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1.
Toxicol Lett ; 303: 67-71, 2019 Mar 15.
Article in English | MEDLINE | ID: mdl-30599194

ABSTRACT

The hepatic cytochrome p450 enzymes 1 A, 2A19 and 2E1 is very important for the elimination of skatole from the body of pigs. Impaired skatole metabolism, results in skatole accumulation, which give rise to off flavor of the meat. Several metabolites of skatole has been identified, however the role of these metabolites in the inhibition of the skatole metabolizing enzymes are not documented. Using microsomes from pigs and fish, we determined the ability of several skatole metabolites to inhibit CYP1 A, CYP2A19 and CYP2E1 dependent activity. Our results show that 2-aminoacetophenone is an inhibitor of porcine CYP2A19 and CYP2E1 activity, but not the piscine orthologues. In conclusion, there is species specific differences in the inhibition of CYP1 A and CYP2A19 dependent metabolism of probe substrates. This is relevant to the evaluation of different model systems and to the reduction of off flavor of meat.


Subject(s)
Cytochrome P-450 CYP2E1 Inhibitors/toxicity , Liver/drug effects , Microsomes, Liver/metabolism , Skatole/toxicity , Acetophenones/toxicity , Animals , Coumarins/toxicity , Cytochrome P-450 CYP1A1/antagonists & inhibitors , Cytochrome P-450 CYP1A1/metabolism , Cytochrome P-450 CYP2E1/metabolism , Cytochrome P-450 Enzyme System/metabolism , Fishes , Liver/metabolism , Male , Microsomes, Liver/drug effects , Nitrophenols/toxicity , Oxazines/toxicity , Red Meat/analysis , Seafood/analysis , Species Specificity , Swine
2.
Toxicol Lett ; 277: 1-8, 2017 Aug 05.
Article in English | MEDLINE | ID: mdl-28571685

ABSTRACT

The role of the cross-talk between nuclear receptors in the regulation of Cytochrome P450 expression in the liver is well-documented. Most studies have focused on the cross-talk between the pregnane X receptor (PXR) and other receptors, such as the constitutive androstane receptor. However, cross-talk between PXRs and aryl hydrocarbon receptors (AhRs) has also been suggested, but reports regarding this cross-talk are conflicting. In the present study, we treated HepaRG and primary human hepatocytes (PHHs) with both a strong (TCDD) and weak (3-methylindole; 3MI) AhR activator to investigate their impact on PXR-regulated expression of CYP3A4. Moreover, we investigated the effect of co-activation of PXR, using rifampicin, and AhR, using TCDD and 3MI, on the regulation of CYP3A4 induction. We also investigated whether knockdown of AhR using siRNA affected the basal expression of PXR and CYP3A4 and induction of CYP3A4 by rifampicin, TCDD and 3MI. The results showed that the treatment of HepaRG cells, but not of PHHs, with AhR activators decreased mRNA expression of CYP3A4 and PXR. Moreover, in both HepaRG and PHHs, AhR activation decreased rifampicin-induced expression of CYP3A4 mRNA. Knock-down of AhR in PHHs increased both basal and rifampicin-induced expression of CYP3A4 mRNA. In conclusion, the presented results suggested that the cross-talk between PXR and AhR plays a role in the regulation of CYP3A4 gene expression.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/agonists , Cytochrome P-450 CYP3A/biosynthesis , Hepatocytes/drug effects , Polychlorinated Dibenzodioxins/toxicity , Receptors, Aryl Hydrocarbon/agonists , Rifampin/pharmacology , Stem Cells/drug effects , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cytochrome P-450 CYP3A/genetics , Cytochrome P-450 CYP3A Inhibitors , Dose-Response Relationship, Drug , Enzyme Induction , Hepatocytes/enzymology , Humans , Pregnane X Receptor , Primary Cell Culture , RNA Interference , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptor Cross-Talk/drug effects , Receptors, Aryl Hydrocarbon/genetics , Receptors, Aryl Hydrocarbon/metabolism , Receptors, Steroid/agonists , Receptors, Steroid/genetics , Receptors, Steroid/metabolism , Signal Transduction , Skatole/toxicity , Stem Cells/enzymology , Transfection
3.
Chemosphere ; 150: 304-310, 2016 May.
Article in English | MEDLINE | ID: mdl-26915592

ABSTRACT

We studied the in vitro metabolism of 3-methylindole (3MI) in hepatic microsomes from fish. Hepatic microsomes from juvenile and adult carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss) were included in the study. Incubation of 3MI with hepatic microsomes revealed the time-dependent formation of two major metabolites, 3-methyloxindole (3MOI) and indole-3-carbinol (I3C). The rate of 3MOI production was similar in both species at both ages. No differences in kinetic parameters were observed (p = 0.799 for Vmax, and p = 0.809 for Km). Production of I3C was detected only in the microsomes from rainbow trout. Km values were similar in juvenile and adult fish (p = 0.957); Vmax was higher in juvenile rainbow trout compared with adults (p = 0.044). In rainbow trout and carp, ellipticine reduced formation of 3MOI up to 53.2% and 81.9% and ketoconazole up to 65.8% and 91.3%, respectively. The formation of I3C was reduced by 53.7% and 51.5% in the presence of the inhibitors ellipticine and ketoconazole, respectively. These findings suggest that the CYP450 isoforms CYP1A and CYP3A are at least partly responsible for 3MI metabolism. In summary, 3MI is metabolised in fish liver to 3MOI and I3C by CYP450, and formation of these metabolites might be species-dependent.


