Survival Cost and Diverse Molecular Mechanisms of Magnaporthe oryzae Isolate Resistance to Epoxiconazole.

Rice blast caused by Magnaporthe oryzae is one of the most destructive diseases on rice worldwide. Epoxiconazole is a 14α-demethylation inhibitor (DMI) with excellent control on rice blast; to date, no resistant isolates have been observed in the field. Four mutants resistant to epoxiconazole were generated from three parental isolates via fungicide adaptation. Resistance was stable after 10 weekly consecutive transfers on fungicide-free medium. Three parameters, including growth rate, sporulation in vitro, and aggressiveness, were significantly lower for mutants compared with their parental isolates, with the exception of the low-resistance isolate. Sporulation and aggressiveness were negatively correlated with effective concentration values for 50% inhibition of mycelial growth for parental isolates and mutants (P < 0.05). Cross-resistance was found between epoxiconazole and prochloraz (ρ = 0.863, P = 0.000) or difenoconazole (ρ = 0.861, P = 0.000). The resistance factor for mutants was positively correlated with the relative expression of MoCYP51A in epoxiconazole treatment (r = 0.977, P = 0.02). In addition, two putative amino acid substitutions in MoCYP51A were found in two resistant mutants: Y126F in the high-resistance mutant and I125L in the low-resistance mutant. Mutation Y126F reduced the affinity of MoCYP51A with epoxiconazole, whereas I125L was not in the binding pocket of epoxiconazole. No amino acid change or overexpression in MoCYP51B was found in any of the mutants studied. To our knowledge, this is the first study to report DMI resistance observed in M. oryzae. The survival cost of M. oryzae resistance to epoxiconazole might be the reason why DMI resistance has not yet emerged in field populations worldwide.

Identification of the fungicide epoxiconazole by virtual screening and biological assessment as inhibitor of human 11ß-hydroxylase and aldosterone synthase.

Humans are constantly exposed to a multitude of environmental chemicals that may disturb endocrine functions. It is crucial to identify such chemicals and uncover their mode-of-action to avoid adverse health effects. 11ß-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) catalyze the formation of cortisol and aldosterone, respectively, in the adrenal cortex. Disruption of their synthesis by exogenous chemicals can contribute to cardio-metabolic diseases, chronic kidney disease, osteoporosis, and immune-related disorders. This study applied in silico screening and in vitro evaluation for the discovery of xenobiotics inhibiting CYP11B1 and CYP11B2. Several databases comprising environmentally relevant pollutants, chemicals in body care products, food additives and drugs were virtually screened using CYP11B1 and CYP11B2 pharmacophore models. A first round of biological testing used hamster cells overexpressing human CYP11B1 or CYP11B2 to analyze 25 selected virtual hits. Three compounds inhibited CYP11B1 and CYP11B2 with IC50 values below 3 µM. The most potent inhibitor was epoxiconazole (IC50 value of 623 nM for CYP11B1 and 113 nM for CYP11B2, respectively); flurprimidol and ancymidol were moderate inhibitors. In a second round, these three compounds were tested in human adrenal H295R cells endogenously expressing CYP11B1 and CYP11B2, confirming the potent inhibition by epoxiconazole and the more moderate effects by flurprimidol and ancymidol. Thus, the in silico screening, prioritization of chemicals for initial biological tests and use of H295R cells to provide initial mechanistic information is a promising strategy to identify potential endocrine disruptors inhibiting corticosteroid synthesis. A critical assessment of human exposure levels and in vivo evaluation of potential corticosteroid disrupting effects by epoxiconazole is required.

Asperlin Stimulates Energy Expenditure and Modulates Gut Microbiota in HFD-Fed Mice.

