A rapid method for assaying the metabolism of 7-ethoxyresorufin by microsomal subcellular fractions

Pohl, Roberta J.; Fouts, James R.

doi:10.1016/0003-2697(80)90505-9

Cited by 788 publications

(179 citation statements)

References 7 publications

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“…The H4IIE EROD assay is a modification of the method of Pohl and Fouts (1980). Cell harvesting and preparation of a microsomal fraction were originally performed similar to the AHH bioassay (Sawyer and Safe, 1982;Keys et al, 1986), but now the reaction is generally carried out in the same microtiter plate used for chemical dosing, based on the work of Kennedy et al (1993).…”

Section: E Measurement Of Cyp1a1 Catalytic Activity (Ahh or Erod)mentioning

confidence: 99%

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The H4IIE Cell Bioassay as an Indicator of Dioxin-like Chemicals in Wildlife and the Environment

Whyte

Schmitt

Tillitt

2004

Critical Reviews in Toxicology

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ABSTRACT:The H4IIE cell bioassay has proven utility as a screening tool for planar halogenated hydrocarbons (PHHs) and structurally similar chemicals accumulated in organisms from the wild. This bioassay has additional applications in hazard assessment of PHH exposed populations. In this review, the toxicological principles, current protocols, performance criteria, and field applications for the assay are described. The H4IIE cell bioassay has several advantages over the analytical measurement of PHHs in environmental samples, but conclusions from studies can be strengthened when both bioassay and analytical chemistry data are presented together. Often, the bioassay results concur with biological effects in organisms and support direct measures of PHHs. For biomonitoring purposes and prioritization of PHH-contaminated environments, the H4IIE bioassay may be faster and less expensive than analytical measurements. The H4IIE cell bioassay can be used in combination with other biomarkers such as in vivo measurements of CYP1A1 induction to help pinpoint the sources and identities of dioxin-like chemicals. The number of studies that measure H4IIE-derived TCDD-EQs continues to increase, resulting in subtle improvements over time. Further experiments are required to determine if TCDD-EQs derived from mammalian cells are adequate predictors of toxicity to non-mammalian species. The H4IIE cell bioassay has been used in over 300 published studies, and its combination of speed, simplicity, and ability to integrate the effects of complex contaminant mixtures makes it a valuable addition to hazard assessment and biomonitoring studies.

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Section: E Measurement Of Cyp1a1 Catalytic Activity (Ahh or Erod)mentioning

confidence: 99%

“…The fluorescence of resorufin, the product of the CYP1A1-catalyzed reaction, is measured with an excitation wavelength of 550 nm and an emission wavelength of 585 nm (Pohl and Fouts, 1980). The EROD assay can also be performed on live H4IIE cells (Clemons et al, 1996;Whyte et al, 1998).…”

Section: E Measurement Of Cyp1a1 Catalytic Activity (Ahh or Erod)mentioning

confidence: 99%

The H4IIE Cell Bioassay as an Indicator of Dioxin-like Chemicals in Wildlife and the Environment

Whyte

Schmitt

Tillitt

2004

Critical Reviews in Toxicology

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“…In the benzo(a)pyrene hydroxylation assay, formation of phenolic benzo(a)pyrene was determined using 3-hydroxybenzo(a)pyrene as a standard (14). The O-dealkylations of 7-ethoxyresorufin and 7-methoxyresorufin were determined by measuring fluorescence of resorufin (15). 7-Ethoxycoumarin O-deethylation was determined by measuring the fluorescence of hydroxycoumarin (16).…”

Section: Methodsmentioning

confidence: 99%

Effects of Wu-chu-yu-tang and Its Component Herbs on Drug-Metabolizing Enzymes

Ueng

Don

Peng

et al. 2002

Japanese Journal of Pharmacology

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ABSTRACT-The compound herbal medicine Wu-chu-yu-tang is used for the treatment of migraine and vomiting accompanying a cold. To assess the interactions of herb and drug metabolism, effects of Wu-chuyu-tang on hepatic and renal cytochrome P450 (CYP), UDP-glucuronosyl transferase (UGT) and glutathione S-transferase (GST) were studied in C57BL /6J mice. Treatment of mice with 5 g /kg per day Wu-chu-yutang for 3 days caused 2.5-fold and 2.9-fold increases of liver microsomal 7-ethoxyresorufin O-deethylation (EROD) and 7-methoxyresorufin O-demethylation activities, respectively. However, CYP activities toward 7-ethoxycoumarin, benzphetamine, N-nitrosodimethylamine, erythromycin and nifedipine, and conjugation activities of UGT and GST were not affected. In kidney, Wu-chu-yu-tang-treatment had no effects on Cyp, UGT and GST activities. Among the four component herbs of Wu-chu-yu-tang, only Evodiae Fructus (Wuchu-yu) extract increased EROD activity and CYP1a2 protein level. In E. Fructus, rutaecarpine, evodiamine and dehydroevodiamine are the main active alkaloids. At the doses corresponding to their contents in Wuchu-yu-tang, rutaecarpine-treatment increased hepatic EROD activity, whereas evodiamine and dehydroevodiamine had no effects. These results demonstrated that ingestion of Wu-chu-yu-tang elevated mouse hepatic Cyp1a2 activity and protein level. E. Fructus and rutaecarpine contributed at least in part to the CYP1a2 induction by Wu-chu-yu-tang. Patients should be cautioned about the drug interaction of Wu-chu-yu-tang and CYP1A2 substrates.Keywords: Wu-chu-yu-tang, Evodiae Fructus, Rutaecarpine, Drug-metabolizing enzyme, Liver Due to the increase in the use of herbal remedies, physicians and pharmacists are very much concerned about the toxicity and drug interactions when using herbal medicines. Several medicinal herbs such as Ginkgo biloba and St John's wort have been reported to show interactions with pharmaceutical drugs (1, 2). Modulation of drug-metabolizing enzymes is one of the main causes of drug interactions (3, 4). Oxidation and conjugation are two main reactions involved in drug metabolism. Cytochrome P450 (CYP)-dependent monooxygenases are the primary oxidation enzyme systems involved in the detoxication and bioactivation of a number of drugs and environmental pollutants (5). The oxidations catalyzed by the monooxygenase system require a CYP enzyme, NADPH-CYP reductase and phospholipids. The CYP family comprises a group of enzymes with broad substrate specificity. This substrate specificity leads to the herb-induced drug interactions with selective CYP substrates. For conjugation reactions, UDPglucuronosyl transferase (UGT) and glutathione S-transferase (GST) are important enzymes, which catalyze the glucuronide and glutathione conjugates formation, respectively. Changes of conjugation activities can also affect the detoxication and excretion of drugs (6).Wu-chu-yu-tang (Goshuyu-to in Japanese Kampo medicine) is a traditional Chinese prescription for the treatment of migraine and vomiting accompanying...