Subject(s)
Carps/metabolism , Microsomes, Liver/metabolism , Oncorhynchus mykiss/metabolism , Skatole/metabolism , Water Pollutants, Chemical/metabolism , Animals , Cytochrome P-450 Enzyme Inhibitors/pharmacology , Cytochrome P-450 Enzyme System/metabolism , Ellipticines/pharmacology , In Vitro Techniques , Indoles/metabolism , Ketoconazole/pharmacology , Liver/drug effects , Liver/metabolism , Metabolic Detoxication, Phase I , Microsomes, Liver/drug effects , Oxindoles , Skatole/toxicity , Species Specificity , Water Pollutants, Chemical/toxicity
4.
Water Res ; 47(2): 870-80, 2013 Feb 01.
Article in English | MEDLINE | ID: mdl-23218246

ABSTRACT

The photolysis of five frequent emerging contaminants (Benzotriazole, Chlorophene, N,N-diethyl-m-toluamide or DEET, Methylindole, and Nortriptyline HCl) was investigated in ultrapure water under monochromatic ultraviolet radiation at 254 nm and by a combination of UV and hydrogen peroxide. The results revealed that the photolysis rates followed first-order kinetics, with rate constant values depending on the nature of the specific compound, the pH, and the presence or absence of the scavenger tert-butanol. Quantum yields were also determined and values in the range of 53.8 × 10⁻³ - 9.4 × 10⁻³ mol E⁻¹ for Benzotriazole, 525 × 10⁻³ - 469 × 10⁻³ mol E⁻¹ for Chlorophene, 2.8 × 10⁻³ - 0.9 × 10⁻³ mol E⁻¹ for DEET, 108 × 10⁻³ - 165 × 10⁻³ mol E⁻¹ for Methylindole, and 13.8 × 10⁻³ - 15.0 × 10⁻³ mol E⁻¹ for Nortriptyline were obtained. The study also found that the UV/H2O2 process enhanced the oxidation rate in comparison to direct photolysis. High-performance liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry (HPLC-ESI-QTOF-MS) technique was applied to the concentrations evaluation and further identification of the parent compounds and their by-products, which allowed the proposal of the degradation pathways for each compound. Finally, in order to assess the aquatic toxicity in the photodegradation of these compounds, the Vibrio fischeri acute toxicity test was used, and the results indicated an initial increase of this parameter in all cases, followed by a decrease in the specific case of Benzotriazole, DEET, Methylindole, and Chlorophene.


Subject(s)
Models, Chemical , Water Pollutants, Chemical/analysis , Water Purification/methods , Water/chemistry , Aliivibrio fischeri/drug effects , Aliivibrio fischeri/growth & development , Anti-Infective Agents, Local/analysis , Anti-Infective Agents, Local/chemistry , Anti-Infective Agents, Local/toxicity , Antidepressive Agents, Tricyclic/analysis , Antidepressive Agents, Tricyclic/chemistry , Antidepressive Agents, Tricyclic/toxicity , DEET/analysis , DEET/chemistry , Dichlorophen/analogs & derivatives , Dichlorophen/analysis , Free Radical Scavengers/chemistry , Hydrogen Peroxide/chemistry , Hydrogen-Ion Concentration , Insect Repellents/analysis , Insect Repellents/chemistry , Insect Repellents/toxicity , Kinetics , Nortriptyline/analysis , Nortriptyline/chemistry , Nortriptyline/toxicity , Oxidants/chemistry , Oxidation-Reduction , Photolysis , Skatole/toxicity , Triazoles/analysis , Triazoles/toxicity , Ultraviolet Rays , Water Pollutants, Chemical/chemistry , Water Pollutants, Chemical/toxicity , tert-Butyl Alcohol/chemistry
5.
Drug Metab Dispos ; 40(4): 642-7, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22228748

ABSTRACT

The aim of this study was to determine whether mouse CYP2A5 and CYP2F2 play critical roles in the bioactivation of 3-methylindole (3MI), a tissue-selective toxicant, in the target tissues, the nasal olfactory mucosa (OM) and lung. Five metabolites of 3MI were identified in NADPH- and GSH-fortified microsomal reactions, including 3-glutathionyl-S-methylindole (GS-A1), 3-methyl-2-glutathionyl-S-indole (GS-A2), 3-hydroxy-3-methyleneindolenine (HMI), indole-3-carbinol (I-3-C), and 3-methyloxindole (MOI). The metabolite profiles and enzyme kinetics of the reactions were compared between OM and lung, and among wild-type, Cyp2a5-null, and Cyp2f2-null mice. In lung reactions, GS-A1, GS-A2, and HMI were detected as major products, and I-3-C and MOI, as minor metabolites. In OM reactions, all five metabolites were detected in ample amounts. The loss of CYP2F2 affected formation of all 3MI metabolites in the lung and formation of HMI, GS-A1, and GS-A2 in the OM. In contrast, loss of CYP2A5 did not affect formation of 3MI metabolites in the lung but caused substantial decreases in I-3-C and MOI formation in the OM. Thus, whereas CYP2F2 plays a critical role in the 3MI metabolism in the lung, both CYP2A5 and CYP2F2 play important roles in 3MI metabolism in the OM. Furthermore, the fate of the reactive metabolites produced by the two enzymes through common dehydrogenation and epoxidation pathways seemed to differ with CYP2A5 supporting direct conversion to stable metabolites and CYP2F2 supporting further formation of reactive iminium ions. These results provide the basis for understanding the respective roles of CYP2A5 and CYP2F2 in 3MI's toxicity in the respiratory tract.


