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Cellular levels and molecular dynamics simulations of estragole DNA adducts point at inefficient repair resulting from limited distortion of the double-stranded DNA helix. Arch Toxicol 2020; 94:1349-1365. [PMID: 32185416 PMCID: PMC7225201 DOI: 10.1007/s00204-020-02695-5] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2019] [Accepted: 03/02/2020] [Indexed: 10/25/2022]
Abstract
Estragole, naturally occurring in a variety of herbs and spices, can form DNA adducts after bioactivation. Estragole DNA adduct formation and repair was studied in in vitro liver cell models, and a molecular dynamics simulation was used to investigate the conformation dependent (in)efficiency of N2-(trans-isoestragol-3'-yl)-2'-deoxyguanosine (E-3'-N2-dG) DNA adduct repair. HepG2, HepaRG cells, primary rat hepatocytes and CHO cells (including CHO wild-type and three NER-deficient mutants) were exposed to 50 μM estragole or 1'-hydroxyestragole and DNA adduct formation was quantified by LC-MS immediately following exposure and after a period of repair. Results obtained from CHO cell lines indicated that NER plays a role in repair of E-3'-N2-dG adducts, however, with limited efficiency since in the CHO wt cells 80% DNA adducts remained upon 24 h repair. Inefficiency of DNA repair was also found in HepaRG cells and primary rat hepatocytes. Changes in DNA structure resulting from E-3'-N2-dG adduct formation were investigated by molecular dynamics simulations. Results from molecular dynamics simulations revealed that conformational changes in double-stranded DNA by E-3'-N2-dG adduct formation are small, providing a possible explanation for the restrained repair, which may require larger distortions in the DNA structure. NER-mediated enzymatic repair of E-3'-N2-dG DNA adducts upon exposure to estragole will be limited, providing opportunities for accumulation of damage upon repeated daily exposure. The inability of this enzymatic repair is likely due to a limited distortion of the DNA double-stranded helix resulting in inefficient activation of nucleotide excision repair.
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Santacroce MP, Iaffaldano N, Zacchino V, Rosato MP, Casalino E, Centoducati G. Effects of Aflatoxin Bi on liver phase I and phase II enzymes inducedin vitroon Sparus aurata hepatocytes primary culture. ITALIAN JOURNAL OF ANIMAL SCIENCE 2016. [DOI: 10.4081/ijas.2012.e60] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
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Fukasawa K, Kagaya S, Maruyama S, Kuroiwa S, Masuda K, Kameyama Y, Satoh Y, Akatsu Y, Tomura A, Nishikawa K, Horie S, Ichikawa YI. A novel compound, NK150460, exhibits selective antitumor activity against breast cancer cell lines through activation of aryl hydrocarbon receptor. Mol Cancer Ther 2014; 14:343-54. [PMID: 25522763 DOI: 10.1158/1535-7163.mct-14-0158] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Antiestrogen agents are commonly used to treat patients with estrogen receptor (ER)-positive breast cancer. Tamoxifen has been the mainstay of endocrine treatment for patients with early and advanced breast cancer for many years. Following tamoxifen treatment failure, however, there are still limited options for subsequent hormonal therapy. We discovered a novel compound, NK150460, that inhibits 17β-estradiol (E2)-dependent transcription without affecting binding of E2 to ER. Against our expectations, NK150460 inhibited growth of not only most ER-positive, but also some ER-negative breast cancer cell lines, while never inhibiting growth of non-breast cancer cell lines. Cell-based screening using a random shRNA library, identified aryl hydrocarbon receptor nuclear translocator (ARNT) as a key gene involved in NK150460's antitumor mechanism. siRNAs against not only ARNT but also its counterpart aryl hydrocarbon receptor (AhR) and their target protein, CYP1A1, dramatically abrogated NK150460's growth-inhibitory activity. This suggests that the molecular cascade of AhR/ARNT plays an essential role in NK150460's antitumor mechanism. Expression of ERα was decreased by NK150460 treatment, and this was inhibited by an AhR antagonist. Unlike two other AhR agonists now undergoing clinical developmental stage, NK150460 did not induce histone H2AX phosphorylation or p53 expression, suggesting that it did not induce a DNA damage response in treated cells. Cell lines expressing epithelial markers were more sensitive to NK150460 than mesenchymal marker-expressing cells. These data indicate that NK150460 is a novel AhR agonist with selective antitumor activity against breast cancer cell lines, and its features differ from those of the other two AhR agonists.
