitutive forms of P450 isolated from untreated trout such as P450s LMC3, LMC4, and LMC5, CYP2M1 (P450 LMC1), and CYP2K1 (P450 LMC2) did not produce any of the DMBA metabolites (except for DMBA-t-8,9-diol by CYP2K1) generated by the trout microsomes. Generation of DMBA-DNA and DMBA-protein adducts in vitro was enhanced by treatment of trout with BNF and by ISF to a lesser extent. Formation of adducts and DMBA diols by BNF-induced liver microsomes and by trout CYP1A1 was completely blocked by the CYP1A inhibitor ellipticine (100 microM). These results suggest that the BNF-inducible trout P450 (CYP1A), not the constitutive P450s, is the major catalyst for the biotransformation of DMBA to metabolites that bind to macromolecules.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1997

CYP1A induction by beta-naphthoflavone, Aroclor 1254, and 2,3,7,8-tetrachlorodibenzo-p-dioxin and its influence on aflatoxin B1 metabolism and DNA adduction in zebrafish.

This study investigated the inductive response of cytochrome P4501A (CYP1A) in the zebrafish (Danio rerio) following exposure to Aroclor 1254, beta-naphthoflavone (betaNF), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and then investigated TCDD modulation of aflatoxin B1 (AFB1) metabolism and hepatic AFB1-DNA adduction. Aroclor 1254 fed at 500 ppm for 1 to 9 days or intraperitoneal (ip) injection of 75-200 mg Aroclor 1254/kg body weight failed to induce CYP1A protein or associated 7-ethoxyresorufin-O-deethylase (EROD) activity. By contrast, dietary betaNF at 500 ppm for 3 or 7 days induced CYP1A protein and EROD activity approximately threefold above controls. A single ip injection of 150 mg/kg betaNF showed maximal induction of CYP1A protein and EROD activity near 24 hr, both of which decreased to control levels during the next 6 days. Single ip administration of 25, 50, 100, or 150 mg betaNF/kg body weight provided dose-responsive increases in CYP1A and EROD activity. Dietary exposure to 0.75 ppm TCDD for 3 days also significantly induced CYP1A and EROD. The effect of TCDD on the metabolism of [3H]AFB1 in zebrafish was then investigated. The major [3H]AFB1 metabolites excreted in water over 24 hr in the control group were aflatoxicol, aflatoxicol-glucuronide, and parent AFB1. By contrast, the predominant metabolites in the TCDD-pretreated group were aflatoxicol-M1-glucuronide, aflatoxicol, aflatoxin M1 plus aflatoxicol-M1 (unresolved), aflatoxicol-glucuronide, and parent AFB1. Surprisingly, hepatic AFB1-DNA adduction was approximately fourfold higher in the TCDD treated group than in controls. This significant difference could not be explained by increased capacity for bioactivation of AFB1 as measured by an in vitro AFB1-exo-8, 9-epoxide trapping assay. However, it was demonstrated that both control and induced zebrafish have high capacity to bioactivate aflatoxin M1 to a reactive intermediate, such that secondary bioactivation of this genotoxic intermediate may be responsible for the increased DNA binding.

Aflatoxin B1↗

PubMed1997

Down regulation of hepatic flavin-containing monooxygenase activity by 17 beta-estradiol in rainbow trout (Oncorhynchus mykiss).

Expression of flavin-containing monooxygenase (FMO) activity has been shown to be directly correlated with salinity in euryhaline fish. Sex steroids play a significant role in osmoregulation in euryhaline fish. The effects of 17 beta-estradiol and testosterone on hepatic FMO activity (N,N-dimethylaniline N-oxygenase; thiourea S-oxygenase) and expression was examined in 6-month-old and 1-year-old rainbow trout (Oncorhynchus mykiss). Fish were treated with 3 mg/kg of each compound on days 1, 4, 7, 10, 13, and 15 with euthanasia on day 19. Treatment of 6-month-old and 1-year-old female rainbow trout with testosterone or estradiol significantly reduced FMO-catalyzed DMA N-oxygenase activity in liver. However, testosterone treatment failed to significantly alter thiourea S-oxidase in 1-year-old males, although 17 beta-estradiol significantly reduced FMO activity. These results parallel those of studies showing repressive effects of sex steroids on branchial chloride cells and Na+K+ATPase of salmonids and provide further evidence of a role for FMO in teleost osmoregulation.

Animals↗

PubMed1997

Beta-naphthoflavone induced CYP1A1 and 1A3 proteins in the liver of rainbow trout (Oncorhynchus mykiss).

