Modeling, Synthesis, and Biological Evaluation of Potential Retinoid-X-Receptor (RXR) Selective Agonists: Analogs of 4-[1-(3,5,5,8,8-Pentamethyl-5,6,7,8-tetrahyro-2-naphthyl)ethynyl]benzoic Acid (Bexarotene) and 6-(Ethyl(4-isobutoxy-3-isopropylphenyl)amino)nicotinic Acid (NEt-4IB).

Five novel analogs of 6-(ethyl)(4-isobutoxy-3-isopropylphenyl)amino)nicotinic acid-or NEt-4IB-in addition to seven novel analogs of 4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethynyl]benzoic acid (bexarotene) were prepared and evaluated for selective retinoid-X-receptor (RXR) agonism alongside bexarotene (1), a FDA-approved drug for cutaneous T-cell lymphoma (CTCL). Bexarotene treatment elicits side-effects by provoking or disrupting other RXR-dependent pathways. Analogs were assessed by the modeling of binding to RXR and then evaluated in a human cell-based RXR-RXR mammalian-2-hybrid (M2H) system as well as a RXRE-controlled transcriptional system. The analogs were also tested in KMT2A-MLLT3 leukemia cells and the EC50 and IC50 values were determined for these compounds. Moreover, the analogs were assessed for activation of LXR in an LXRE system as drivers of ApoE expression and subsequent use as potential therapeutics in neurodegenerative disorders, and the results revealed that these compounds exerted a range of differential LXR-RXR activation and selectivity. Furthermore, several of the novel analogs in this study exhibited reduced RARE cross-signaling, implying RXR selectivity. These results demonstrate that modification of partial agonists such as NEt-4IB and potent rexinoids such as bexarotene can lead to compounds with improved RXR selectivity, decreased cross-signaling of other RXR-dependent nuclear receptors, increased LXRE-heterodimer selectivity, and enhanced anti-proliferative potential in leukemia cell lines compared to therapeutics such as 1.

Development of Bexarotene Analogs for Treating Cutaneous T-Cell Lymphomas.

Bexarotene, a drug approved for treatment of cutaneous T-cell lymphoma (CTCL), is classified as a rexinoid by its ability to act as a retinoid X receptor (RXR) agonist with high specificity. Rexinoids are capable of inducing RXR homodimerization leading to the induction of apoptosis and inhibition of proliferation in human cancers. Numerous studies have shown that bexarotene is effective in reducing viability and proliferation in CTCL cell lines. However, many treated patients present with cutaneous toxicity, hypothyroidism, and hyperlipidemia due to crossover activity with retinoic acid receptor (RAR), thyroid hormone receptor (TR), and liver X receptor (LXR) signaling, respectively. In this study, 10 novel analogs and three standard compounds were evaluated side-by-side with bexarotene for their ability to drive RXR homodimerization and subsequent binding to the RXR response element (RXRE). In addition, these analogs were assessed for proliferation inhibition of CTCL cells, cytotoxicity, and mutagenicity. Furthermore, the most effective analogs were analyzed via qPCR to determine efficacy in modulating expression of two critical tumor suppressor genes, ATF3 and EGR3. Our results suggest that these new compounds may possess similar or enhanced therapeutic potential since they display enhanced RXR activation with equivalent or greater reduction in CTCL cell proliferation, as well as the ability to induce ATF3 and EGR3. This work broadens our understanding of RXR-ligand relationships and permits development of possibly more efficacious pharmaceutical drugs. Modifications of RXR agonists can yield agents with enhanced biological selectivity and potency when compared to the parent compound, potentially leading to improved patient outcomes.

Non-invasive assessment of central venous pressure in heart failure: a systematic prospective comparison of echocardiography and Swan-Ganz catheter.

