Natural Product Skatole Ameliorates Lipotoxicity-Induced Multiple Hepatic Damage under Hyperlipidemic Conditions in Hepatocytes.

Skatole (3-methylindole, 3MI) is a natural-origin compound derived from plants, insects, and microbial metabolites in human intestines. Skatole has an anti-lipid peroxidation effect and is a biomarker for several diseases. However, its effect on hepatocyte lipid metabolism and lipotoxicity has not been elucidated. Hepatic lipotoxicity is induced by excess saturated free fatty acids in hyperlipidemia, which directly damages the hepatocytes. Lipotoxicity is involved in several metabolic diseases and hepatocytes, particularly affecting nonalcoholic fatty liver disease (NAFLD) progression. NAFLD is caused by the accumulation of fat by excessive free fatty acids (FFAs) in the blood and is accompanied by hepatic damage, such as endoplasmic reticulum (ER) stress, abnormal glucose and insulin metabolism, oxidative stress, and lipoapoptosis with lipid accumulation. Hepatic lipotoxicity causes multiple hepatic damages in NAFLD and has a directly effect on the progression from NAFLD to nonalcoholic steatohepatitis (NASH). This study confirmed that the natural compound skatole improves various damages to hepatocytes caused by lipotoxicity in hyperlipidemic conditions. To induce lipotoxicity, we exposed HepG2, SNU-449, and Huh7 cells to palmitic acid, a saturated fatty acid, and confirmed the protective effect of skatole. Skatole inhibited fat accumulation in the hepatocytes, reduced ER and oxidative stress, and recovered insulin resistance and glucose uptake. Importantly, skatole reduced lipoapoptosis by regulating caspase activity. In conclusion, skatole ameliorated multiple types of hepatocyte damage induced by lipotoxicity in the presence of excess free fatty acids.

A single nucleotide polymorphism in the CYP2E1 gene promoter affects skatole content in backfat of boars of two commercial Duroc-sired crossbred populations.

The prevention of unpleasant boar taint is the main reason for castration of male piglets. This study aimed to investigate how the malodorous compound skatole is affected by a single nucleotide polymorphism (g.2412 C>T at -586 ATG) in the porcine cytochrome p450 II E1 (CYP2E1) gene. 119 boars of two commercial Duroc-sired crossbred populations raised at different farms were investigated. Skatole and androstenone in backfat averaged 114±125 ng/g and 1206±895 ng/g melted fat, respectively. The frequency of the genotypes CC, CT, and TT was 25, 52, and 23%, respectively. CC boars had the highest average skatole levels (175 ng/g) compared to CT (92 ng/g) and TT (93 ng/g). Applying suggested sensory threshold levels for skatole (>150 ng/g) and androstenone (>2000 ng/g), 30% of the carcasses may be unacceptably tainted while the proportion of tainted carcasses is significantly higher within genotype CC (56.7%) compared to genotypes CT (24.3%) and TT (14.8%). Effective reduction of tainted carcasses appears feasible applying marker assisted selection.

Consumer studies on sensory acceptability of boar taint: a review.

Boar taint can be found in meat from boars and affects consumer acceptability of pork. The aim of this review is: (1) to describe different aspects of the existing methodologies used in consumer studies when evaluating boar taint from a sensory point of view, (2) to draw conclusions on different studies regarding the acceptability of meat from entire males, and (3) to discuss a possible harmonization of the different aspects to be considered when performing consumer studies on boar taint. This paper focuses on different aspects of studies previously carried out such as the country of assessment, the location of the test, the cooking procedure, the type of meat samples evaluated, the attributes and scales used, consumer profile, the results obtained, and the effect of androstenone sensitivity of the consumers on boar meat acceptability. A discussion on the possibility of a harmonization of the different aspects is also performed and final remarks and considerations have been drawn.

Predicting the likelihood of developing boar taint: early physical indicators in entire male pigs.

