A fish-based diet intervention improves endothelial function in postmenopausal women with type 2 diabetes mellitus: A randomized crossover trial

Objective

The beneficial effects of fish and n-3 polyunsaturated fatty acids (PUFAs) consumption on atherosclerosis have been reported in numerous epidemiological studies. However, to the best of our knowledge, the effects of a fish-based diet intervention on endothelial function have not been investigated. Therefore, we studied these effects in postmenopausal women with type 2 diabetes mellitus (T2DM).

Materials/Methods

Twenty-three postmenopausal women with T2DM were assigned to two four-week periods of either a fish-based diet (n-3 PUFAs ≧ 3.0 g/day) or a control diet in a randomized crossover design. Endothelial function was measured with reactive hyperemia using strain-gauge plethysmography and compared with the serum levels of fatty acids and their metabolites. Endothelial function was determined with peak forearm blood flow (Peak), duration of reactive hyperemia (Duration) and flow debt repayment (FDR).

Results

A fish-based dietary intervention improved Peak by 63.7%, Duration by 27.9% and FDR by 70.7%, compared to the control diet. Serum n-3 PUFA levels increased after the fish-based diet period and decreased after the control diet, compared with the baseline (1.49 vs. 0.97 vs. 1.19 mmol/l, p < 0.0001). There was no correlation between serum n-3 PUFA levels and endothelial function. An increased ratio of epoxyeicosatrienoic acid/dihydroxyeicosatrienoic acid was observed after a fish-based diet intervention, possibly due to the inhibition of the activity of soluble epoxide hydrolase.

Conclusions

A fish-based dietary intervention improves endothelial function in postmenopausal women with T2DM. Dissociation between the serum n-3 PUFA concentration and endothelial function suggests that the other factors may contribute to this phenomenon.     

Inhibition of soluble epoxide hydrolase enhances the anti-inflammatory effects of aspirin and 5-lipoxygenase activation protein inhibitor in a murine model

Inflammation is a multi-staged process whose expansive phase is thought to be driven by acutely released arachidonic acid (AA) and its metabolites. Inhibition of cyclooxygenase (COX), lipoxygenase (LOX), or soluble epoxide hydrolase (sEH) is known to be anti-inflammatory. Inhibition of sEH stabilizes the cytochrome P450 (CYP450) products epoxyeicosatrienoic acids (EETs). Here we used a non-selective COX inhibitor aspirin, a 5-lipoxygenase activation protein (FLAP) inhibitor MK886, and a sEH inhibitor t-AUCB to selectively modulate the branches of AA metabolism in a lipopolysaccharide (LPS)-challenged murine model. We used metabolomic profiling to simultaneously monitor representative AA metabolites of each branch. In addition to the significant crosstalk among branches of the AA cascade during selective modulation of COX, LOX, or sEH, we demonstrated that co-administration of t-AUCB enhanced the anti-inflammatory effects of aspirin or MK886, which was evidenced by the observations that co-administration resulted in favorable eicosanoid profiles and better control of LPS-mediated hypotension as well as hepatic protein expression of COX-2 and 5-LOX. Targeted disruption of the sEH gene displayed a parallel profile to that produced by t-AUCB. These observations demonstrate a significant level of crosstalk among the three major branches of the AA cascade and that they are not simply parallel pathways. These data illustrate that inhibition of sEH by both pharmacological intervention and gene knockout enhances the anti-inflammatory effects of aspirin and MK886, suggesting the possibility of modulating multiple branches to achieve better therapeutic effects.     

Inhibition of soluble epoxide hydrolase contributes to the anti-inflammatory effect of antimicrobial triclocarban in a murine model

The increasing use of the antimicrobial triclocarban (TCC) in personal care products (PCPs) has resulted in concern regarding environmental pollution. TCC is a potent inhibitor of soluble epoxide hydrolase (sEH). Inhibitors of sEH (sEHIs) are anti-inflammatory, anti-hypertensive and cardio-protective in multiple animal models. However, the in vivo effects anticipated from a sEHI have not been reported for TCC. Here we demonstrated the anti-inflammatory effects in vivo of TCC in a murine model. TCC was employed in a lipopolysaccharide (LPS)-challenged murine model. Systolic blood pressure, plasma levels of several inflammatory cytokines and chemokine, and metabolomic profile of plasma oxylipins were determined. TCC significantly reversed LPS-induced morbid hypotension in a time-dependent manner. TCC significantly repressed the increased release of inflammatory cytokines and chemokine caused by LPS. Furthermore, TCC significantly shifted the oxylipin profile in vivo in a time-dependent manner towards resolution of inflammation as expected from a sEHI. These results demonstrated that at the doses used TCC is anti-inflammatory in the murine model. This study suggests that TCC may provide some benefits in humans in addition to its antimicrobial activities due to its potent inhibition of sEH. It may be a promising starting point for developing new low volume high value applications of TCC. However these biological effects also caution against the general over use of TCC in PCPs.     

