Effects of vehicle, diet and gender on the expression of PMP70- and CYP2K1/2M1-like proteins in the mummichog.

The mummichog (Fundulus heteroclitus) has been shown to be responsive to peroxisome proliferating agents (PPAs). Peroxisomes function as important sites for fatty acid beta-oxidation. Peroxisome proliferation by PPAs or starvation can lead to changes in the size and number of peroxisomes and the expression of omega-hydroxylases (CYP2K1/2M1 in rainbow trout). Mummichogs were subjected to 96 h fasting or 96 h recovery from fasting. Expression of PMP70- and CYP2K1/2M1-like proteins in vehicle-treated or non-treated controls was compared in both males and females. Fasting and vehicle produced decreases in PMP70- and CYP2K1/2M1-like proteins in both males and females. In reproductive females, decreases due to fasting and vehicle treatment were greater than in female fish that were not gravid. Recovery from fasting resulted in levels of CYP2K1/2M1 near control levels in males while in recovered females, about 2-fold higher levels compared to controls were noted. These results indicate that gender, reproductive status and diet can produce changes in the expressed levels of peroxisomal PMP70 and microsomal CYP2K1/2M1-like proteins in the mummichog.

Immunodetection of hepatic peroxisomal PMP70 as an indicator of peroxisomal proliferation in the mummichog, Fundulus heteroclitus.

Peroxisomes are important sites for beta-oxidative fatty acid metabolism and peroxidative detoxification. Agents causing peroxisomal proliferation have been associated with reproductive and developmental toxicity and hepatocarcinogenesis. Female mummichog (Fundulus heteroclitus) were exposed to waterborne 2,4-dichlorophenoxyacetic acid (2,4-D), a model peroxisome proliferator, at sublethal concentrations of 0.01, 0.10, and 1.00 ppm or dimethyl sulfoxide (DMSO) vehicle for 7, 14, or 21 days. A polyclonal antibody to rat PMP70 protein (70 kDa peroxisomal membrane protein, a major component of peroxisomes and member of the ABC transporter superfamily) was used for Western blotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to determine possible peroxisome proliferation. Significant increases of an approximately 70-kDa protein band recognized by anti-PMP70 were observed on all days, especially at the highest exposure concentration. The data suggest that immunoassay of PMP70 is a useful biomarker assay for peroxisome proliferation, and may be applicable to a wide range of species. The response also suggests that this assay could be used for measuring chronic exposures to environmental peroxisome proliferating agents.

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.

Use of 7-alkoxyphenoxazones, 7-alkoxycoumarins and 7-alkoxyquinolines as fluorescent substrates for rainbow trout hepatic microsomes after treatment with various inducers.

Various fluorescent substrates have been used as specific indicators of induction or activity of different cytochrome P450 isozymes in both fish and mammalian species. In an attempt to identify additional definitive fluorescent substrates for use in fish, we examined a series of 7-alkoxyphenoxazones, 7-alkoxycoumarins and 7-alkoxyquinolines as substrates in O-dealkylation assays with hepatic microsomes from rainbow trout (Oncorhynchus mykiss). Microsomes were prepared after 48 hr of treatment with beta-naphthoflavone (beta-NF), pregnenolone-16 alpha-carbonitrile (PCN), phenobarbital (PB), isosafrole (ISF), or dexamethasone (DEX). Total P450 spectra were obtained, and spectral binding studies were performed. Microsomal O-dealkylation rates were greater after ISF treatment than after beta-NF treatment for 7-methoxy-, 7-ethoxy-, 7-propoxy- and 7-benzyloxyphenoxazones but not for 7-butoxyphenoxazone. DEX treatment resulted in a significant elevation of pentoxyphenoxazone metabolism (about a 144-fold increase) compared with microsomes induced by beta-NF (11-fold) and ISF (37-fold). The rates of dealkylation of the alkoxyphenoxazones by ISF-treated microsomes occurred in the following order: methoxy > ethoxy > propoxy > benzxyloxy > butoxy > pentoxy. When beta-NF-treated microsomes were used, the 7-alkoxyphenoxazones were metabolized as follows: methoxy > ethoxy > propoxy > butoxy > benzyloxy = pentoxy, while the order of metabolism of the 7-alkoxycoumarins was: ethoxy >> butoxy > propoxy = methoxy > benzyloxy > pentoxy. None of the other treatments significantly increased the rate of metabolism of any of the alkoxycoumarins. Treatment with beta-NF did not significantly elevate the rate of metabolism of any of the alkoxyquinolines. DEX treatment produced significant elevations in the rate of metabolism of benzyloxy-, ethoxy-, and butoxy- = pentoxy- = propoxyquinoline, in that order. ISF treatment significantly elevated the rate of metabolism of benzyloxy-, methoxy- and butoxyquinoline, in that order. These results suggest that some of these new fluorescent substrates can be used to characterize induction of rainbow trout hepatic microsomal monooxygenase activity by ISF and DEX, in addition to the commonly used ethoxyphenoxazone and ethoxycoumarin for the characterization of induction by beta-NF or other 3-methylcholanthrene-type P450 inducers. Distinction between ISF-type and beta-NF-type inducers in rainbow trout hepatic microsomes may best be made using 7-methoxycoumarin as a substrate. Distinction between ISF-type and DEX-type inducers and between beta-NF-type and DEX-type inducers may best be made using 7-methoxyphenoxazone as a substrate.(ABSTRACT TRUNCATED AT 400 WORDS)