Three potential early-age predictors of which boars are likely to develop boar taint (testes volume, skin lesions and dirtiness) were measured on 102 boars every fortnight from 10 weeks of age until slaughter. These predictors were correlated with the level of boar taint according to the hot iron method and the concentrations of skatole and androstenone as determined by chemical analysis. The chance of no/low boar taint according to the hot iron method decreased with higher testes volume (weeks 22 and 24) and increased with skin lesion score (weeks 12, 16 and 18). For the concentrations of androstenone and skatole, the strongest correlation was found with testes volume in week 12. Skin lesions in week 16 were negatively correlated with skatole levels. Dirtiness was negatively correlated with skatole concentrations (week 18) but positively correlated with androstenone concentrations (weeks 20 and 22). Testes volume has the greatest potential for predicting the likelihood of developing boar taint.

Relationship between olfactory function and olfactory neuronal population in C57BL6 mice injected intraperitoneally with 3-methylindole.

OBJECTIVE: It is not known how many olfactory receptor neurons should be intact to maintain olfaction in mouse models treated with 3-methylindole. The aim of this study is to investigate the relationship between a simple olfactory test outcome and the olfactory neuronal population. STUDY DESIGN: Mouse model. SETTING: Animal laboratory of the Seoul National University Bundang Hospital. SUBJECTS AND METHODS: Olfactory dysfunction was induced by intraperitoneal injection of 3-methylindole in 38 six-week-old female C57BL6 mice. Olfactory function was evaluated by a food-finding test following 72-hour starvation. The olfactory neuronal population was quantified by olfactory marker protein (OMP) expression. RESULTS: The average time for finding food was 8.1 seconds in control mice. It was 13.4, 84.4, 90.1, and 111.4 seconds for mice injected with 100, 200, 300, and 400 µg/g of 3-methylindole, respectively. Harvesting the whole olfactory neuroepithelium, densitometric analysis showed significant decrease of OMP in the 300- and 400-µg/g groups as compared with controls (18.8% and 17.5% of relative density, respectively). In the olfactory bulb, there was a significant decrease of OMP in the 200-, 300-, and 400-µg/g groups (44.5%, 37.0%, and 9.0% of relative density, respectively). The food-finding time had a significant reverse correlation with the relative density of OMP both in the olfactory bulb and in the olfactory neuroepithelium. CONCLUSION: Our study showed that olfactory impairment was correlated with olfactory neuronal population in mice treated with 3-methylindole. The food-finding test would be a useful tool that could be easily performed without special training in the 3-methylindole-treated C57BL6 anosmic mouse model.

3-Methylindole metabolites induce lung CYP1A1 and CYP2F1 enzymes by AhR and non-AhR mechanisms, respectively.

3-Methylindole (3MI) is a highly selective pneumotoxicant that is present in abundant amounts (as high as 1.4 mug/cigarette) in cigarette smoke. Several human cytochrome P450 enzymes that are expressed in lung, such as CYP1A1, CYP2F1, CYP2A13, and CYP4B1, catalyze the dehydrogenation of 3MI to the reactive intermediate 3-methyleneindolenine, which alkylates DNA and induces cell death through apoptosis. In addition, 3MI potently damages DNA at low concentrations (observable at 0.1 muM). However, it seemed possible that 3MI could induce the levels of P450 enzymes, so transcription and translation of 1A1 and 2F1 genes were measured in primary normal human bronchial epithelial cells. In this study, 3MI-induced DNA damage at the 10 muM concentration was ameliorated when P450 turnover was inactivated with the cytochrome P450 suicide substrate inhibitor 1-aminobenzotriazole. Thus, the observed DNA damage was cytochrome P450-dependent. Quantitative real-time polymerase chain reaction analysis revealed both concentration- and time-dependent increases in CYP1A1 and CYP2F1 transcription by the same 3MI concentrations that damaged DNA. Aryl hydrocarbon receptor (AhR) activation lead to CYP1A1 induction. Treatment with 3MI in combination with the AhR antagonist alpha-naphthoflavone prevented 3MI-mediated CYP1A1 induction, indicating that the induction was AhR-dependent. Conversely, CYP2F1 induction did not appear to require activation of AhR. These intriguing findings show that not only is induction of 1A1 and 2F1 caused by 3MI metabolites, rather than 3MI itself, but transcriptional activation of these pulmonary genes occurs through disparate mechanisms. Thus, the induction process, and subsequent increased bioactivation of 3MI to toxic intermediates, is a facile process that might enhance the acute toxicity and/or mutagenicity of this chemical.