Subject(s)
Aryl Hydrocarbon Hydroxylases/physiology , Cytochrome P-450 Enzyme System/physiology , Lung/metabolism , Olfactory Mucosa/metabolism , Skatole/metabolism , Animals , Aryl Hydrocarbon Hydroxylases/genetics , Biotransformation , Chromatography, High Pressure Liquid , Cytochrome P-450 CYP2A6 , Cytochrome P-450 Enzyme System/genetics , Cytochrome P450 Family 2 , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Microsomes/metabolism , Skatole/pharmacokinetics , Skatole/toxicity , Tandem Mass Spectrometry
6.
Chem Res Toxicol ; 23(11): 1682-90, 2010 Nov 15.
Article in English | MEDLINE | ID: mdl-20795680

ABSTRACT

3-Methylindole (3MI) is a preferential pneumotoxicant found in cigarette smoke. A number of lung-expressed human cytochrome P450 enzymes, including 1A1, 2F1, and 2A13, catalyze the metabolism of 3MI to reactive intermediates that fragment DNA, measured with the Comet assay to assess DNA damage, in a cytochrome P450-dependent manner in primary normal human lung cells in culture, but the mutagenesis of 3MI has been controversial. In the present study, the mutagenic potential of 3MI was compared to the prototypical cigarette smoke carcinogens benzo(a)pyrene (B(a)P) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). 3MI, B(a)P, and NNK were incubated with the Salmonella typhimurium strain TA98, which is known to detect the most common subtype of cigarette smoke-induced mutagenicity, frameshift mutations in DNA, and with Salmonella typhimurium strain TA100, which detects base pair substitution mutants, with five sources of P450-mediated bioactivation: rat liver S9, human lung microsomes, recombinant CYP2A13, purified CYP2F3, and recombinant CYP1A1. Only B(a)P was mutagenic in TA100, and it was bioactivated by human lung microsomes and rat liver S9 sources of P450s. However, with the TA98 strain, CYP1A1, CYP2A13, CYP2F3, and human lung microsomes bioactivated 3MI to highly mutagenic intermediates, whereas neither human nor rat liver S9 subcellular fractions formed mutagenic intermediates from 3MI. Quantitative Western blot analysis verified that all three respiratory enzymes were present in human lung microsomes in widely varying amounts. These results indicate that metabolism of 3MI by human lung-expressed cytochrome P450 enzymes but not hepatic P450s elicits equivalent or higher mutagenicity than the prototype cigarette smoke mutagens B(a)P and NNK and indicates that 3MI is a likely human pulmonary carcinogen.


Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Lung/enzymology , Mutagens/toxicity , Skatole/toxicity , Animals , Aryl Hydrocarbon Hydroxylases/metabolism , Benzo(a)pyrene/chemistry , Benzo(a)pyrene/toxicity , Cells, Cultured , Cytochrome P-450 Enzyme System/genetics , DNA Damage , Humans , Liver/metabolism , Microsomes/enzymology , Microsomes/metabolism , Mutagenicity Tests , Mutagens/chemistry , Nitrosamines/chemistry , Nitrosamines/toxicity , Rats , Ribosomal Protein S9 , Ribosomal Proteins/genetics , Ribosomal Proteins/metabolism , Salmonella typhimurium/drug effects , Skatole/chemistry , Smoking
7.
Am J Rhinol Allergy ; 24(2): 121-5, 2010.
Article in English | MEDLINE | ID: mdl-20338109

ABSTRACT

BACKGROUND: This study was performed to investigate whether statins can enhance the recovery of the olfactory epithelium (OE) damaged by 3-methylindole (3MI), an olfactotoxicant, and to compare the effects with those of steroids. METHODS: Randomized placebo-controlled trial was performed. Fifty-four healthy female Sprague-Dawley rats (aged 9-10 weeks and weighing 160-180 g each) were randomly allocated to the statin-treated, steroid-treated, or control groups. Olfactory loss was induced using i.p. injection of 3MI in adult rats. Atorvastatin (10 mg/kg for 4 weeks), prednisolone (1 mg/kg for 2 weeks), or normal saline (1 cc for 4 weeks) was then administered per os with a gastric tube. Hematoxylin and eosin (H&E) staining and immunohistochemical staining were performed to evaluate the change of thickness and the arrangement of the OE, and immunoreactivity to protein gene product (PGP) 9.5 and proliferating cell nuclear antigen (PCNA). RESULTS: The statin-treated group showed the earliest increase of the thickness of the OE (p = 0.002 at 7 days after 3MI injection) and the immunoreactivity to PCNA (p = 0.032 at 7 days after 3MI injection) among the three groups. The immunoreactivity to PGP 9.5 showed significantly better improvement at the 7th and 28th days after 3MI injection compared with the steroid-treated or control groups (p = 0.002 and p < 0.001, respectively). CONCLUSION: Statins might enhance the proliferation and neuroregenesis of the OE after 3MI injection.