Assessing hemodynamics, especially central venous pressure (CVP), is essential in heart failure (HF). Right heart catheterization (RHC) is the gold-standard, but non-invasive methods are also needed. However, the role of 2-dimensional echocardiography (2DE) remains uncertain, and 3-dimensional echocardiography (3DE) is not always available. This study investigated standardized and breathing-corrected assessment of inferior vena cava (IVC) volume using echocardiography (2DE and 3DE) versus CVP determined invasively using RHC. Sixty consecutive HF patients were included (82% male, age 54 ± 11 years, New York Heart Association class 2.23 ± 0.8, ejection fraction 46 ± 18.4%, brain natriuretic peptide 696.93 ± 773.53 pg/mL). All patients underwent Swan-Ganz RHC followed by 2DE and 3DE, and IVC volume assessment. On 2DE, mean IVC size was 18.3 ± 5.5 mm and 13.8 ± 6 mm in the largest deflection and shortest distention, respectively. Mean CVP from RHC was 9.3 ± 5.3 mmHg. Neither 2DE nor 3DE showed acceptable correlation with invasively measured CVP; IVC volume acquisition showed optimal correlation with RHC CVP (0.64; 95% confidence interval 0.46-0.77), with better correlation when mitral valve early diastole E wave and right ventricular end-diastolic diameter were added. Using a CVP cut-point of 10 mmHg, receiver operating characteristic curve showed true positivity (specificity) of 0.90 and sensitivity of 62% for predicting CVP. A validation study confirmed these findings and verified the high predictive value of IVC volume assessment. Neither 2DE nor 3DE alone can reliably mirror CVP, but IVC volume acquisition using echocardiography allows non-invasive and adequate approximation of CVP. Correlation with invasively measured pressure was strongest when CVP is > 10 mmHg.

Bioconcentration of xenobiotics in trout bile: a proposed monitoring aid for some waterborne chemicals.

A technique is proposed for the monitoring of certain xenobiotic pollutants in suspect aquatic enviornments by fish bile analysis. Bile removed from rainbow trout (Salmo gairdneri) exposed to nine different radioactive compounds in vivo contained concentrations of radioactivity greater than those in the surrounding water. Bile-to-water radioactivity ratios as high as 10,000: 1 were found after 24-hour exposures. The results of these experiments suggest that analysis of bile of wild or caged fish from a suspect site may be useful as a qualitative monitoring aid for certain types of xenobiotics in water.

Effect of dietary 2(3)-tert-butyl-4-hydroxyanisole on the metabolism and action of estradiol and estrone in female CD-1 mice.

Administration of 0.75% 2(3)-tert-butyl-4-hydroxyanisole (BHA) in AIN-76A diet to female CD-1 mice for 3 weeks increased liver microsomal glucuronidation of estradiol, estrone, 4-aminophenol, and 4-nitrophenol by 103, 187, 162, and 92%, respectively (at pH 7.4). The overall rate of NADPH-dependent metabolism of estradiol and estrone by liver microsomes of BHA-treated animals as determined by substrate disappearance was increased by 20-40% over that by liver microsomes from control animals. The rate of 2-hydroxylation of estradiol and estrone (the major metabolic pathway) was increased by 24-38%, the rate of formation of 6alpha-hydroxyestradiol plus 6beta-hydroxyestradiol was increased by 90-115%, and the rate of 6beta-hydroxyestrone formation (a minor metabolite formed in liver microsomes from control mice) was increased by approximately 370% over controls. In contrast, BHA administration had little or no effect on the liver microsomal formation of 4- and 16alpha-hydroxylated estradiol and estrone metabolites. Measurable levels of estradiol and estrone were observed in the serum and uterus of ovariectomized CD-1 mice at 30 min after a single i.p. injection of 100 or 300 ng of estradiol or estrone, and these levels were decreased by 30-60% in animals fed a 0.75% BHA diet for 18 days prior to the injection of estrogen. Feeding a 0.75% BHA-supplemented diet to ovariectomized CD-1 mice for 18 days inhibited the uterotropic effect of estradiol or estrone (45 or 75 ng/mouse, i.p. once daily for 3 days) as compared to the response of animals fed the control diet. BHA administration also inhibited estradiol- or estrone-stimulated [3H]thymidine incorporation into uterine DNA. In conclusion, feeding a 0.75% BHA-supplemented diet to female CD-1 mice for 2-3 weeks increased the activities of liver microsomal enzymes that catalyze uridine 5'-diphosphoglucuronic acid-dependent glucuronidation and NADPH-dependent oxidation of estradiol and estrone, enhanced the in vivo metabolism of these estrogens, and inhibited their uterotropic action.