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Affiliation(s)
- Kazuteru Fukasawa
- Pharmaceutical Research Laboratories, Research and Development Group, Nippon Kayaku Co., Ltd., Kita-ku, Tokyo, Japan. Department of Urology, Graduate School of Medicine, Juntendo University, Bunkyo-ku, Tokyo, Japan.
| | - Shigehide Kagaya
- Pharmaceutical Research Laboratories, Research and Development Group, Nippon Kayaku Co., Ltd., Kita-ku, Tokyo, Japan
| | - Sakiko Maruyama
- Pharmaceutical Research Laboratories, Research and Development Group, Nippon Kayaku Co., Ltd., Kita-ku, Tokyo, Japan
| | - Shunsuke Kuroiwa
- Pharmaceutical Research Laboratories, Research and Development Group, Nippon Kayaku Co., Ltd., Kita-ku, Tokyo, Japan
| | - Kuniko Masuda
- Pharmaceutical Research Laboratories, Research and Development Group, Nippon Kayaku Co., Ltd., Kita-ku, Tokyo, Japan
| | - Yoshio Kameyama
- Pharmaceutical Research Laboratories, Research and Development Group, Nippon Kayaku Co., Ltd., Kita-ku, Tokyo, Japan
| | - Yoshitaka Satoh
- Pharmaceutical Research Laboratories, Research and Development Group, Nippon Kayaku Co., Ltd., Kita-ku, Tokyo, Japan
| | - Yuichi Akatsu
- Pharmaceutical Research Laboratories, Research and Development Group, Nippon Kayaku Co., Ltd., Kita-ku, Tokyo, Japan
| | - Arihiro Tomura
- Pharmaceutical Research Laboratories, Research and Development Group, Nippon Kayaku Co., Ltd., Kita-ku, Tokyo, Japan
| | - Kiyohiro Nishikawa
- Pharmaceutical Research Laboratories, Research and Development Group, Nippon Kayaku Co., Ltd., Kita-ku, Tokyo, Japan
| | - Shigeo Horie
- Department of Urology, Graduate School of Medicine, Juntendo University, Bunkyo-ku, Tokyo, Japan
| | - Yuh-ichiro Ichikawa
- Pharmaceutical Research Laboratories, Research and Development Group, Nippon Kayaku Co., Ltd., Kita-ku, Tokyo, Japan
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Carvajal M. Transformación de la aflatoxina B1 de alimentos, en el cancerígeno humano, aducto AFB1-ADN. TIP REVISTA ESPECIALIZADA EN CIENCIAS QUÍMICO-BIOLÓGICAS 2013. [DOI: 10.1016/s1405-888x(13)72082-5] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/23/2022] Open
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Wills LP, Jung D, Koehrn K, Zhu S, Willett KL, Hinton DE, Di Giulio RT. Comparative chronic liver toxicity of benzo[a]pyrene in two populations of the atlantic killifish (Fundulus heteroclitus) with different exposure histories. ENVIRONMENTAL HEALTH PERSPECTIVES 2010; 118:1376-81. [PMID: 20501349 PMCID: PMC2957915 DOI: 10.1289/ehp.0901799] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/09/2009] [Accepted: 05/25/2010] [Indexed: 05/04/2023]
Abstract
BACKGROUND The Atlantic Wood Industries Superfund site on the Elizabeth River (ER) in Portsmouth, Virginia, is contaminated with polycyclic aromatic hydrocarbons (PAHs) derived from creosote. Embryos and larvae of ER killifish (Fundulus heteroclitus) are refractory to the induction of enzymes regulated by the aryl hydrocarbon receptor including cytochrome P4501A (CYP1A) and are resistant to PAH-induced lethality and teratogenicity. However, adult ER killifish show a greater prevalence of hepatic and pancreatic tumors compared with those from reference sites. OBJECTIVES We used controlled laboratory studies to determine if ER killifish are more or less sensitive to PAH-induced chronic hepatic toxicity than killifish from an uncontaminated site. METHODS Larvae from the ER and a reference site on King's Creek (KC) were subjected to two 24-hr aqueous exposures of benzo[a]pyrene (BaP; 0-400 µg/L). At various time points, larvae were analyzed for CYP1A activity, BaP concentrations, nuclear and mitochondrial DNA damage, and liver pathology. RESULTS CYP1A activity was induced by BaP in KC but not ER larvae, and KC larvae demonstrated a greater reduction in whole-body concentrations of BaP over time. Mitochondrial and nuclear DNA lesion frequency increased significantly in BaP-exposed KC larvae, but not in ER larvae. Nine months postexposure, KC juveniles exhibited significantly more hepatic foci of cellular alteration and only KC juveniles developed hepatocellular carcinomas. CONCLUSIONS In addition to acquiring the heritable resistance to the acute teratogenic effects of PAHs, ER fish appear to have concomitantly developed resistance to chronic effects, including cancer.