1. Two CYP1A proteins, designated HAP 1 and HAP 2, were isolated from the liver of the beta-naphthoflavone (BNF)-treated rainbow trout. The proteins were initially resolved by chromatography on a DEAE sepharose column and were further purified by hydroxyl-apatite chromatography. 2. Both HAP 1 and HAP 2 proteins exhibited high 7-ethoxyresorufin, methoxy-resorufin and phenacetin O-dealkylase activities and were good catalysts for the oxidation of 7,12-dimethylbenz[a]anthracene (DMBA). No qualitative difference was observed between the two proteins in their ability to catalyse the formation of the individual metabolites of DMBA. 3. The two purified proteins showed identical amino acid sequence for the first 13 amino acids. However, the 14th amino acid was valine for HAP 1 protein and alanine for HAP 2 protein. 4. Alignment of the amino acid sequences showed that HAP 1 protein was identical to the deduced protein of the previously reported trout CYP1A2 (renamed CYP1A1) gene for the first 24 amino acids at the N-terminal region. HAP 2 protein corresponded to the deduced protein sequence of CYP1A3 gene for the first 14 amino acids. However, unlike the deduced sequences of CYP1A1 and 1A3 the N-terminal methionine was absent in the purified proteins. 5. We conclude that HAP 1 and HAP 2 are the products of the CYP1A1 and CYP1A3 genes respectively, and are found in the liver of the BNF-treated rainbow trout.

Amino Acid Sequence↗

PubMed1997

The regiospecific hydroxylation of lauric acid by rainbow trout (Oncorhynchus mykiss) cytochrome P450s.

We have reexamined the hydroxylation of [1-14C]-lauric acid by trout liver microsomes and reconstituted trout P450s using a new HPLC system that gave an improved separation of hydroxylauric acids. Under these conditions, hepatic microsomes from yearling juvenile trout were shown to form omega-, (omega-1)-, (omega-2)-, (omega-3)-, (omega-4)-, (omega-5)-, and (omega-6)-OH lauric acid oxidation products (12-OH, 11-OH, 10-OH, 9-OH, 8-OH, 7-OH, and 6-OH lauric acid, respectively) as verified by GC/MS analysis. Microsomes from male and female juvenile trout liver formed (omega-1)-OH lauric acid as the major metabolite (23-29% of total radioactivity) and no major differences were observed between males and females. By contrast, liver microsomes from 3-year-old sexually mature trout had substantially lower lauric acid hydroxylase activity than juvenile microsomes and produced small quantities of only the (omega-1)-, (omega-2)-, and (omega-6)-hydroxylation products. Moreover, microsomes from sexually mature female trout had markedly lower lauric acid hydroxylase activity than those from the sexually mature male trout. Rat liver microsomes were quite catalytically active but formed mostly the omega- and omega-1 lauric acid oxidation products. Lauric acid metabolism also was analyzed in reconstituted systems containing purified juvenile trout LMC1 (CYP2M1) and LMC2 (CYP2K1) and with hepatic microsomes from juvenile trout in the presence of rabbit polyclonal antibodies raised against the two purified trout P450s. CYP2M1 catalyzed the (omega-6)-hydroxylation of lauric acid while the trout CYP2K1 form produces mainly (omega-1)-OH lauric acid together with a smaller quantity of the (omega-2)-hydroxylation product. All of the trout and rat radiometric lauric acid metabolism results were confirmed by direct mass spectrometric analysis of derivatized lauric acid metabolism mixtures. Use of direct mass spectrometric analysis for the hydroxylated lauric acids offers considerable advantages since the method did not require use of a radioactive fatty acid, completely separated all of the lauric acid hydroxylation products, confirmed identification of each metabolite, and was more sensitive than the radiometric analysis method.

Animals↗

PubMed1996

Dieldrin pretreatment alters [14C]dieldrin and [3H]7,12-dimethylbenz[a]anthracene uptake in rainbow trout liver slices.