Subject(s)
Heptanoic Acids/administration & dosage , Olfactory Mucosa/drug effects , Olfactory Mucosa/pathology , Pyrroles/administration & dosage , Animals , Atorvastatin , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Female , Olfactory Mucosa/metabolism , Prednisolone/administration & dosage , Proliferating Cell Nuclear Antigen/genetics , Proliferating Cell Nuclear Antigen/metabolism , Rats , Rats, Sprague-Dawley , Regeneration/drug effects , Skatole/toxicity , Ubiquitin Thiolesterase/genetics , Ubiquitin Thiolesterase/metabolism
8.
J Comp Pathol ; 143(2-3): 209-12, 2010.
Article in English | MEDLINE | ID: mdl-20153869

ABSTRACT

A herd of red deer experienced a small outbreak of atypical interstitial pneumonia on two occasions 3 years apart. The first occasion involved the death of two of a group of 70 hinds and the second outbreak involved a single death in a similar sized group. On both occasions a number of the adults exhibited increased respiratory effort, particularly on exercise. Both outbreaks occurred in July, a few days after moving the herd from a close grazed grass paddock to an aftermath paddock, which had regrown to a good sward. The history and pathology is reminiscent of 3-methyl indole toxicity in cattle.


Subject(s)
Deer , Disease Outbreaks , Lung Diseases, Interstitial/veterinary , Animals , Lung Diseases, Interstitial/epidemiology , Lung Diseases, Interstitial/pathology , Skatole/toxicity , United Kingdom/epidemiology
9.
Toxicol Sci ; 112(1): 59-67, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19700606

ABSTRACT

Previous work has shown that bioactivation of the cigarette smoke pneumotoxicant 3-methylindole (3MI) by pulmonary cytochrome P450 enzymes is directly associated with formation of DNA adducts. Here, we present evidence that normal human lung epithelial cells, exposed to low micromolar concentrations of 3MI, showed extensive DNA damage, as measured by the comet assay, with similar potency to the prototypical genotoxic agents, doxorubicin and irinotecan. The DNA damage caused by 3MI was predominantly caused by single-strand breaks. Furthermore, we show that this damage decreased with time, given a subtoxic concentration, with detectable DNA fragmentation peaking 4 h after exposure and diminishing to untreated levels within 24 h. Pretreatment with an inhibitor of poly(ADP-ribose) polymerase 1 (PARP1), NU1025, nearly doubled the DNA damage produced by 5 microM 3MI, implying that PARP1, which among other activities, functions to repair single-strand breaks in DNA, repaired at least some of the 3MI-induced DNA fragmentation. A key cellular response to DNA damage, phosphorylation, and nuclear localization of p53 was seen at subtoxic levels of 3MI exposure. 3MI was highly mutagenic, with essentially the same potency as the prototype carcinogen, benzo[a]pyrene, only when a lung-expressed CYP2F3 enzyme was used to dehydrogenate 3MI to its putative DNA-alkylating intermediate. Conversely, a rat liver S9 metabolic system did not bioactivate 3MI to its mutagenic intermediate(s). Concentrations higher than 25 microM caused apoptosis, which became extensive at 100 microM, similar to the response seen with 10 microM doxorubicin. Our findings indicate that there is a low concentration window in which 3MI can cause extensive DNA damage and mutation, without triggering apoptotic defenses, reinforcing the hypothesis that inhaled 3MI from cigarette smoke may be a potent lung-selective carcinogen.


Subject(s)
Carcinogens/toxicity , Lung Neoplasms/chemically induced , Mutagens/toxicity , Skatole/toxicity , Apoptosis/drug effects , Blotting, Western , Bronchi/cytology , Bronchi/drug effects , Bronchi/metabolism , Cells, Cultured , DNA Damage , DNA Repair , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Humans , Mutagenicity Tests , Salmonella typhimurium/genetics , Tumor Suppressor Protein p53/metabolism
10.
Am J Rhinol ; 22(3): 292-6, 2008.
Article in English | MEDLINE | ID: mdl-18588762

ABSTRACT

BACKGROUND: Olfactory loss is a challenging disease. Although glucocorticoid is sometimes used for the treatment of anosmia, it has been reported that it potentiated neural damage in the early phase of treatment. This study is designed to identify the effect of ginkgo biloba, an antioxidant that acts as a free radical scavenger, in the treatment of olfactory injury aggravated by dexamethasone. METHODS: Anosmia mouse model was induced by i.p. injection of 3-methylindole (3-MI). Twenty-five mice were divided into one control group without anosmia and four anosmia treatment groups (given treatments of dexamethasone and/or ginkgo biloba). The effects of treatment were evaluated by behavioral test, Western blot, and immunohistochemistry 2 weeks after 3-MI injection. RESULTS: Induction of anosmia was confirmed by behavioral tests. The thickness and cell number of olfactory neuroepithelium were decreased more significantly in the dexamethasone treatment group than in the combination treatment group. The expression of olfactory marker protein (OMP) in olfactory epithelium was more decreased also in the dexamethasone treatment group than in the combination treatment group. The expression of OMP was decreased significantly in the olfactory bulbs of anosmia groups but there were no differences between the anosmia treatment groups. CONCLUSION: Dexamethasone treatment was associated with further deterioration of olfactory injury by 3-MI and it was recovered by combination treatment of dexamethasone and ginkgo biloba. The antioxidant effect of ginkgo biloba might play a role in restoration of olfactory loss and it was effective only when oxidative stress is maximized by dexamethasone.