Asperlin is a marine-derived, natural product with antifungal, anti-inflammatory and anti-atherosclerotic activities. In the present study, we showed that asperlin effectively prevented the development of obesity in high-fat diet (HFD)-fed mice. Oral administration of asperlin for 12 weeks significantly suppressed HFD-induced body weight gain and fat deposition without inhibiting food intake. Hyperlipidemia and liver steatosis were also substantially ameliorated. A respiratory metabolism monitor showed that asperlin efficiently increased energy expenditure and enhanced thermogenic gene expression in adipose tissue. Accordingly, asperlin-treated mice showed higher body temperature and were more tolerant of cold stress. Meanwhile, asperlin also increased the diversity and shifted the structure of gut microbiota. Oral administration of asperlin markedly increased the relative abundance of Bacteroidetes, leading to a higher Bacteroidetes-to-Fimicutes ratio. The HFD-induced abnormalities at both phylum and genus levels were all remarkably recovered by asperlin. These results demonstrated that asperlin is effective in preventing HFD-induced obesity and modulating gut microbiota. Its anti-obesity properties may be attributed to its effect on promoting energy expenditure.

Human and ecological risk assessment of a crop protection chemical: a case study with the azole fungicide epoxiconazole.

Conventional risk assessments for crop protection chemicals compare the potential for causing toxicity (hazard identification) to anticipated exposure. New regulatory approaches have been proposed that would exclude exposure assessment and just focus on hazard identification based on endocrine disruption. This review comprises a critical analysis of hazard, focusing on the relative sensitivity of endocrine and non-endocrine endpoints, using a class of crop protection chemicals, the azole fungicides. These were selected because they are widely used on important crops (e.g. grains) and thereby can contact target and non-target plants and enter the food chain of humans and wildlife. Inhibition of lanosterol 14α-demethylase (CYP51) mediates the antifungal effect. Inhibition of other CYPs, such as aromatase (CYP19), can lead to numerous toxicological effects, which are also evident from high dose human exposures to therapeutic azoles. Because of its widespread use and substantial database, epoxiconazole was selected as a representative azole fungicide. Our critical analysis concluded that anticipated human exposure to epoxiconazole would yield a margin of safety of at least three orders of magnitude for reproductive effects observed in laboratory rodent studies that are postulated to be endocrine-driven (i.e. fetal resorptions). The most sensitive ecological species is the aquatic plant Lemna (duckweed), for which the margin of safety is less protective than for human health. For humans and wildlife, endocrine disruption is not the most sensitive endpoint. It is concluded that conventional risk assessment, considering anticipated exposure levels, will be protective of both human and ecological health. Although the toxic mechanisms of other azole compounds may be similar, large differences in potency will require a case-by-case risk assessment.

Assessment of the roles of P-glycoprotein and cytochrome P450 in triptolide-induced liver toxicity in sandwich-cultured rat hepatocyte model.

Triptolide (TP), a main bioactive component of Tripterygium wilfordii Hook F., is a promising agent for treatment of autoimmune diseases. However, a high incidence of dose-limiting hepatotoxicity was observed in the clinic. Sandwich-cultured rat hepatocyte model was used in this study to identify the involvement of P-glycoprotein (P-gp) in TP disposition and to evaluate TP-induced hepatotoxicity after modulation of P-gp by the known inhibitors, ritonavir and tariquidar, and known inducers, phenobarbital, quercetin, and H(2)O(2). Our data showed that biliary clearance of TP reduced 73.7% and 84.2% upon treatment of ritonavir (25 µM) and tariquidar (5 µM), respectively. In contrast, increases of 346%, 280%, and 273% in biliary clearance of TP were observed with treatment of phenobarbital (1.0 mM), quercetin (20 µM), and H(2)O(2) (0.5 mM), respectively. The TP-induced hepatotoxicity increased by twofold when CYP activity was blocked by 1-aminobenzotriazole, suggesting that CYP and P-gp may both contribute to the detoxification of TP in the SCRH model. In addition, hepatotoxicity and the expression of apoptosis proteins Bax and Bcl-2 were correlated qualitatively with the TP exposure duration and its intracellular concentration, which, in turn, can be modulated by P-gp inhibitors or inducers. Our results for the first time demonstrated that in addition to CYP-mediated metabolism, P-gp also plays an important role in the disposition of TP and TP-induced hepatotoxicity. Thus, the modulation of canalicular P-gp has a potential to cause drug-drug interaction between TP and the coadministered P-gp inhibitors or inducers in the clinic.