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Affiliation(s)
- Lauren P. Wills
- Nicholas School of the Environment, Duke University, Durham, North Carolina, USA
- Department of Pharmaceutical and Biomedical Sciences, South Carolina College of Pharmacy, Medical University of South Carolina, Charleston, South Carolina, USA
| | - Dawoon Jung
- Nicholas School of the Environment, Duke University, Durham, North Carolina, USA
- Department of Physiology, Dartmouth Medical School, Hanover, New Hampshire, USA
| | - Kara Koehrn
- Nicholas School of the Environment, Duke University, Durham, North Carolina, USA
| | - Shiqian Zhu
- Department of Pharmacology and Environmental Toxicology Research Program, University of Mississippi, University, Mississippi, USA
| | - Kristine L. Willett
- Department of Pharmacology and Environmental Toxicology Research Program, University of Mississippi, University, Mississippi, USA
| | - David E. Hinton
- Nicholas School of the Environment, Duke University, Durham, North Carolina, USA
| | - Richard T. Di Giulio
- Nicholas School of the Environment, Duke University, Durham, North Carolina, USA
- Address correspondence to R.T. Di Giulio, Nicholas School of the Environment, Duke University, Box 90328, Durham, NC 27708 USA. Telephone: (919) 613-8024. Fax: (919) 668-1799. E-mail:
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Coulombe RA, Klein PJ, Hall JO. Toxicol Appl Pharmacol 2003; 189:152. [DOI: 10.1016/s0041-008x(03)00104-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register]
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Klein PJ, Van Vleet TR, Hall JO, Coulombe RA. Effects of dietary butylated hydroxytoluene on aflatoxin B1-relevant metabolic enzymes in turkeys. Food Chem Toxicol 2003; 41:671-8. [PMID: 12659720 DOI: 10.1016/s0278-6915(02)00332-0] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
Abstract
We have shown previously that the extreme sensitivity of turkeys to aflatoxin B(1) (AFB(1)) is due to a combination of efficient AFB(1) activation by cytochrome P450s (CYPs) 1A and deficient detoxification by glutathione S-transferases (GSTs). Phenolic antioxidants such as butylated hydroxytoluene (BHT) have been shown to be chemoprotective in some animal models due, in part, to modulation of AFB(1)-relevant phase I and/or phase II activities, and we wished to determine whether BHT has a similar effect in turkeys. Ten-day-old male turkeys were maintained on diets amended with 1000 or 4000 ppm of BHT for 10 days, then sampled. Hepatic microsomal CYP 1A activity as well as conversion of AFB(1) to the putative toxic metabolite, the exo-AFB(1)-8,9-epoxide (AFBO), were significantly lower compared with control. Conversely, dietary BHT significantly increased activities of several isoforms of hepatic cytosolic GST, as well quinone oxidoreductase (QOR). Western immunoblotting confirmed that dietary BHT increased expression of homologues to rodent GST isoforms Yc1, Yc2 and Ya. There was, however, no observable BHT-related increase in GST-mediated specific conjugation with microsomally-generated AFBO. In total, our data indicates that dietary BHT modulates a variety of AFB(1)-relevant phase I and phase II enzymes, while having no measurable effect towards specific AFB(1) detoxification by GST.