Subject(s)
Antioxidants/therapeutic use , Dexamethasone/therapeutic use , Ginkgo biloba , Glucocorticoids/therapeutic use , Olfaction Disorders/drug therapy , Phytotherapy/methods , Plant Preparations/therapeutic use , Animals , Behavior, Animal/drug effects , Blotting, Western , Cell Count , Disease Models, Animal , Drug Therapy, Combination , Female , Follow-Up Studies , Immunohistochemistry , Mice , Mice, Inbred BALB C , Neuroepithelial Cells/drug effects , Neuroepithelial Cells/pathology , Olfaction Disorders/chemically induced , Olfaction Disorders/physiopathology , Olfactory Bulb/drug effects , Olfactory Bulb/metabolism , Olfactory Bulb/pathology , Olfactory Marker Protein/biosynthesis , Olfactory Mucosa/drug effects , Olfactory Mucosa/innervation , Olfactory Mucosa/metabolism , Olfactory Nerve/drug effects , Olfactory Nerve/metabolism , Olfactory Nerve/pathology , Skatole/toxicity , Treatment Outcome
12.
Vet Pathol ; 40(4): 363-70, 2003 Jul.
Article in English | MEDLINE | ID: mdl-12824507

ABSTRACT

Response to 3-methylindole (3MI) varies among species. Mice recover from 3MI-induced bronchiolar epithelial injury but sustain persistent olfactory mucosal injury with scarring and epithelial metaplasia. In contrast, 3MI induces obliterative bronchiolitis in horses and ponies, but olfactory mucosal injury has not been reported. To evaluate the effect of 3MI on equine olfactory mucosa, ponies were dosed orally with 100 mg 3MI/kg (n = 9) or corn oil vehicle (n = 6). All ponies treated with 3MI developed obliterative bronchiolitis with mild olfactory injury. By 3 days after 3MI dosing, olfactory epithelium appeared disorganized with decreased and uneven surface height and scalloping of the basement membrane zone. Epithelial cells of Bowman's glands were hypertrophic. Proliferation of olfactory epithelium and Bowman's glands was supported by an increased mitotic index and positive immunohistochemical staining for proliferating cell nuclear antigen as compared with controls. The activity of 11beta-hydroxysteroid dehydrogenase, an olfactory mucosal cytosolic enzyme localized to sustentacular and Bowman's glandular epithelial cells, was concurrently decreased. By 9 days postdosing, olfactory mucosal lesions had lessened. Results indicate that 3MI transiently injures equine olfactory mucosa without the extensive necrosis, scarring, or metaplasia seen in murine olfactory mucosa or in equine bronchiolar epithelium.


Subject(s)
Horse Diseases/chemically induced , Horse Diseases/pathology , Olfactory Mucosa/drug effects , Olfactory Mucosa/pathology , Skatole/toxicity , Animals , Epithelial Cells/drug effects , Epithelial Cells/enzymology , Epithelial Cells/pathology , Horse Diseases/enzymology , Horses , Nose Diseases/chemically induced , Nose Diseases/enzymology , Nose Diseases/pathology , Olfactory Mucosa/enzymology
13.
Toxicol Sci ; 71(2): 229-36, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12563108

ABSTRACT

Transfected BEAS-2B cells that express different cytochrome P450 enzymes were used to assess whether human bronchial epithelial cell lines are target cells for 3-methylindole (3MI)-induced damage. Four different transfected BEAS-2B lines overexpressing P450s 2A6, 3A4, 2F1, and 2E1 (B-CMV2A6, B-CMV3A4, B-CMV2F1, and B-CMV2E1), respectively, were compared. The B-CMV2F1 and B-CMV3A4 cells were the most susceptible to 3MI-mediated cytotoxicity, measured by leakage of lactate dehydrogenase into the medium after a 48-h incubation. The toxicity was ameliorated by pretreatment with 1-aminobenzotriazole (ABT). Depletion of glutathione with diethylmaleate decreased the onset and increased the extent of cell death with 3MI. Thus, 3MI is cytotoxic to immortalized bronchial epithelial cells overexpressing 2F1 without concomitant depletion of GSH, but depletion of GSH modestly enhances the cytotoxicity of 3MI to human lung cells. Additional studies clearly demonstrated that a low concentration of 3MI (10 micro M) induced apoptosis in BEAS-2B cells that was measured by DNA fragmentation, and apoptosis was inhibited by the presence of ABT. The B-CMV2F1 cells overexpressing 2F1 demonstrated increased apoptosis (measured by Annexin-V binding) at 24 h with 100 micro M 3MI. Therefore, CYP2F1 in human bronchial epithelial lung cells may bioactivate 3MI to 3-methyleneindolenine, which induces programmed cell death at relatively low concentrations. Human lung cells may be susceptible to this prototypical pneumotoxicant.