Effects of phenobarbital, dexamethasone, and 3-methylcholanthrene administration on the metabolism of 17 beta-estradiol by liver microsomes from female rats.

Female rats were treated with phenobarbital, dexamethasone, 3-methylcholanthrene, clofibrate, or isoniazid to induce different hepatic cytochromes P-450. The profile of hydroxylated metabolites of estradiol (E2) formed by liver microsomes was then determined using a new HPLC method for the separation of hydroxylated estrogen metabolites. Inhibition of liver microsomal E2 metabolism by monoclonal antibodies raised against specific cytochrome P-450 isozymes was also evaluated. Treatment of immature or adult female rats with phenobarbital caused a 3-fold increase in the 2-hydroxylation of E2 and a more than 5-fold increase in liver microsomal hydroxylation of E2 at the 4-, 6 alpha, 6 beta-, and 14 alpha-positions. Monoclonal antibody directed toward CYP2B1/2B2 completely inhibited the 6 alpha- and 6 beta-hydroxylation of E2 and partially inhibited the 2-hydroxylation of E2 by liver microsomes from phenobarbital-treated adult female rats. Antibodies directed toward CYP3A1/3A2 completely inhibited the 4- and 14 alpha-hydroxylation of E2 by these liver microsomes. Treatment of immature or adult female rats with dexamethasone resulted in a 2- to 3-fold increase in the microsomal 2-hydroxylation of E2 and a several-fold increase in the hydroxylation of E2 at the 4-, 6 beta-, 7 alpha-, and 14 alpha-positions. A substantial increase in the formation of two unidentified nonpolar metabolite peaks (UK1 and UK2) was also observed. A monoclonal antibody directed against CYP3A1/3A2 markedly inhibited the 2-, 4-, and 14 alpha-hydroxylation of E2 by liver microsomes from adult female rats treated with dexamethasone. Antibody directed against CYP2B1/2B2 inhibited only the 6 beta-hydroxylation of E2 by these microsomes. Treatment of immature or adult female rats with 3-methylcholanthrene resulted in a several-fold increase in the metabolism of E2 to 7 alpha-hydroxyestradiol (7 alpha-OH E2) and 15 alpha-OH E2, but there was a substantial decrease in the formation of 16 alpha-OH E2. Treatment with 3-methylcholanthrene caused a small increase in 2-hydroxylation (< or = 50%) in liver microsomes from immature or adult female rats, whereas a substantial increase in 6 alpha-hydroxylation was seen in liver microsomes from adult female rats. A monoclonal antibody directed toward CYP1A1 partially inhibited the 6 alpha-hydroxylation of E2 and the formation of the 7 alpha-OH E2/15 alpha-OH E2 peak by microsomes from adult female rats treated with 3-methylcholanthrene, but the 2-hydroxylation of E2 was not inhibited. Treatment of adult female rats with clofibrate increased the 2- and 4-hydroxylation of E2 by about 2-fold and by more than 6-fold, respectively. Isoniazid treatment had little or no effect on the metabolism of E2. The data demonstrate that prototype inducers of cytochrome P-450 can substantially alter the profile of hepatic E2 metabolism in female rats. Our results suggest that inducers of environmental relevance may also have an impact on E2 metabolism and homeostasis in humans.

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)

Sex differences in cytochrome P-450 isozyme composition and activity in kidney microsomes of mature rainbow trout.

Kidney microsomes from sexually mature male, as opposed to female, rainbow trout displayed an approximately 20-fold higher cytochrome P-450 specific content, NADPH-cytochrome c reductase activity, and rates of hydroxylation of lauric acid, testosterone, progesterone and aflatoxin B1. Little or no sex difference in metabolism was observed with benzo[a]pyrene or benzphetamine as substrates. A similar pattern was observed in hepatic microsomes from these fish, but the difference was much less striking (approximately 2-fold higher activity in males). Juvenile trout (both sexes) possessed activities intermediate between mature males and females. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of kidney and liver microsomes of juvenile and sexually mature male and female trout suggested that the striking sex difference in kidney could be due to the high amount of trout P-450 isozyme LM2 in sexually mature males. Immunoquantitation of LM2, performed by Western Blotting and immunostaining with rabbit anti-trout LM2-IgG, confirmed that mature male kidney contained much higher levels of P-450 LM2 than juvenile or female kidney, or even of liver microsomes of all three groups. The amount of P-450 LM2 in mature female kidney microsomes was barely detectable. The high amount of LM2 in male trout kidney is consistent with the high activity of these microsomes towards lauric acid and aflatoxin B1, which have been shown previously to be preferentially metabolized by trout P-450 LM2. It is suggested that rainbow trout may serve as an alternative to the rat as an animal model for the study of sex-dependent differences in cytochromes P-450.