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Affiliation(s)
- P J Klein
- Department of Veterinary Sciences, and Graduate Program in Toxicology, Utah State University, Logan 84322-4620, USA
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Galvano F, Piva A, Ritieni A, Galvano G. Dietary strategies to counteract the effects of mycotoxins: a review. J Food Prot 2001; 64:120-31. [PMID: 11198434 DOI: 10.4315/0362-028x-64.1.120] [Citation(s) in RCA: 172] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
We reviewed various dietary strategies to contain the toxic effects of mycotoxins using antioxidant compounds (selenium, vitamins, provitamins), food components (phenolic compounds, coumarin, chlorophyll and its derivatives, fructose, aspartame), medicinal herbs and plant extracts, and mineral and biological binding agents (hydrated sodium calcium aluminosilicate, bentonites, zeolites, activated carbons, bacteria, and yeast). Available data are primarily from in vitro studies and mainly focus on aflatoxin B1, whereas much less information is available about other mycotoxins. Compounds with antioxidant properties are potentially very efficacious because of their ability to act as superoxide anion scavengers. Interesting results have been obtained by food components contained in coffee, strawberries, tea, pepper, grapes, turmeric, Fava tonka, garlic, cabbage, and onions. Additionally, some medicinal herbs and plant extracts could potentially provide protection against aflatoxin B1 and fumonisin B1. Activated carbons, hydrated sodium calcium aluminosilicate, and bacteria seem to effectively act as binders. We conclude that dietary strategies are the most promising approach to the problem, considering their limited or nil interference in the food production process. Nevertheless, a great research effort is necessary to verify the in vivo detoxification ability of the purposed agents, their mode of action, possible long-term drawbacks of these detoxification-decontamination procedures, and their economical and technical feasibility.
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Affiliation(s)
- F Galvano
- Dipartimento di Scienze e Tecnologie Agroforestali ed Ambientali, Universita di Reggio, Gallina di Reggio Calabria, Italy.
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Whyte JJ, Jung RE, Schmitt CJ, Tillitt DE. Ethoxyresorufin-O-deethylase (EROD) activity in fish as a biomarker of chemical exposure. Crit Rev Toxicol 2000; 30:347-570. [PMID: 10955715 DOI: 10.1080/10408440091159239] [Citation(s) in RCA: 477] [Impact Index Per Article: 19.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
Abstract
This review compiles and evaluates existing scientific information on the use, limitations, and procedural considerations for EROD activity (a catalytic measurement of cytochrome P4501A induction) as a biomarker in fish. A multitude of chemicals induce EROD activity in a variety of fish species, the most potent inducers being structural analogs of 2,3,7,8-tetracholordibenzo-p-dioxin. Although certain chemicals may inhibit EROD induction/activity, this interference is generally not a drawback to the use of EROD induction as a biomarker. The various methods of EROD analysis currently in use yield comparable results, particularly when data are expressed as relative rates of EROD activity. EROD induction in fish is well characterized, the most important modifying factors being fish species, reproductive status and age, all of which can be controlled through proper study design. Good candidate species for biomonitoring should have a wide range between basal and induced EROD activity (e.g., common carp, channel catfish, and mummichog). EROD activity has proven value as a biomarker in a number of field investigations of bleached kraft mill and industrial effluents, contaminated sediments, and chemical spills. Research on mechanisms of CYP1A-induced toxicity suggests that EROD activity may not only indicate chemical exposure, but also may also precede effects at various levels of biological organization. A current research need is the development of chemical exposure-response relationships for EROD activity in fish. In addition, routine reporting in the literature of EROD activity in standard positive and negative control material will enhance confidence in comparing results from different studies using this biomarker.