Negative control of cytochrome P450 1A1 (CYP1A1) by glucocorticoids in rainbow trout liver.

1. In rainbow trout liver, dexamethasone caused a reduction in cytochrome P450 1A1 proteins but no change in P450 3A proteins as measured by Western blots using specific anti P450 1A1 and P450 3A sera. 2. After dexamethasone administration, the level of P450 1A1 was down to 14% and 8% of the control values at 24 and 48 hr, respectively. 3. There was no change in P450 1A1 mRNA abundance at 24 hr but a decrease to 45% of the control value was observed at 48 hr after dexamethasone. 4. There is, therefore, a differential response between P450 1A1 and P450 3A to dexamethasone in the trout liver. 5. The initial decrease in P450 1A1 protein probably occurs at the translational level while both translational and transcriptional levels are involved 48 hr after dexamethasone administration.

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)

Effect of acrylamide monomer on hepatic CYP1A1 monooxygenase induction in rainbow trout.

1. Rainbow trout hepatic microsomes were pre-incubated in vitro with 1 pM, 1 nM, and 1 microM acrylamide for 20 or 30 min and ethoxyresorufin-O-deethylase (EROD) was measured. In vitro pre-incubation did not produce a significant decrease in EROD catalytic activity. 2. The effects of 50 ppm acrylamide monomer on hepatic CYP1A1 mRNA and CYP1A1 isozyme steady state levels in rainbow trout were determined after 6, 10, and 14 days of exposure. 3. Acrylamide monomer produced a 35% increase, 51% decrease and 140% increase in CYP1A1 mRNA at 6, 10 and 14 days, respectively, while at the same time CYP1A1 isozyme levels were decreased 12%, 67%, and 62% and EROD activity was decreased 33%, 90%, and 86%, respectively. 4. This indicates that acrylamide treatment may result in either a change in the translation of CYP1A1 mRNA or an isozyme selective inactivation of CYP1A1 resulting in loss of CYP1A1 apoprotein. 5. The effect of acrylamide treatment on hepatic CYP1A1 induction was determined using 10 or 14 day treatment with 50 ppm acrylamide monomer in a flowthrough exposure and induction with beta-naphthoflavone (beta-NF; 100 mg/kg i.p.) for 1 or 4 days. 6. Acrylamide and 1-day beta-NF had no effect on CYP1A1 mRNA levels when compared to 1-day beta-NF treatment alone, but acrylamide and 4-day beta-NF resulted in a 74% decrease in CYP1A1 mRNA compared to 4-day beta-NF treatment alone.(ABSTRACT TRUNCATED AT 250 WORDS)

A comparison of hepatic P450 induction in rat and trout (Oncorhynchus mykiss): delineation of the site of resistance of fish to phenobarbital-type inducers.

1. A comprehensive approach was taken to delineate the site of refractivity of trout to phenobarbital-type (PB-type) hepatic monoxygenase (MO) inducers. 2. Model inducers beta-naphthoflavone (BNF; 3-MC-type), and PB as well as the polychlorinated biphenyl isomers, 3,4,5,3',4',5'-hexachlorobiphenyl (3,4,5-HCB; 3-MC-type) and 2,4,5,2',4',5'-hexachlorobiphenyl (2,4,5-HCB; PB-type) were used to assess MO activities, total cytochromes P450, and [35S]-methionine incorporation into de novo synthesized microsomal protein in both trout and rats. 3. In rainbow trout immunodetection of P450 isozymes and nucleic acid hybridization of rainbow trout P(1)450 mRNA using pfP(1)450-3' (trout 3-MC-inducible, P450IA1 gene) and genomic DNA using pfP(1)450-3' or pSP450-oligo (rat PB-inducible, P450IIB1 gene) cDNAs were carried out. 4. In rainbow trout, PB and 2,4,5-HCB do not increase hepatic MO activities, total cytochromes P450, de novo synthesis of microsomal protein, levels of P450 isozymes, or levels of P(1)450 mRNA. 5. Rainbow trout have, within their genome, DNA with sequence(s) similar to rat P450IIB1, but inducibility of this P450 in trout by PB-type inducers is lacking.