Subject(s)
Bronchi/drug effects , Cytochrome P-450 Enzyme System/metabolism , Respiratory Mucosa/drug effects , Skatole/toxicity , Apoptosis/drug effects , Bronchi/enzymology , Bronchi/pathology , Cell Line, Transformed , Cell Survival/drug effects , Cytochrome P-450 Enzyme System/genetics , DNA Fragmentation/drug effects , Dose-Response Relationship, Drug , Drug Antagonism , Glutathione/antagonists & inhibitors , Glutathione/metabolism , Humans , Isoenzymes , L-Lactate Dehydrogenase/metabolism , Maleates/pharmacology , RNA, Messenger/metabolism , Respiratory Mucosa/enzymology , Respiratory Mucosa/pathology , Reverse Transcriptase Polymerase Chain Reaction , Transfection , Triazoles/pharmacology
14.
Ultrastruct Pathol ; 27(1): 13-21, 2003.
Article in English | MEDLINE | ID: mdl-12554532

ABSTRACT

Olfactory mucosa from C57BL/6N mice was examined by transmission electron microscopy at 0.5, 1, 2, 4, 6, 24, 48, and 72 h after 400 mg 3-methylindole (3MI)/kg ip, and at 7, 14, and 21 days after 300 mg 3MI/kg or vehicle. Degeneration was evident in epithelial cells of Bowman's glands and olfactory sustentacular cells by 0.5 h, but not in neurons until 24 h, by which time necrosis was fully developed in sustentacular cells and epithelium of Bowman's glands. Sustentacular cells and neurons detached from the basal lamina by 48 h. Lamina proprial fibroblasts were hypertrophied by 72 h and, with collagen fibrils, formed the bulk of the mucosa at 7 days. By 21 days, fibroblasts were less conspicuous, but Bowman's glands were rarely observed and were lined by epithelial cells without secretory granules. Mucosal epithelium was reconstituted, but disorderly and lacked olfactory differentiation. Mice treated with 3MI were more likely than control mice to taste water treated with isoamyl acetate (odorant) and quinine monohydrochloride (aversive tastant). Though olfactory neurons were initially spared, their absence in the regenerated epithelium explains lingering olfactory deficits in murine 3MI toxicosis.


Subject(s)
Olfaction Disorders/chemically induced , Olfactory Mucosa/drug effects , Regeneration/drug effects , Skatole/toxicity , Smell/drug effects , Animals , Dose-Response Relationship, Drug , Female , Mice , Microscopy, Electron , Necrosis , Olfaction Disorders/physiopathology , Olfactory Mucosa/physiopathology , Olfactory Mucosa/ultrastructure , Smell/physiology , Time Factors
15.
Toxicol Appl Pharmacol ; 175(1): 68-75, 2001 Aug 15.
Article in English | MEDLINE | ID: mdl-11509028

ABSTRACT

A magnetic resonance imaging (MRI) technique was developed to identify mucosal damage to the nasal passages of mice resulting from exposure to respiratory toxicants. 3-Methylindole (3-MI) was chosen as a model nasal toxicant because systemic administration of this compound in mice results in a well-characterized necrotizing nasal lesion that is restricted to the olfactory mucosa. MRI technology allows imaging of the same mice before and at time points after injection. In addition, morphological alterations and increases in the area of sinus cavity airspace can be followed as a function of dose and time following exposure. For 3-MI, the cross-sectional area of the sinus airspaces increased by 1.7-fold in mice injected with 200 mg/kg and 2.6-fold in mice injected with 300 mg/kg at 3 days after injection. Alterations in the nasal turbinates lined by olfactory mucosa were identified 1, 3, and 6 days postadministration of 3-MI using MRI. Postmortem histological examination of the nasal tissue confirmed the intranasal location and distribution of the 3-MI-induced lesions observed by MRI. MRI can be a useful technique to identify toxicant-induced mucosal injury in the nasal passages at an in-plane resolution less than 60 microm.


Subject(s)
Nasal Cavity/drug effects , Nasal Mucosa/injuries , Skatole/toxicity , Animals , Magnetic Resonance Imaging , Male , Mice , Mice, Inbred C57BL , Nasal Cavity/pathology
16.
Toxicology ; 145(1): 39-49, 2000 Apr 07.
Article in English | MEDLINE | ID: mdl-10771130

ABSTRACT

An in vitro model of the rat nasal cavity has been used to compare the responses of nasal tissues in vitro, using loss of intracellular ATP and potassium as indices of toxicity, with the pathological changes occurring following in vivo exposure to four test compounds. Turbinates were incubated in vitro with the test compounds for 4 h, for 24 h or for 4 h followed by 20 h in fresh medium. Titanium dioxide caused little or no loss of ATP in either olfactory epithelium (OE) or respiratory epithelium (RE). Sodium carbonate decreased olfactory, but not respiratory ATP, while acetic acid and 3-methylindole markedly decreased ATP in both tissues. Intracellular potassium concentrations were generally affected to a lesser degree. In vivo, no morphological changes were observed in the nasal cavity following inhalation exposure to either titanium dioxide or sodium carbonate. Inhalation of acetic acid resulted in a very focal lesion in the RE of the dorsal meatus of level 1, while administration of 3-methylindole by intraperitoneal injection caused severe degeneration of OE. In further experiments olfactory turbinates were exposed to a range of concentrations (0-100 mM) of sodium carbonate, acetic acid and 3-methylindole for 4 h and ATP concentrations determined. Concentration-dependent decreases in ATP were observed for sodium carbonate and 3-methylindole, with EC(50) values estimated as 2.57 and 0.91 mM, respectively. Acetic acid only decreased ATP significantly at the 100-mM concentration. In summary, this in vitro model has predicted the nasal toxicity of several compounds, including both direct-acting agents (sodium carbonate, acetic acid) and one requiring metabolic activation (3-methylindole). However, the lack of airflow-dependent dosimetry, results in some lack of discrimination between the different regions of the nasal cavity and may make this model overly sensitive.