Sphingolipids as new biomarkers for assessment of delayed-type hypersensitivity and response to triptolide.

BACKGROUND: Hypersensitivity diseases are associated with many severe human illnesses, including leprosy and tuberculosis. Emerging evidence suggests that the pathogenesis and pathological mechanisms of treating these diseases may be attributable to sphingolipid metabolism. METHODS: High performance liquid chromatography-tandem mass spectrometry was employed to target and measure 43 core sphingolipids in the plasma, kidneys, livers and spleens of BALB/c mice from four experimental groups: control, delayed-type hypersensitivity (DTH) model, DTH+triptolide, and control+triptolide. Orthogonal partial least squares discriminant analysis (OPLS-DA) was used to identify potential biomarkers associated with variance between groups. Relationships between the identified biomarkers and disease markers were evaluated by Spearman correlation. RESULTS: As a treatment to hypersensitivity disease, triptolide significantly inhibit the ear swelling and recover the reduction of splenic index caused by DTH. The sphingolipidomic result revealed marked alterations in sphingolipid levels between groups that were associated with the effects of the disease and triptolide treatment. Based on this data, 23 potential biomarkers were identified by OPLS-DA, and seven of these biomarkers correlated markedly with the disease markers (p<0.05) by Spearman correlation. CONCLUSIONS: These data indicate that differences in sphingolipid levels in plasma and tissues are related to DTH and treatment with triptolide. Restoration of proper sphingolipid levels may attribute to the therapeutic effect of triptolide treatment. Furthermore, these findings demonstrate that targeted sphingolipidomic analysis followed by multivariate analysis presents a novel strategy for the identification of biomarkers in biological samples.

Assessment of the anti-obesity effects of the TNP-470 analog, CKD-732.

The systemic treatment with angiogenesis inhibitor has been shown to result in weight reduction and adipose tissue loss in various models of obesity. To verify the mechanism of CKD-732 (TNP-470 analog) against obesity, we evaluated CKD-732's peripheral and central anti-obesity effects. CKD-732 was injected subcutaneously (s.c.) for 7 days in various animal models and intracerebroventricularly (i.c.v.) in arcuate nucleus (ARC) lesion mice, ob/ob mice, and normal littermates. Modulation of the hypothalamic neuropeptide mRNAs after i.c.v. injection was evaluated in ARC lesion mice and normal littermates. A conditioned taste aversion (CTA) was performed using lithium chloride (LiCl) as a positive control agent in Long-Evans Tokushima Otsuka and Otsuka Long-Evans Tokushima fatty (OLETF) rats. As a result, 7 days of CKD-732 s.c. injection reduced the cumulative food intake and the body weight significantly in both treated obese (e.g. 114.8 +/- 13.4 g vs 170.7 +/- 20.6 g, 7.9 +/- 0.5% decrease vs 0.3 +/- 2.2% decrease; in treated OLETF rat versus control OLETF rat, P < 0.01 respectively) and non-obese models. Epididymal and mesenteric fat pads, and the size of adipocytes were significantly decreased in treated rats. A single i.c.v. injection decreased food intake and body weight in ARC lesion mice and ob/ob mice but not in normal littermates. Unexpectedly, the hypothalamic neuropeptide mRNAs were not altered by single i.c.v. injection. CKD-732 also induced a dose-dependent CTA comparable with LiCl injection, which is a commonly used agent to produce a CTA. In conclusion, CKD-732 causes significant body weight and appetite reduction, possibly by decreasing adiposity directly and inducing central anorexia, which is partly explained by a CTA. These results should be carefully verified to assess the utility of CKD-732 as an anti-obesity drug.