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“…Homogenate was initially centrifuged at 14000g for 20 min in a cold centrifuge (Remi) and supernatant was then further centrifuged at 10, 5000 g for 1 h in an ultracentrifuge (Sorvall). The microsomes obtained from liver homogenate were washed and resuspended in cold phosphate buffer (pH 7.4, 0.1 M) and used for determination of the effect of TEO on the dealkylation of methoxy resorufin by 7-methoxyresorufin-O-demethylase (MROD), CYPIA2, pentoxy resorufin by 7-pentoxyresorufin-O-depentylase (PROD), CYP2B1/2 and ethoxy resorufin by 7-ethoxyresorufin-O-deethylase (EROD), CYP1A1 (Pohl and Fouts, 1980;Nerurkar et al, 1993).…”

Section: Effect Of Teo On the Inhibition Of Various Isoforms Of Cytocmentioning

confidence: 99%

Chemopreventive Activity of Turmeric Essential Oil and Possible Mechanisms of Action

Liju¹,

Jeena²,

Kuttan³

2014

Asian Pacific Journal of Cancer Prevention

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Asian Pac J Cancer Prev, 15 (16), 6575-6580 IntroductionMany human cancers are caused by chemical carcinogens, such as polycyclic aromatic hydrocarbons, heterocyclic amines, aromatic amines etc present in our environment. Continued exposure of these substances to human cells leads to genomic instability, including repair deficiency and accumulation of genetic alteration (Ames, 1983). Mutation is a major factor in carcinogenesis and incidence of cancer may be reduced by decreasing the rate of mutations induced by various chemical mutagens. Many carcinogens are activated through Phase I (cytochrome p450) enzymes present in endoplasmic reticulum of the liver cells. Induction of Phase II detoxification enzymes, such as glutathione S-transferase and UDP-glucuronyl transferase, is another mechanisms of protection against carcinogenesis (Piengchai et al., 2011). Modulating these enzymes may reduce cancer incidence.Spices which are widely used as food ingredient exhibits different pharmacological properties. Essential oils from spices are volatile compounds produced as secondary metabolites. The essential oil from Curcuma longa rhizome is a complex mixture obtained by steam distillation. A total number of 12 components of the essential oil are identified by GC-MS analysis and the principal compounds include ar-turmerone (61%), Department of Biochemistry, Amala Cancer Research Centre, Kerala, India *For correspondence: amalacancerresearch@gmail. com, amalaresearch@hotmail.com AbstractThis study aimed to evaluate the antimutagenic and anticarcinogenic activity of turmeric essential oil as well as to establish biochemical mechanisms of action. Antimutagenicity testing was accomplished using strains and known mutagens with and without microsomal activation. Anticarcinogenic activity was assessed by topical application of 7, 12 -dimethylbenz[a]anthracene (DMBA) as initiator and 1% croton oil as promoter for the induction of skin papillomas in mice. Inhibition of p450 enzymes by TEO was studied using various resorufins and aminopyrene as substrate. Turmeric essential oil (TEO) showed significant antimutagenic activity (p<0.001) against direct acting mutagens such as sodium azide (NaN 3 ), 4-nitro-O-phenylenediamine (NPD) and N-methyl-N-nitro N'nitrosoguanine (MNNG). TEO was found to have significant antimutagenic effect (>90%) against mutagen needing metabolic activation such as 2-acetamidoflourene (2-AAF). The study also revealed that TEO significantly inhibited (p<0.001) the mutagenicity induced by tobacco extract to Salmonella TA 102 strain. DMBA and croton oil induced papilloma development in mice was found to be delayed and prevented significantly by TEO application. Moreover TEO significantly (P<0.001) inhibited isoforms of cytochrome p450 (CYP1A1, CYP1A2, CYP2B1/2, CYP2A, CYP2B and CYP3A) enzymes in vitro, which are involved in the activation of carcinogens. Results indicated that TEO is antimutagenic and anticarcinogenic and inhibition of enzymes (p450) involved in the activation of carcinogen is one of its mecha...

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A rapid method for assaying the metabolism of 7-ethoxyresorufin by microsomal subcellular fractions

Cited by 788 publications

References 7 publications

The H4IIE Cell Bioassay as an Indicator of Dioxin-like Chemicals in Wildlife and the Environment

The H4IIE Cell Bioassay as an Indicator of Dioxin-like Chemicals in Wildlife and the Environment

Effects of Wu-chu-yu-tang and Its Component Herbs on Drug-Metabolizing Enzymes

Chemopreventive Activity of Turmeric Essential Oil and Possible Mechanisms of Action

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