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Affiliation(s)
- J J Whyte
- U.S. Geological Survey (USGS) Cooperative Fish and Wildlife Research Unit, University of Missouri-Columbia, Columbia Environmetal Research Center (CERC), 65201, USA
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Klein PJ, Buckner R, Kelly J, Coulombe RA. Biochemical basis for the extreme sensitivity of turkeys to aflatoxin B(1). Toxicol Appl Pharmacol 2000; 165:45-52. [PMID: 10814552 DOI: 10.1006/taap.2000.8926] [Citation(s) in RCA: 69] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Poultry are the most susceptible food animal species to the toxic effects of the mycotoxin aflatoxin B(1) (AFB(1)). Feed contaminated with even small amounts of AFB(1) results in significant adverse health effects in poultry. The purpose of this study was to explain the biochemical mechanism(s) for this extreme sensitivity. We measured microsomal activation of AFB(1) to the AFB(1)-8,9-epoxide (AFBO), the putative toxic intermediate, as well as cytosolic glutathione S-transferase (GST)-mediated detoxification of AFBO, in addition to other hepatic phase I and phase II enzyme activities, in 3-week-old male Oorlop strain turkeys. Liver microsomes prepared from these turkeys activated AFB(1) in vitro with an apparent K(m) of 109 microM and a V(max) of 1.25 nmol/mg/min. Preliminary evidence for the involvement of cytochromes P450 (CYP) 1A2 and, to a lesser extent, 3A4 for AFB(1) activation was assessed by the use of specific mammalian CYP inhibitors. The possible presence of avian orthologues of these CYPs was supported by activity toward ethoxyresorufin and nifedipine, as well as by Western immunoblotting using antibodies to human CYPs. Cytosol prepared from turkey livers exhibited GST-mediated conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) and 3,4-dichloronitrobenzene (DCNB), but at a much lower rate than that observed in other species. Western immunoblotting indicated the presence of alpha and sigma class GSTs and another AFB(1)-detoxifying enzyme, AFB(1)-aldehyde reductase (AFAR). Turkey liver cytosol also had quinone oxidoreductase (QOR) activity. Importantly, cytosol exhibited no measurable GST-mediated detoxification of microsomally activated AFB(1), indicating that turkeys are deficient in the most crucial AFB(1)-detoxification pathway. In total, our data indicate that the extreme sensitivity of turkeys to AFB(1) may be attributed to a combination of efficient AFB(1) activation and deficient detoxification by phase II enzymes, such as GSTs.
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Affiliation(s)
- P J Klein
- Department of Animal, Dairy, and Veterinary Sciences, Utah State University, Logan, Utah, 84322-4620, USA
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Abstract
AIM: To study the influence of inducers BNF and PB on the stere oselective metabolism of propranolol in rat hepatic microsomes.
METHODS: Phase I metabolism of propranolol was studied by using the microsomes induced by BNF and PB and the non-induced microsome as the control. The enzymatic kinetic parameters of propranolol enantiomers were calculated by regression analysis of Lineweaver-Burk plots. Propranolol concentrations we re assayed by HPLC.
RESULTS: A RP-HPLC method was developed to determine propranol ol concentration in rat hepatic microsomes. The linearity equations for R (+)pr opranolol and S (-)propranolol were A = 705.7C + 311.2C (R = 0.9987) and A = 697.2C+311.4C (R = 0.9970) respectively. Recoveries of each enant iomer were 98.9%, 99.5%, 101.0% at 60 μmol/L, 120 μmol/L, 240 μmol/L respectively. At the concentration level of 120 μmol/L, propranolol enantiomers were metabolized at different rates in different microsomes. The concentration ratio R (+)/S (-) of control and PB induced microsomes increased with time, whereas that of microsome induced by BNF decreased. The assayed enzyme parameters were: 1. Km. Control group: R (+)30 ± 8, S (-) 18 ± 5; BNF group: R (+) 34 ± 3, S (-)39 ± 7; PB group: R (+)38 ± 17, S (-) 36 ± 10. 2. Vmax. Control group: R (+)1.5 ± 0.2, S (-)2.9 ± 0.3; BNF group: R (+)3.8 ± 0.3, S (-)3. 3 ± 0.5 ; PB group: R (+)0.07 ± 0.03, S (-)1.94 ± 0. 07. 3. Clint. Control group: R (+)60 ± 3, S (-) 170 ± 30; BNF group: R (+)111.0 ± 1, S (-) 84 ± 5; PB group: R (+)2.0 ± 2, S (-)56.0 ± 1. The enzyme parameters compared with unpaired t tests showed that no stereoselectivity was observed in enzymatic affinity of three microsomes to enantiomers and their catalytic abilities were quite different and had stereoselectivities. Compared with the control, microsome induced by BNF enhanced enzyme activity to propranolol R (+)enantiomer, and microsome induced by PB showed less enzyme activity to propranolol S (-)-enan tiomer which remains the same stereoselectivities as that of the control.