Modulation of cytochrome-derived epoxyeicosatrienoic acids pathway: a promising pharmacological approach to prevent endothelial dysfunction in cardiovascular diseases?

Progress in methods of investigating endothelial function in humans has led to the demonstration that endothelial dysfunction is an early process involved in the pathophysiology of cardiovascular diseases, and represents a new independent therapeutic target that may help to improve patient health. The administration of antioxidant, anti-hypertensive, lipid lowering or antidiabetic agents appear insufficient to fully restore the normal functions of the vascular endothelium and specific therapeutic strategies are still lacking. In this context, one emerging promising pharmacological approach to prevent endothelial dysfunction is to restore epoxyeicosatrienoic acids (EETs) pathway. EETs are eicosanoids synthesized by endothelial cytochrome epoxygenases that contribute to the regulation of endothelium-dependent dilatation, vascular inflammation, cell proliferation, angiogenesis and hemostasis. Moreover, it has been shown in vivo in humans that EETs act as endothelium-derived hyperpolarizing factors to regulate the vascular tone in both resistance and conduit arteries. In various cardiovascular disorders such as arterial hypertension, a decrease in EETs availability, due to an increased degradation by soluble epoxide hydrolase (sEH), is a deleterious mechanism that contributes to endothelial dysfunction and promotes cardiovascular inflammation and remodeling. Subsequently, the use of sEH inhibitors, which have been shown to decrease blood pressure, limit ischemic injury and prevent hypertrophy in various animal models, appears to be an attractive opportunity to restore endothelial function. Future research will be necessary to demonstrate that sEH inhibitors can prevent endothelial dysfunction in human arteries, which may help to prevent the development of cardiovascular complications and improve cardiovascular prognosis in patients.     

Acute doxorubicin cardiotoxicity alters cardiac cytochrome P450 expression and arachidonic acid metabolism in rats

Doxorubicin (DOX) is a potent anti-neoplastic antibiotic used to treat a variety of malignancies; however, its use is limited by dose-dependent cardiotoxicity. Moreover, there is a strong correlation between cytochrome P450 (CYP)-mediated arachidonic acid metabolites and the pathogenesis of many cardiovascular diseases. Therefore, in the current study, we have investigated the effect of acute DOX toxicity on the expression of several CYP enzymes and their associated arachidonic acid metabolites in the heart of male Sprague-Dawley rats. Acute DOX toxicity was induced by a single intraperitoneal injection of 15 mg/kg of the drug. Our results showed that DOX treatment for 24 h caused a significant induction of CYP1A1, CYP1B1, CYP2C11, CYP2J3, CYP4A1, CYP4A3, CYP4F1, CYP4F4, and EPHX2 gene expression in the heart of DOX-treated rats as compared to the control. Similarly, there was a significant induction of CYP1A1, CYP1B1, CYP2C11, CYP2J3, CYP4A, and sEH proteins after 24 h of DOX administration. In the heart microsomes, acute DOX toxicity significantly increased the formation of 20-HETE which is consistent with the induction of the major CYP ω-hydroxylases: CYP4A1, CYP4A3, CYP4F1, and CYP4F4. On the other hand, the formation of 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs) was significantly reduced, whereas the formation of their corresponding dihydroxyeicosatrienoic acids was significantly increased. The decrease in the cardioprotective EETs can be attributed to the increase of sEH activity parallel to the induction of the EPHX2 gene expression in the heart of DOX-treated rats. In conclusion, acute DOX toxicity alters the expression of several CYP and sEH enzymes with a consequent alteration in arachidonic acid metabolism. These results may represent a novel mechanism by which this drug causes progressive cardiotoxicity.     