Chronic dexamethasone treatment potentiates insult to olfactory receptor cells produced by 3-methylindole.

The effect of chronic dexamethasone treatment on damage to olfactory receptor cells produced by 3-methylindole (3-MI) was examined. Twelve rats were injected, every other day, with dexamethasone (1.5 mg/kg, i.p.), and 12 rats with saline alone. Injections began 1 week before and continued, in different rats, from 1 to 4 weeks after a single intraperitoneal administration of 150 mg/kg 3-MI. One, two, three, and four weeks after exposure to 3-MI, different groups of rats, three specimens per each treatment condition, received bilateral application of horseradish peroxidase to the olfactory mucosa and were subsequently sacrificed. Anterograde labeling of primary afferents, i.e., an inverse correlate of the degree of cellular damage, was quantitatively determined by measuring the mean optical density (MOD) of staining in sections of the olfactory bulb. In saline-injected rats, the MOD values were 27.0, 46.6, 87.1, and 104.7 for one, two, three, and four post-3-MI weeks, respectively. The corresponding values in the dexamethasone-treated rats were 15.7, 29.7, 87.5, and 110.5. The MOD values of the dexamethasone-injected rats were significantly lower than those of the saline-injected rats for post-3-MI weeks 1 and 2, indicative of stronger damage to olfactory receptor cells in the rats treated with the glucocorticoid. The data suggest that dexamethasone potentiates the 3-MI olfactotoxicity during the first 2 weeks after insult. This effect, at least partly, may be due to the inducing action of dexamethasone on the cytochrome P450 responsible for metabolic bioactivation of 3-MI.

Effect of melengestrol acetate on development of 3-methylindole-induced pulmonary edema and emphysema in sheep.

The involvement of melengestrol acetate (MGA) in susceptibility to developing pulmonary edema and emphysema following oral administration of 3-methylindole (3MI) was investigated using 10 Suffolk ewes receiving 0 or 0.15 mg of MGA daily (n = 5). Blood, urine and ruminal fluid were collected immediately prior to 3MI dosing (0.2 g/kg BW) and 1, 2, 3, 4, 5, 6, 12 and 24 h (blood); 3, 6, 9, 12 and 15 h (urine) and 1, 2, 3 and 12 h (ruminal fluid) afterward. Ewes receiving MGA experienced earlier (P < 0.05) onset of respiratory distress than the control ewes (2.5 vs 4 h), and upon euthanasia at 96 h, their lung weight relative to body weight tended (P < 0.10) to be lower. Ruminal 3MI concentrations did not differ between treatments (P > 0.05). Ewes receiving MGA had higher (P < 0.05) concentrations of 3MI metabolites in plasma prior to dosing than did control ewes, and these values tended to remain higher throughout the sampling period. Immunoreactivity assays indicated more pneumotoxin present in the lungs of MGA-treated ewes than controls. Lung damage was apparently more acute and accelerated in the MGA-treated ewes than in the controls. Urinary 3MI mercapturate concentrations differed (control > MGA-treated, P < 0.05) at 9, 12, and 15 h, but this difference was not apparent when urinary production (as estimated by creatinine concentration) was considered. The implications of these findings for MGA-treated feedlot heifers are currently under investigation.

Structural and biochemical changes in lungs of 3-methylindole-treated rats.

Effects of a single dose of 3-methylindole (3-MI) (250 mg/kg intraperitoneally) were studied at different times ranging from 12 hours to 2 weeks post-treatment (PT). Microscopic study revealed mild Clara cell injury 24 hours PT and mucus hyperplasia 24 hours to 2 weeks PT. Diffuse type I alveolar epithelial cell necrosis occurred at 48 hours, followed by type II cell hyperplasia. Septal edema and accumulation of interstitial and capillary polymorphonuclear leukocytes and perivascular mixed mononuclear inflammatory cells accompanied the injury and repair. A gradual resolution of lesions with persistent mononuclear inflammatory cellular clusters at septal junctions, focal septal fibrosis, and accumulation of alveolar macrophages was evident at 1 and 2 weeks PT. Collagen, measured as hydroxyproline, in 3-MI-treated rats was significantly increased to 130% and 139% of control (3.0 mg/lung) at 1 and 2 weeks PT, respectively. Biphasic peaks of plasma 6-keto-prostaglandin F1 alpha occurred at 12 to 24 hours and at 96 hours PT with 3-MI and thromboxane B2 was elevated 12, 48, and 96 hours PT. Right ventricular/left ventricular and septal weight was increased to 120% and 140% of the control 1 and 2 weeks PT. We concluded that 3-MI induces alveolar septal injury in the rat with relatively complete repair of the alveolar epithelium and residual mild focal septal fibrosis and pulmonary hypertension 2 weeks PT. Arachidonic acid-derived mediators and inflammation are associated with 3-MI-induced lung injury.