CONCLUSION: Enzyme activity centers of the microsome were changed in composition and regioselectivity after the induction of BNF and PB, and the stereoselectivities of propranolol cytochrome P450 metabolism in rat hepatic microsomes were likely due to the stereoselectivities of the catalyzing function in enzyme. CYP-1A subfamily induced by BNF exhibited pronounced contribution to propranolol metabolism with stereoselectivity to R (+)-enantiomer. CYP-2B subfamily induced by PB exhibited moderate contribution to propranolol metabolism, but still had the stereoselectivity of S (-)-enantiomer.
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Ottinger CA, Kaattari SL. Long-term immune dysfunction in rainbow trout (Oncorhynchus mykiss) exposed as embryos to aflatoxin B1. FISH & SHELLFISH IMMUNOLOGY 2000; 10:101-106. [PMID: 10938727 DOI: 10.1006/fsim.1999.0227] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/23/2023]
Affiliation(s)
- C A Ottinger
- Department of Environmental Sciences, School of Marine Science, Virginia Institute of Marine Science, College of William and Mary, Gloucester Point 23062, USA
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Breinholt V, Arbogast D, Loveland P, Pereira C, Dashwood R, Hendricks J, Bailey G. Chlorophyllin chemoprevention in trout initiated by aflatoxin B(1) bath treatment: An evaluation of reduced bioavailability vs. target organ protective mechanisms. Toxicol Appl Pharmacol 1999; 158:141-51. [PMID: 10406929 DOI: 10.1006/taap.1999.8696] [Citation(s) in RCA: 44] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Chlorophyllin (CHL) is known to inhibit DNA adduction and hepatocarcinogenesis in trout when administered at doses up to 4000 ppm in the diet with aflatoxin B(1) (AFB(1)). The principal protective mechanism is believed to involve CHL:AFB(1) complex formation, which may reduce systemic carcinogen absorption. However, mechanisms operative within the target organ in situ have not been ruled out. The present study used alternative CHL and AFB(1) exposures as well as hepatic metabolism studies to distinguish these mechanisms. Duplicate lots of 150 rainbow trout each were initiated by brief water bath exposure to 0.1 ppm AFB(1), with or without 500 ppm CHL in the water. The addition of 500 ppm CHL to the water bath, under conditions where AFB(1) is calculated to be >99% sequestered as the CHL:AFB(1) complex, reduced hepatic AFB(1)-DNA adduction by 95% and reduced hepatocarcinogenesis from 20.5% to 2%, compared with exposure to AFB(1) alone. Inclusion of 500 ppm CHL in the water bath also significantly reduced total body burden and hepatic levels of AFB(1) as well as AFB(2), a structural analogue of AFB(1) unable to directly form the 8,9-epoxide proximate electrophile but equally capable of complexing with CHL. By contrast, internal target organ CHL loading by pretreatment of trout with 4000 ppm dietary CHL for 7 days prior to (and 2 days following) AFB(1) waterbath exposure had no effect on AFB(1)-DNA adduction or tumorigenicity. Dietary CHL up to 8000 ppm had no effect on hepatic CYP2K1, CYP1A, glutathione transferase, UDP-glucuronosyl transferase, or, with one exception, the relative ratios among hepatic AFB(1) metabolites in vivo. These results support the hypothesis that CHL:AFB(1) complex formation and reduced systemic AFB(1) bioavailability is a principal mechanism for CHL chemoprevention in this model and that in situ target organ inhibitory mechanisms are relatively insignificant.