Monte Carlo simulations assessing the risk of salmonellosis from consumption of almonds.

Recent outbreaks of salmonellosis associated with raw almonds have raised awareness of this food as a vector for foodborne illness. We performed a quantitative assessment of the risk of contracting salmonellosis from consumption of raw almonds, accounting for factors that become important after almonds reach the processor. We estimated the risk associated with the consumption of raw almonds and the risk reduction associated with almonds treated with a theoretical 5-log reduction process or treated with propylene oxide using a standard commercial process. Probability distributions were chosen to describe the chance of almond contamination and the effects of storage time, storage temperature, and processing from currently available data. A beta-Poisson model for the dose-response relationship for Salmonella was obtained from published literature. The simulation estimated a 78% chance of one or more cases of salmonellosis per year from consumption of raw almonds. The application of a commercial propylene oxide treatment reduced this risk to 0.01%. Hypothetical 5-log reduction treatments with different standard deviations (+/-1, +/-0.5, +/-0.1, and +/-0) reduced the predicted yearly risk of salmonellosis to 0.69, 0.35, 0.30, and 0.21%, respectively. These results suggest that the risk of one or more U.S. cases of salmonellosis per year from consumption of raw almonds can be reduced from 78% to less than 1% by using a process achieving a 5-log reduction in Salmonella with a process standard deviation as large as 1 log unit or by using a commercial propylene oxide treatment.

Health and economic consequences of sevelamer use for hyperphosphatemia in patients on hemodialysis.

OBJECTIVES: The safety and efficacy of sevelamer hydrochloride in binding phosphate in patients with end-stage renal disease and its ability to attenuate the progression of cardiac calcification have been well documented but not the longer-term health and economic implications. Thus, a model of the predicted long-term consequences of sevelamer compared with calcium-based binders (acetate and carbonate) was developed. METHODS: Long-term cardiovascular implications of 1 year of treatment with phosphate binders in patients on hemodialysis are estimated based on the patient's demographics, comorbidities, and physiologic and renal parameters. The initial calcification score and expected changes over 1 year are derived using regression equations developed from the Treat-to-Goal study and translated to cardiovascular disease risk based on equations developed from a long-term cohort study. In this article, the implications of cardiovascular disease for life expectancy and medical costs are accounted for from a US payer perspective. RESULTS: The cardioprotective effect of sevelamer over 1 year is estimated to result in a 12% reduction in cardiovascular events compared with calcium acetate. In a population of 100 patients, the savings of 205,600 dollars accrued due to avoiding nine cardiovascular events with sevelamer, largely offset the increased binder costs, leading to a favorable cost-effectiveness ratio of about 2200 dollars per (discounted) life-year gained. CONCLUSIONS: Although both binders provide equivalent phosphate binding capacity, the results indicate that the advantage of 1 year of treatment with sevelamer in attenuating the progression of calcification has important clinical and economic consequences, suggesting that this provides good value for money.

13C/31P NMR assessment of mitochondrial energy coupling in skeletal muscle of awake fed and fasted rats. Relationship with uncoupling protein 3 expression.