Cloned rainbow trout liver P(1)450 complementary DNA as a potential environmental monitor.

A technique is proposed for the biological monitoring of pollutants in aquatic environments by use of a complementary DNA (cDNA) probe. The induction of hepatic cytochrome P(1)450 mRNA has been investigated utilizing pfP(1)450-3', a 3'-specific 1.5 kb cDNA clone derived from 3-methylcholanthrene-inducible mRNA of rainbow trout. A time course of induction of both the hybridizable mRNA and hepatic monooxygenase catalytic activity in rainbow trout with a known inducer in fish, beta-naphthoflavone, was studied. The cDNA probe was also shown to hybridize with induced mRNA of brook trout, scup, garter snake, painted turtle, and rat demonstrating the suitability of the probe for examining induction of mRNA in various species. The results of these experiments suggest that the cDNA probe may be useful as a biological monitoring tool for determining the presence and effects of chemical pollutants which are inducers of hepatic microsomal monooxygenase activity. The probe may have the potential to be applied as an early warning system in the monitoring of water quality.

Induction of cytochrome P-450 mRNA in rainbow trout: in vitro translation and immunodetection.

The time course of induction of the rainbow trout microsomal hepatic monooxygenase (MO) system was examined by determination of levels of mRNA and corresponding levels of catalytic activity. Animals were pretreated with beta-naphthoflavone (beta-NF, ip, 100 mg/kg) and terminated at 0, 2, 6, 18, and 48 hr postinjection. Levels of mRNA were determined by immunoprecipitation of in vitro translation products. Levels of mRNA coding for the cytochrome P-450 LM4b isozyme were maximally increased (13-fold) at 18 hr and had decreased almost to pretreatment levels by 48 hr post-treatment. This was in contrast to the catalytic activity in which ethoxyresorufin-O-deethylase (EROD) and ethoxycoumarin-O-deethylase (ECOD) were significantly elevated at both 18 hr (25- and 5-fold, respectively) and 48 hr (46- and 8-fold, respectively). Pretreatment with beta-NF (ip, 100 mg/kg) or 2,4,5,2',4',5'-hexachlorobiphenyl (6-CB, ip, 150 mg/kg) for 18 hr resulted in significant differences in levels of mRNA in only the beta-NF-treated group. The LM2 P-450 isozyme could not be detected by immunoprecipitation with anti-LM2 IgG in trout treated with these same inducers. The results suggest a difference between the time course of induction of the mRNA for cytochrome P-450 LM4b isozyme and the induction of catalytic activity. Under the detection system utilized, the results suggest that the phenobarbital-like inducer, 6-CB, does not induce cytochrome activity nor does it induce the mRNA for cytochrome P-450 LM4b isozyme.

Regulation of plasma 1,25-(OH)2-D3 by phosphate: evidence against a role for total or acid-soluble renal phosphate content.

In order to evaluate a possible role for tissue phosphate or phosphorylated compounds in mediating the increase in plasma 1,25-(OH)2-D3 levels during dietary phosphate deprivation, measurements of total and acid-soluble renal cortical phosphate content have been made in both intact and hypophysectomized (hypox) rats eating a normal diet and also after four days of dietary phosphate deprivation. Similar measurements were also made in phosphate-deprived hypophysectomized rats replaced with growth hormone (GH). Total and acid-soluble renal cortical phosphate content averaged 81 +/- 8 mumol/g and 4.1 +/- 0.6 mumol/g, respectively, in intact rats eating the normal diet and were not significantly altered after phosphate deprivation despite a fall in plasma phosphate of about 40% and a fourfold increase in plasma 1,25-(OH)2-D3 levels. Total and acid-soluble renal cortical phosphate content levels were higher in hypox rats, averaging 92 +/- 8 mumol/g and 4.9 +/- 0.7 mumol/g, respectively, but also did not change after phosphate deprivation. Replacement of phosphate-deprived hypox rats with GH resulted in a further fall in plasma phosphate and a significant increase in plasma 1,25-(OH)2-D3 levels, but there was no change in either total or acid-soluble renal cortical phosphate content. The distribution of organophosphorus compounds in the acid-soluble phosphate fraction in these experiments was also evaluated using 31P NMR spectrometry. Although there appeared to be an increase in the total concentration of organophosphorus compounds after phosphate deprivation, this effect was not altered by hypophysectomy or by replacement of phosphate-deprived hypox rats with GH.(ABSTRACT TRUNCATED AT 250 WORDS)