Propranolol-induced alterations in rat erythrocyte membrane fluidity and apparent phase-transition temperatures. A depth-dependent process.

Propranolol-induced alterations of membrane structure were studied in rat erythrocytes using electron spin resonance techniques. Propranolol produced a concentration-dependent change in membrane fluidity in hydrophobic membrane regions, while producing virtually no change in hydrophilic membrane regions. The changes were associated with depth-dependent alterations in "apparent" phase-transition profiles and transition temperatures. The effects of propranolol on these membrane characteristics were similar to those produced by cholesterol. Propranolol fluidized erythrocyte membranes in a depth-specific fashion, by virtue of its association with the rigid phospholipid acyl chains and cholesterol sterol rings in the hydrophilic regions of the membrane, which produced distant perturbations within the hydrophilic regions of the membrane.

Cytochrome P-450 monooxygenase systems in aquatic species: carcinogen metabolism and biomarkers for carcinogen and pollutant exposure.

High levels of polynuclear aromatic hydrocarbon (PAH) carcinogens commonly occur in aquatic systems where neoplasms arise in fish and other animals. Enzymes that transform PAHs can act in initiating these diseases and can indicate the contamination of fish by carcinogens and other pollutants. Cytochrome P-450 has similar roles in activating PAH carcinogens in fish and mammalian species. PAHs and many chlorinated hydrocarbons, e.g., polychlorinated biphenyls (PCBs) induce a form of cytochrome P-450 in fish that is the primary catalyst of PAH metabolism. The induction of this P-450 in fish can accelerate the disposition of hydrocarbons, but can also enhance the formation of carcinogenic derivatives of PAHs. Invertebrates have lower rates of PAH metabolism than fish. These rates are not obviously inducible by exposure to PAHs or PCBs. The lower rates of foreign compound metabolism contribute to higher pollutant residue levels in bivalve mollusks (clams, mussels, etc.) than in fish and may limit the involvement of some procarcinogens (requiring activation) in disease processes in invertebrates. The induction of P-450 forms can indicate the exposure of fish to PAHs, PCBs, and other toxic compounds. This is not restricted to carcinogens. Environmental induction has been detected in fish from contaminated areas by use of catalytic assay, antibodies to fish P-450, and cDNA probes that hybridize with P-450 messenger RNA. Application of these methods can provide sensitive biological monitoring tools that can detect environmental contamination of fish by some carcinogens and tumor promoters.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship between biotransformation and the toxicity and fate of xenobiotic chemicals in fish.

Many of the biotransformation reactions which have been described for xenobiotic substances in mammals have been demonstrated in fish in both in vitro and in vivo experiments. Several of these biotransformation reactions have been shown to occur in fish at rates which are sufficient to have significant effects on the toxicity and residue dynamics of selected chemicals. Inhibition of these reactions can lead to increased toxicity and bioaccumulation factors for certain chemicals. Several classes of compounds, including some polychlorinated biphenyls, are metabolized slowly, and their disposition in fish may not be influenced to any great extent by biotransformation. Metabolites of compounds which are biotransformed rapidly may appear in certain fish tissues, and in many instances these are not accounted for by conventional residue analysis methods. Microsomal mixed-function oxidases in several species of fish have been demonstrated to be induced by specific polycyclic aromatic hydrocarbons and by exposure of fish to crude oil. Induction of these enzymes in fish can result in both qualitative and quantitative differences in the metabolic disposition of xenobiotics to which fish are exposed.

Induction of monooxygenation in rainbow trout by polybrominated biphenyls: a comparative study.