Subject(s)
Nasal Mucosa/drug effects , Acetic Acid/toxicity , Adenosine Triphosphate/analysis , Animals , Carbonates/toxicity , Dose-Response Relationship, Drug , In Vitro Techniques , Male , Nasal Mucosa/chemistry , Nasal Mucosa/pathology , Potassium/analysis , Rats , Rats, Wistar , Skatole/toxicity , Titanium/toxicity
17.
Am J Vet Res ; 60(5): 563-70, 1999 May.
Article in English | MEDLINE | ID: mdl-10328425

ABSTRACT

OBJECTIVE: To evaluate the potential synergy between bovine respiratory syncytial virus (BRSV) and 3-methylindole (3MI) in inducing respiratory disease in cattle. ANIMALS: 20 mixed-breed beef calves. PROCEDURE: A 2 X 2 factorial design was used, with random assignment to the following 4 treatment groups: unchallenged control, BRSV challenge exposure (5 X 10(4) TCID50 by aerosolization and 5.5 X 10(5) TCID50 by intratracheal inoculation), 3MI challenge exposure (0.1 g/kg of body weight, PO), and combined BRSV-3MI challenge exposure. Clinical examinations were performed daily. Serum 3MI concentrations, WBC counts, PCV, total plasma protein, and fibrinogen concentrations were determined throughout the experiment. Surviving cattle were euthanatized 7 days after challenge exposure. Pulmonary lesions were evaluated at postmortem examination. RESULTS: Clinical respiratory disease was more acute and severe in cattle in the BRSV-3MI challenge-exposure group than in cattle in the other groups. All 5 cattle in this group and 3 of 5 cattle treated with 3MI alone died or were euthanatized prior to termination of the experiment. Mean lung displacement volume was greatest in the BRSV-3MI challenge-exposure group. Gross and histologic examination revealed that pulmonary lesions were also most severe for cattle in this group. CONCLUSIONS AND CLINICAL RELEVANCE: Feedlot cattle are commonly infected with BRSV, and 3MI is produced by microflora in the rumen of all cattle. Our results suggest that there is a synergy between BRSV and 3MI. Thus, controlling combined exposure may be important in preventing respiratory disease in feedlot cattle.


Subject(s)
Cattle Diseases/physiopathology , Lung/physiopathology , Respiratory Syncytial Virus Infections/veterinary , Respiratory Syncytial Virus, Bovine , Skatole/toxicity , Animals , Cattle , Cattle Diseases/pathology , Factor Analysis, Statistical , Lung/drug effects , Lung/pathology , Organ Size/drug effects , Respiratory Mechanics/drug effects , Respiratory Syncytial Virus Infections/pathology , Respiratory Syncytial Virus Infections/physiopathology , Skatole/blood , Skatole/pharmacokinetics
18.
Exp Lung Res ; 25(2): 165-81, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10188110

ABSTRACT

The role of type II epithelial cell proliferation in repair of diffuse alveolar epithelial injury was examined using two pneumotoxicants, 3-methylindole (3-MI) and monocrotaline (MCT). It was hypothesized that if MCT inhibits type II epithelial cell mitosis, then pulmonary fibrosis would result after diffuse 3-MI-induced type I alveolar cell injury in rats preadministered MCT. Four groups of rats were given vehicle control, MCT, 3-MI, or MCT and 3-MI. Lungs from rats killed 4 days post-treatment were examined subjectively and quantitatively by light and electron microscopy. Proliferative stimulus was estimated by bromodeoxyuridine (BrdU) incorporation. Lungs from rats killed 2 weeks post-treatment were evaluated by light microscopy. At 4 days, the number of type II cells in the lungs of 3-MI-treated rats was 3 times greater than in the lungs of the dually (MCT/3-MI) treated rats which was the same as the control rat lungs. There was no significant difference between the MCT/3-MI-treated rats and the 3-MI-treated rats with regard to the percentage of denuded alveolar basement membrane. The number of BrdU-labeled type II epithelial cells was increased above the control in both 3-MI-treated groups, but was greater in the 3-MI-treated rat lungs than in the lungs of the MCT/3-MI-treated rats. The average type II cell volume in dually treated rats was 3 times the volume in the control animals and 50% greater than that in 3-MI-treated rats. Transmission electron microscopy of the lungs of the MCT/3-MI-treated rats demonstrated flattened hypertrophic type II cells over large portions of the basement membrane. The light microscopic appearance and collagen staining of the lungs of the dually treated rats were similar to the negative control rat lungs 2 weeks after dosing with 3-MI. This suggests that despite a proliferative stimulus, MCT inhibits type II cell division after diffuse alveolar type I cell injury, but that type II cell migration and coverage of the basal lamina proceed. Results of this study suggest that coverage of the denuded basal lamina by any method is sufficient to prevent interstitial alveolar fibrosis.