We previously demonstrated that pretreatment of rainbow trout with the organochlorine insecticide dieldrin altered in vivo disposition of a subsequent [14C]dieldrin dose. This was not explained by changes in total lipid content or the activity of common xenobiotic metabolizing enzymes. We hypothesized that dieldrin induced hepatic proteins responsible for organochlorine (OC) sequestration, transport, or excretion and that these changes reflected an adaptive response of trout to OC exposure. Here, uptake of 1.18 microM [14C]-dieldrin by precision cut liver slices was increased by dieldrin pretreatment of rainbow trout. Uptake of 0.118 and 1.18 microM [3H]-7,12-dimethylbenz[a]anthracene (DMBA) and efflux of 0.118 microM [3H]DMBA were significantly increased in slices from dieldrin-pretreated trout. Liver slice uptake of 10 but not 1.18 microM [3H]-estradiol and [3H]cholic acid was significantly increased by dieldrin pretreatment. There were no such significant differences for [3H]cholesterol, [3H]cholesterol-oleate, or [3H]oleic acid uptake. Dieldrin pretreatment did not alter hepatic microsomal metabolism of [3H]DMBA or [14C]benzo[a]pyrene or content of six cytochrome P450 isozymes, as quantitated by Western Blot analysis. These results provide further evidence that altered disposition of [14C]dieldrin and [3H]DMBA in dieldrin-pretreated trout was not explained by microsomal enzyme induction but reflected altered processes integral to hepatocellular transmembrane kinetics. These changes may have important implications for OC bioaccumulation by rainbow trout and demonstrate an interaction between dieldrin and DMBA in the absence of cytochrome P450 system induction.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1995

Temperature-modulated incidence of aflatoxin B1-initiated liver cancer in rainbow trout.

Rainbow trout (initial weight of 4 or 5 g) were acclimated at a cool, 11.0 degrees C (C), a warm, 18.0 degrees C (W), or an intermediate temperature 14.5 degrees C (I) for 1 month. There was a slight difference in hepatic microsomal content of one of six cytochrome P450 isozymes between acclimation groups. Monounsaturated fatty acids in hepatic phosphotidylethanolamine but not phosphotidylcholine increased at lower acclimation temperatures. Saturated fatty acid content decreased with temperature for both phospholipid classes. Fish were exposed to 0.08-0.12 ppm waterborne aflatoxin B1 (AFB1) for 30 min at respective acclimation temperatures or after acute temperature shifts (24 hr) and reared for 9 months at C, I, or W. With exposure concentrations which delivered equivalent target organ doses, trout acclimated, exposed, and reared at C, I, or W had liver tumor incidences of 4, 35, and 61%, respectively. The average number of tumors per liver increased from 1.25-1.34 at C to 2.46-2.66 at W. There were no temperature-dependent differences in tumor diameter. When C- and W-acclimated fish were AFB1 exposed and reared at I, tumor incidence was 12.5% for W-I-shifted fish and 26.5% for C-I-shifted fish. This was consistent with previous work which demonstrated acute downward temperature shift reduced [3H]AFB1 adduction to hepatic DNA. Tumor incidence and multiplicity data suggested manipulation of temperature permitted selective modulation of cancer initiation and promotion in rainbow trout.

Aflatoxin B1↗

PubMed1995

Inhibition of in vitro aflatoxin B1-DNA binding in rainbow trout by CYP1A inhibitors: alpha-naphthoflavone, beta-naphthoflavone and trout CYP1A1 peptide antibody.

Rainbow trout cytochrome P450 (CYP)1A detoxifies aflatoxin B1 (AFB1) to aflatoxin M1 (AFM1), whereas CYP2K1 activates AFB1 to AFB1-8,9-epoxide. We report that alpha-naphthoflavone (ANF) and beta-naphthoflavone (BNF) both strongly inhibit CYP1A-mediated ethoxyresorufin O-deethylase (EROD) activity (Ki = 9.1 +/- 0.8 and 7.6 +/- 1.1 nM, respectively). These inhibitors (selective for mammalian CYP1A at low concentrations), as well as rabbit polyclonal antibody to a trout CYP1A1 peptide (residues 277-294), also strongly inhibited trout microsome-catalyzed AFB1-DNA binding and lauric acid (omega-1) hydroxylation in vitro, reactions previously established to be CYP2K1-dependent. ANF at 0.5, 5, 50 and 500 microM inhibited liver microsome-catalyzed AFB1-DNA binding by 22, 58, 84 and 91%, respectively, whereas BNF at the same concentrations inhibited 22, 74, 78 and 81%, respectively. The CYP1A1 peptide and CYP2K1 polyclonal antibodies (10 mg IgG/mg microsomal protein) inhibited AFB1-DNA binding by 84 and 66%, respectively, compared with pre-immune IgG. Lauric acid (omega-1) hydroxylation was inhibited 61% by 5 microM ANF, 69% by 5 microM BNF and 100% by either antibody at 12 mg IgG/mg microsomal protein. These results demonstrate that mammalian CYP1A inhibitors also inhibit trout microsomal AFB1-DNA binding and lauric acid (omega-1) hydroxylation, catalyzed primarily by CYP2K1. In the absence of evidence that trout CYP1A can catalyze AFB1-DNA binding, the results suggest configuration similarities at, or near, the active sites for these two fish enzymes that result in antibody crossreaction and loss of the inhibitor specificity observed with mammalian CYP1A.

Aflatoxin B1↗