Two commercial polychlorinated biphenyl mixtures (Aroclor 1254 and Aroclor 1242) and one polybrominated biphenyl mixture (FireMaster BP-6) were examined for their abilities to induce hepatic microsomal monooxygenation in rainbow trout (Salmo gairdneri). Pretreatment of rainbow trout with Aroclors 1254 and 1242 (150 mg/kg IP) resulted in an approximate 10-fold induction of arylhydrocarbon (benzo[a]pyrene) hydroxylation, ethoxycoumarin-O-deethylation and ethoxyresorufin-O-deethylation within 7 days after injection. These enzyme activities remained elevated above control values for at least 2-3 weeks. Administration of FireMaster BP-6 (150 mg/kg IP) also resulted in an induction of several monooxygenase activities. Arylhydrocarbon (benzo[a]pyrene) hydroxylation, ethoxycoumarin-O-deethylation and ethoxyresorufin-O-deethylation were increased by 6-, 3,- and 25-fold, respectively. Only the latter two activities remained elevated two weeks post-injection. Ethylmorphine-N-demethylation was unaffected by the polyhalogenated biphenyls. Significant increases in P-450 hemoprotein were not observed after pretreatment with any of the polyhalogenated biphenyls studied.

Biotransformation and induction: implications for toxicity, bioaccumulation and monitoring of environmental xenobiotics in fish.

Biotransformation of xenobiotics in fish occurs by many of the same reactions as in mammals. These reactions have been shown to affect the bioaccumulation, persistence, residue dynamics, and toxicity of select chemicals in fish. P-450-dependent monooxygenase activity of fish can be induced by polycyclic aromatic hydrocarbons, but phenobarbital-type agents induce poorly, if at all. Fish monooxygenase activity exhibits ideal temperature compensation and sex-related variation. Induction of monooxygenase activity by polycyclic aromatic hydrocarbons can result in qualitative as well as quantitative changes in the metabolic profile of a chemical. Induction can also alter toxicity. In addition, multiple P-450 isozymes have been described for several fish species. The biotransformation products of certain chemicals have been related to specific P-450 isozymes, and the formation of these products can be influenced by induction. Exposure of fish to low levels of certain environmental contaminants has resulted in induction of specific monooxygenase activities and monitoring of such activities has been suggested as a means of identifying areas of pollutant exposure in the wild.

Dimethyl formamide (DMFA) and ethylene glycol (EG) are estrogenic in rainbow trout.

During our studies on the effects of environmental estrogenic chemicals on vitellogenin gene expression in fish, dimethyl formamide (DMFA) and ethylene glycol (EG) were used as solvents to dissolve and deliver hydrophobic beta-estradiol or nonylphenol. Control groups were injected with water only. It was surprising to find that in certain doses, Vg mRNA from control fish can be induced by DMFA or EG alone. This suggested that DMFA and EG are weak estrogens.

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.

Effects of estrogen and nonylphenol on the post-transcriptional regulation of vitellogenin gene expression.

Estrogenic chemicals have drawn considerable attention over the past several years because they are known to be responsible for increased breast and prostate cancer. Vitellogenin expression driven by estrogen has been becoming a model for studying estrogenic effects in aquatic species. For the first time, we showed evidence that, without stimulation, vitellogenin mRNA precursor is expressed in both male and female immature fish. After 4 h, in fish treated with estradiol, the vitellogenin mRNA was synthesized and the precursor mRNA began to disappear. The environmental chemical, nonylphenol, showed the same effect on the vitellogenin gene expression as estrogen. It is suggested that estrogen and nonylphenol may be involved in a post-transcriptional regulation process -- possibly in the initiation of pre-mRNA splicing.

Induction of cytochrome P-450-dependent monooxygenase systems in embryos and eleutheroembryos of the killfish Fundulus heteroclitus.