Subject(s)
Carcinogens/toxicity , Epithelial Cells/drug effects , Epithelial Cells/pathology , Monocrotaline/toxicity , Pulmonary Alveoli/drug effects , Pulmonary Alveoli/pathology , Pulmonary Fibrosis/chemically induced , Pulmonary Fibrosis/pathology , Skatole/toxicity , Animals , Bromodeoxyuridine/metabolism , Cell Division/drug effects , Disease Models, Animal , Epithelial Cells/ultrastructure , Immunohistochemistry , Microscopy, Electron , Pulmonary Alveoli/ultrastructure , Rats , Rats, Inbred F344
19.
Chem Res Toxicol ; 11(11): 1326-31, 1998 Nov.
Article in English | MEDLINE | ID: mdl-9815193

ABSTRACT

Cytochrome P450 enzymes can potentially oxygenate 3-methylindole to form 2,3-epoxy-3-methylindoline which could rearrange to the stable metabolite 3-methyloxindole or open to form 3-hydroxy-3-methylindolenine, a putative electrophilic imine. The purpose of the current work was to determine if the imine was formed, and to characterize it via its adducts with thiol nucleophiles. Thiols were added to incubations of goat lung microsomes with 3-methylindole and deuterated analogues of 3-methylindole to trap the imine intermediate as its thioether conjugates. The N-acetylcysteine conjugate of 3-hydroxy-3-methylindolenine was detectable by LC/MS, but a molecular ion was not observed because the adduct rapidly dehydrated to form the 2-substituted indole. However, the imine was S-alkylated, and the intermediate carbinol was intramolecularly trapped using thioglycolic acid as a trapping agent that induced cyclocondensation to a lactone. The retention of one atom of deuterium from [2-2H]-3-methylindole and three from 3-[2H3-methyl]indole substantiated the mechanism in which the lactone adduct was produced by sulfur addition to either 3-hydroxy-3-methylindolenine or the epoxide. Tandem mass spectrometry of the lactone adduct produced a daughter ion spectrum consistent with this adduct. These studies demonstrated the existence of a new reactive intermediate of 3-methylindole, 3-hydroxy-3-methylindolenine, which may play a role in the pneumotoxicity of this chemical.


Subject(s)
Imines/chemistry , Lung Diseases/chemically induced , Skatole/chemistry , Sulfides/chemistry , Animals , Chromatography, Liquid , Goats , Lung/metabolism , Mass Spectrometry , Microsomes/metabolism , Skatole/toxicity , Thioglycolates/chemistry
20.
Chem Res Toxicol ; 11(7): 741-9, 1998 Jul.
Article in English | MEDLINE | ID: mdl-9671536

ABSTRACT

The existence of a cytochrome P450-dependent 2,3-epoxide of the potent pneumotoxin 3-methylindole was indirectly confirmed using stable isotope techniques and mass spectrometry. Determination of hydride shift and incorporation of labeled oxygen in 3-methyloxindole and 3-hydroxy-3-methyloxindole, metabolites that may be in part dependent on the presence of the epoxide, were utilized as indicators of the epoxide's existence. One mechanism for the formation of 3-methyloxindole involves cytochrome P450-mediated epoxidation followed by ring opening requiring a hydride shift from C-2 to C-3. Through incubations of goat lung microsomes with [2-2H]-3-methylindole, the retention of 2H in 3-methyloxindole was found to be 81%, indicating a majority of the oxindole was produced by the mechanism described above. 3-Hydroxy-3-methylindolenine is an imine reactive intermediate that could be produced by ring opening of the 2,3-epoxide. The imine may be oxidized to 3-hydroxy-3-methyloxindole by the cytosolic enzyme aldehyde oxidase. Activities of this putative detoxification enzyme were determined in both hepatic and pulmonary tissues from goats, rats, mice, and rabbits, but the activities could not be correlated to the relative susceptibilities of the four species to 3-methylindole toxicity. The 18O incorporation into either 3-methyloxindole or 3-hydroxy-3-methyloxindole from both 18O2 and H218O was determined. The 18O incorporation into 3-methyloxindole from 18O2 was 91%, strongly implicating a mechanism requiring cytochrome P450-mediated oxygenation. Incorporation of 18O into 3-hydroxy-3-methyloxindole indicated that the alcohol oxygen originated from molecular oxygen, also implicating an epoxide precursor. These studies demonstrate the existence of two new reactive intermediates of 3-methylindole and describe the mechanisms of their formation and fate.


Subject(s)
Epoxy Compounds/chemistry , Indoles/chemistry , Skatole/chemistry , Aldehyde Oxidase , Aldehyde Oxidoreductases/metabolism , Animals , Chromatography, Liquid , Cytochrome P-450 Enzyme System/metabolism , Gas Chromatography-Mass Spectrometry , Goats , Male , Mice , Microsomes/chemistry , Microsomes/metabolism , Oxygen Radioisotopes , Rabbits , Rats , Skatole/toxicity
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