Pulmonary function changes in goats given 3-methylindole orally.

Six adult goats were given 0.2 g of 3-methylindole (3MI)/kg of body weight orally. Lung mechanics and ventilatory function were obtained before 3MI and 2, 4, 6, 24, 48, and 72 hours after 3MI administration. Clinical signs were also monitored. Lungs were removed for gross and microscopic examination and for morphometric analysis from goats that died spontaneously or were killed with an overdose of pentobarbital after 72 hours. The major pulmonary function changes observed included a marked decrease in dynamic lung compliance with a moderate increase in airway resistance, a concomitant hypoxemia, sustained increase in respiratory frequency, a progressive decrease in tidal volume and alveolar ventilation, and increased dead space to tidal volume ratio. A mild metabolic acidosis was also noticed.

3-Methylindole-induced pulmonary injury in lambs. A comparison with acute respiratory distress syndrome (ARDS).

Five to six month-old lambs of two different Norwegian breeds (Dala and Rygja) were dosed with 3-methyl indole (3MI) and DL-tryptophan (DL-T), and examined clinically, hematologically and post mortem. Results were compared with the findings in Acute Respiratory Distress Syndrome (ARDS), a condition of acute dyspnea occurring in 5-6 month-old lambs (of Dala and Spael breeds) after they have been moved from mountain pastures onto lush aftermath in September. The results show that such young Norwegian lambs are susceptible to 3MI-dosing, and that the symptoms and lesions were similar to those described earlier in connection with 3MI-dosing in cattle and adult sheep. The results clearly demonstrate that 3-MI-induced pulmonary injury in lambs differs from ARDS as far as both symptoms, hematology and pathology are concerned.

Ultrastructural pathology of 3-methylindole-induced pneumotoxicity in cattle. I. Glycogen accumulation in the alveolar type II cells and tubular myelin accumulation in the alveoli.

3-Methylindole (3MI) was orally administered as a single dose of 0.1-0.2 gm/kgm which then produced acute pulmonary edema in cattle. Electronmicroscopic examination of lung tissue collected after 72 hours revealed vesiculation and desquamation of alveolar type I cells as well as hyperplasia of the alveolar type II cells. Hyaline membrane formation, along with massive accumulation of tubular myelin in the alveolar spaces, was observed. Pulmonary capillaries were occluded with microthrombi and leukocytes infiltrated the interstitium as well as the alveoli. Accumulation of large aggregates of glycogen in the alveolar type II cells were the most unusual changes seen in the experimental animals. It is suggested that accumulation of tubular myelin is a structural manifestation of surfactant inactivation by the plasma proteins. Furthermore, the disease process may be complicated by a compositional or quantitative defect in the surfactant produced by the alveolar type II cells, which are pathologically loaded with glycogen.

Effects of 3-methylindole in cattle.

1 Rapid intravenous injection of 3-methylindole (3-MI) was shown to induce an anaphylactoid-like reaction in calves. 2 This was suggested by the reduction in response to a repeat dose of 3-MI, by the reduction of effects in the presence of antagonists to the putative mediators of anaphylaxis in cattle and by the production of signs similar to those seen in experimentally induced bovine anaphylaxis. 3 The plasma half-life of 3-MI was short (14.4 min) and, since absorption of 3-MI from the rumen is known to be slow, the extent of formation of 3-MI from L-tryptophan in the rumen would have to be substantial if 3-MI is to be considered the causative agent of 'fog fever', an acute respiratory distress syndrome seen in cattle.