Microsomal aryl hydrocarbon hydroxylase (AHH) activity was highly inducible by polychlorinated biphenyls (PCBs) in Fundulus embryos, and stages prior to the appearance of the liver rudiment were competent to respond to these inducers. Consistent with previous observations, basal AHH activity in whole eleutheroembryo microsomes was shown to increase about 9-fold within 24 h of hatching. Aminopyrine N-demethylase (APD) activity also increased with time after hatching. However, the increase in APD activity was much less than that of AHH activity, suggesting a post-hatching change in basal cytochrome P-450 isozyme composition. Also associated with hatching was an increase in the sensitivity to PCBs as inducers of AHH activity. The ED50 for induction of AHH activity in eleutheroembryos was estimated to be only one-third to one-fourth that in embryos. This developmental increase in the sensitivity to PCBs was not due to a redistribution of PCBs between the yolk and tissues with yolk absorption, and was not simply age-dependent, but appeared to require hatching. An additional change in the monooxygenase system associated with hatching was that microsomal NADPH-cytochrome c reductase activity was not inducible by PCBs prior to hatching, but was modestly inducible after hatching. High performance liquid chromatographic (HPLC) analysis of benzo[a]-pyrene (BP) metabolites formed by microsomes from control and PCB-treated eleutheroembryos demonstrated production of dihydrodiols in the 7,8- and 9,10-positions of the benzo-ring. The formation of these metabolites was completely inhibited by the epoxide hydrolase (EH) inhibitor, trichloropropene oxide, indicating the presence of EH in Fundulus eleutheroembryos. Furthermore, these results indicate the Fundulus eleutheroembryos probably can activate BP to its ultimate carcinogenic form, the 7,8-dihydrodiol-9,10-epoxide, and induction of AHH activity by PCBs is likely to increase the rate of formation of activated metabolites from BP and related compounds. However, during the most active period of organogenesis, prior to hatching, basal AHH activity was low, and prehatching stages were relatively insensitive to cytochrome P-450 inducers. The combination of these effects may help to protect these stages from damage from activated metabolites.

Metabolism of nonylphenol by rat and human microsomes.

The in vitro metabolism of [14C]-nonylphenols (NPs) by rat hepatic microsomes in vitro was examined. Product formation was NADPH dependent and inhibited by the cytochrome P450 inhibitors, piperonyl butoxide and SKF525. Hepatic microsomes isolated from various inducer-treated rats (including beta naphthoflavone, phenobarbital, ethanol, dexamethasone, and clofibrate which selectively induce CYP1A, 2B, 2E, 3A and 4A, respectively) all metabolized NPs. Only microsomes from phenobarbital-treated rats exhibited a significantly higher activity towards NPs and showed a different profile of NP metabolites compared to control, untreated rats. Microsomes from human CYP2B6 transfected cells with endogenous NADPH-P450 reductase activity but not microsomes from the non-transfected parent cells metabolized NPs. The metabolism of NPs using microsomes from phenobarbital-treated rats was inhibited by 4-amino-2, 6-dinitro-1-t-butylxylene, a specific CYP2B enzyme inhibitor. Addition of a general anti-CYP2B sera to the reaction mixture attenuated the enzyme activity of microsomes from phenobarbital-treated rats to metabolize NPs. This metabolic reaction was, however, insensitive to a specific anti-CYP2B1 sera that had been shown to inhibit enzyme activities attributed only to CYP2BI suggesting that the CYP2B2 pathway is predominant in NP metabolism. The results indicate that hepatic cytochrome P450 enzyme(s) can metabolize NPs and that CYP2B isozymes are probably involved.

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.

Direct thermal desorption-gas chromatography and gas chromatography-mass spectrometry profiling of hop (Humulus lupulus L.) essential oils in support of varietal characterization.

The use of direct thermal desorption-gas chromatography-mass spectrometry (DTD-GC-MS) and DTD-GC-flame ionization detection (DTD-GC-FID) for characterization of hop essential oils is described. Four hop varieties (Nugget, Galena, Willamette, and Cluster) from the Yakima valley (Yakima, WA) 1998 harvest were analyzed by DTD-GC-MS and DTD-GC-FID methodology. Approximately 1 g of hops was needed for the analysis. Hop samples were prepared for GC-MS and/or GC-FID profiling in approximately 20 min. More than 100 volatile compounds have been identified and quantified for each hop variety. The results were found to be in good agreement with conventional steam distillation-extraction (SDE) data. A calibration curve for determination of essential oil content in hops by DTD-GC-FID has been generated. Quantitation of hop oil content by DTD-GC-FID was shown to be in good agreement with conventional SDE data. The recovery of key oil components valuable for varietal identification was demonstrated to be highly reproducible and characteristic of each variety analyzed when DTD-GC-FID was used for analysis.