Rapid simultaneous analysis of cyclooxygenase, lipoxygenase and cytochrome P-450 metabolites of arachidonic and linoleic acids using high performance liquid chromatography/mass spectrometry in tandem mode

Eicosanoids are oxidized arachidonate-derived lipid products generated by cyclooxygenase, lipoxygenase and cytochrome P-450 pathways. They are involved in diverse processes in health and disease and they are highly bioactive. Gas chromatography and enzyme immunoassays were used to quantify these mediators in the past. However, the recent availability of high-sensitivity liquid chromatography-mass spectrometry has provided a new approach for quantification that minimizes the sample size and the required preparation. This paper describes a rapid and simple technique for the simultaneous quantitative analysis of prostaglandin (PG) E₂ and PGJ₂; leukotrienes (LT) B₄ and D₄; 5-, 12-, 15- and 20-hydroxyeicosatetraenoic acids (HETEs); 13-hydroxyoctadecadienoic acid (13-HODE); 5,6-, 8,9-, 11,12- and 14,15-epoxyeicosatrienoic acids (EETs); and 11,12- and 14,15-dihydroxieicosatrienoic acids (DHETs) in cell culture supernatants and urine. We simultaneously analyzed 14 arachidonic acid metabolites representative from the three pathways, together with 13-HODE, a linoleic-derived product. Solid phase extraction was used for the sample preparation. The recoveries obtained ranged from 25% to 100%, depending on the metabolites. The LC/MS/MS method used the gradient on a C₁₈ column and electrospray ionization in negative ion detection mode. The method was optimized for sensitivity and for separation within 20 min. The linear ranges of the calibration curves were 0.1-200 ng/ml for PGE₂, PGJ₂, LTB₄, 5-HETE, 12-HETE, 15-HETE, 13-HODE, 11,12-EET, 11,12-DHET and 14,15-DHET, and 1-200 ng/ml for LTD₄, 20-HETE, 5,6-EET, 8,9-EET and 14,15-EET. The advantages of this method include minimal sample preparation, high sensitivity and elimination of the problem associated with thermal instability in gas chromatography analysis.     

Time-dependent expression pattern of cytochrome P450 epoxygenases and soluble epoxide hydrolase in normal human placenta

CYP2C and CYP2?J enzymes, commonly named as cytochrome P450 (CYP) epoxygenases, convert arachidonic acid to four regioisomeric epoxyeicosatrienoic acids (EETs), biologically active eicosanoids with many functions in organism. EETs are rapidly hydrolysed to less active dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (sEH). We investigated spatio-temporal expression pattern of CYP2C8, CYP2C9, CYP2?J2 and sEH in normal human placenta by immunohistochemical method. In the villous trophoblast, CYP2C8 was the most abundant protein. Its expression is higher than the CYP2C9 and CYP2?J2 in the cytotrophoblast in the embryonic stage of development and remains higher in syncytiotrophoblast of term placenta. Unlike to CYP2C8, CYP2C9 and CYP2?J2 expression decrease in term placenta. sEH expression increases with gestation age and is strictly limited to cytotrophoblast in embryonic and foetal stages of the development. Moreover, CYP2C8 shows more intensive staining than the other protein monitored in Hofbauer cells in villous stroma. Specific information regarding the exact role of EETs and DHETs functions in a normal placenta is still unknown. Based on CYP epoxygenases and sEH localization and well known information about the functions of placental structures during development, we suggest that these enzymes could play different roles in various cell populations in the placenta. As the placenta is absolutely crucial for prenatal development, arachidonic acid is essential part of human nutrient and CYP epoxygenases expression can be affected by xenobiotics, further investigation of the exact role of CYP epoxygenases, sEH, and their metabolites in normal pregnancy and under pathological conditions is needed.     

Yeast Saccharomyces cerevisiae devoid of Cu, Zn-superoxide dismutase as a cellular model to study acrylamide toxicity

Acrylamide is known as a cytotoxic and genotoxic component of starch-containing heat-processed food. We demonstrate that yeast Saccharomyces cerevisiae may be used as a cellular model to examine the biochemical mechanisms of acrylamide toxicity. We found that acrylamide causes impairment of growth of the yeast deficient in Cu, Zn-superoxide dismutase (Δsod1) in a concentration-dependent manner. This growth inhibitory effect is not due to cell death but to decreased cell vitality and proliferative capacity. Treatment of the Δsod1 yeast with acrylamide induced generation of increased reactive oxygen species and depletion of glutathione. The toxicity of acrylamide for yeast cells may be abolished by antioxidants (ascorbate, cysteine, N-acetylcysteine, glutathione and dithiothreitol) or lowering oxygen content in the atmosphere.