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Affiliation(s)
- V Breinholt
- Marine/Freshwater Biomedical Sciences Center, Oregon State University, Corvallis, Oregon, 97331, USA
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Raj HG, Parmar VS, Jain SC, Goel S, Singh A, Gupta K, Rohil V, Tyagi YK, Jha HN, Olsen CE, Wengel J. Mechanism of biochemical action of substituted 4-methylbenzopyran-2-ones. Part II: Mechanism-based inhibition of rat liver microsome-mediated aflatoxin B1-DNA binding by the candidate antimutagen 7,8-diacetoxy-4-methylcoumarin. Bioorg Med Chem 1998; 6:1895-904. [PMID: 9839019 DOI: 10.1016/s0968-0896(98)00111-4] [Citation(s) in RCA: 40] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023]
Abstract
7,8-Diacetoxy-4-methylcoumarin (DAMC), with no prerequisite for oxidative biotransformation has been reported to produce suicide inactivation of microsomal cytochrome P-450-catalysed formation of aflatoxin B1-8,9-oxide that binds to DNA. Parenteral administration of DAMC to rats caused significant inhibition of AFB1 binding to hepatic DNA in vivo as well as AFB1-induced micronuclei formation in bone marrow cells. These results highlight the antimutagenic potential of DAMC.
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Affiliation(s)
- H G Raj
- Department of Biochemistry, V. P. Chest Institute, University of Delhi, India
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Williams DE, Lech JJ, Buhler DR. Xenobiotics and xenoestrogens in fish: modulation of cytochrome P450 and carcinogenesis. Mutat Res 1998; 399:179-92. [PMID: 9672659 DOI: 10.1016/s0027-5107(97)00255-8] [Citation(s) in RCA: 77] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
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.
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Affiliation(s)
- D E Williams
- Marine/Freshwater Biomedical Sciences Center, Oregon State University, Corvallis 97331-6602, USA.
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Troxel CM, Buhler DR, Hendricks JD, Bailey GS. 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. Toxicol Appl Pharmacol 1997; 146:69-78. [PMID: 9299598 DOI: 10.1006/taap.1997.8219] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
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.
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Affiliation(s)
- C M Troxel
- Toxicology Program, Oregon State University, Corvallis, Oregon, 97331, USA
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Troxel CM, Reddy AP, O'Neal PE, Hendricks JD, Bailey GS. In vivo aflatoxin B1 metabolism and hepatic DNA adduction in zebrafish (Danio rerio). Toxicol Appl Pharmacol 1997; 143:213-20. [PMID: 9073610 DOI: 10.1006/taap.1996.8058] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
The zebrafish (Danio rerio) is assuming prominence in developmental genetics research. By comparison, little is known of tumorigenesis and nothing is known of carcinogen metabolism in this species. This study evaluated the ability of zebrafish to metabolize a well-characterized human carcinogen, aflatoxin B1 (AFB1), to phase I and phase II metabolites and assessed hepatic AFB1-DNA adduction in vivo. Fish i.p. injected with 50-400 micrograms [3H]AFB1/kg body wt displayed a linear dose response for hepatic DNA binding at 24 hr. AFB1-DNA adduct levels among treatments showed no statistical difference over the period from 1 to 21 days after injection, suggesting poor adduct repair in this species. DNA binding in female fish was 1-7-fold higher than that in males (p < 0.01). An in vitro AFB1 metabolism assay verified that zebrafish liver extracts oxidize AFB1 to the 8,9-epoxide proximate electrophile (Km = 79.0 +/- 16.4 microM, Vmax = 11.7 +/- 1.4 pmol/min/mg protein at 28 degrees C). The excretion of AFB1 and its metabolites was also examined by HPLC. As is typical of other fish studied, major metabolites excreted were aflatoxicol (AFL) and aflatoxicol-glucuronide (AFL-g), followed by unreacted AFB1. AFL appeared as early as 5 min after injection, whereas AFL-g was a significant metabolite after 18 hr. This study shows that in vivo administration of AFB1 to zebrafish results in moderate adduction of the carcinogen to liver DNA and that zebrafish have the capacity for both phase I and phase II metabolism of AFB1. The approximate fourfold difference between rainbow trout and zebrafish AFB1-DNA covalent binding index appears insufficient to explain the relative resistance of zebrafish to dietary AFB1 hepatocarcinogenicity.
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Affiliation(s)
- C M Troxel
- Department of Food Science and Technology, Oregon State University, Corvallis 97331, USA
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