To examine the relationship between mitochondrial energy coupling in skeletal muscle and change in uncoupling protein 3 (UCP3) expression during the transition from the fed to fasted state, we used a novel noninvasive (31)P/(13)C NMR spectroscopic approach to measure the degree of mitochondrial energy coupling in the hind limb muscles of awake rats before and after a 48-h fast. Compared with fed levels, UCP3 mRNA and protein levels in the gastrocnemius increased 1.7- (p < 0.01) and 2.9-fold (p < 0.001), respectively, following a 48-h fast. Tricarboxylic acid cycle flux measured using (13)C NMR as an index of mitochondrial substrate oxidation was 212 +/- 23 and 173 +/- 25 nmol/g/min (p not significant) in the fed and 48-h fasted groups, respectively. Unidirectional ATP synthesis flux measured using (31)P NMR was 79 +/- 15 and 57 +/- 9 nmol/g/s (p not significant) in the fed and 48-h fasted groups, respectively. Mitochondrial energy coupling as expressed by the ratio of ATP synthesis to tricarboxylic acid cycle flux was not different between the fed and fasted states. To test the hypothesis that UCP3 may be involved in the translocation of long chain free fatty acids (FFA) into the mitochondrial matrix under conditions of elevated FFA availability, [U-(13)C]palmitate/albumin was administered in a separate group of rats with (+) or without (-) etomoxir (an inhibitor of carnitine palmitoyltransferase I). The ratio of glutamate enrichment ((+) etomoxir/(-) etomoxir) in the hind limb muscles was the same between groups, indicating that UCP3 does not appear to function as a translocator for long chain FFA in skeletal muscle following a 48-h fast. In summary, these data demonstrate that despite a 2-3-fold increase in UCP3 mRNA and protein expression in skeletal muscle during the transition from the fed to fasted state, mitochondrial energy coupling does not change. Furthermore, UCP3 does not appear to have a major role in FFA translocation into the mitochondria. The physiological role of UCP3 following a 48-h fast in skeletal muscle remains to be elucidated.

Modulating factors of individual sensitivity to diepoxybutane: sister chromatid exchanges induced in vitro in human lymphocytes.

Spontaneous and diepoxybutane (DEB)-induced sister chromatid exchanges (SCEs) were examined in cultured peripheral lymphocytes (PBL) from 122 healthy donors. SCE-inducing activity under defined experimental conditions and individual sensitivity to genotoxic stress were assessed. SCE means distribution appeared asymmetrical, identifying about 22% of subjects characterized by a 'high-respondent' phenotype with more than 111 SCEs/cell. Confounding factors, such as smoking habit, wine and coffee consumption, work activity and hematological factors, showed a limited capacity to affect individual SCE responsiveness, however hemoglobin and uric acid seemed to antagonize DEB genotoxicity.

Assessment of the energetic role of endogenous substrates in the tail artery of rat by means of enzyme inhibitors.

The rat tail artery during a 180 min incubation period in a medium containing glucose plus oxfenicine (an inhibitor of fatty acid oxidation) did not show changes in the contractile responses to adrenaline. In a substrate-free medium the extent of the contractions underwent a slight decrease during the last 60 min of incubation. When the substrate-free medium contained 2-deoxyglucose (an inhibitor of glycolysis and glycogenolysis) or oxfenicine, the decline of the contractile activity developed faster and attained a similar extent with each inhibitor. When the substrate-free medium contained 2-deoxyglucose together with oxfenicine or methylpalmoxirate (an inhibitor of fatty acid oxidation) the arteries displayed a pronounced and early fall in the contraction strength. These data suggest that in the presence of glucose the reserve substrates are not necessary as fuel source for the arterial contractions. However, in substrate-free conditions they constitute an important energy source. Furthermore, glycogen and triacylglycerol share the supply of energy and there does not seem to be any other reserve material in the smooth muscle of the rat tail artery.

Use of three-way differential staining and liquid holding for the assessment of individual repair capacity.

Many studies on DNA repair mechanisms in mammalian cells have used liquid holding (LH) recovery to evaluate premutational damage repair. We used human peripheral lymphocytes (HPL) to assess damage reduction during the G0 phase. This technique was matched with the three-way differential (TWD) staining that allows identification of sister-chromatid exchanges (SCE) per cell cycle in third metaphases. By adopting this approach, the persistence of diepoxybutane (DEB)-induced lesions during subsequent cycles and individual repair capacity in LH conditions were measured. Our results show that most DEB-induced damage was repaired during the first cell cycle; a large part of lesions were removed during LH recovery, demonstrating G0 HPL repair capacity.