Mechanistic insights on the effect of crocin, an active ingredient of saffron, on atherosclerosis in apolipoprotein E knockout mice.

BACKGROUND: We investigated the effect of crocin treatment on atherosclerosis and serum lipids in apolipoprotein E knockout (ApoE-/-) mice, focusing on the expression of endothelial nitric oxide synthase (eNOS) and hypoxia-induced factor-1 alpha (HIF-1α). METHODS: Sixty-two animals were divided into two groups and randomly allocated to crocin (100 mg/kg/day) in drinking water or no crocin. All mice were maintained on standard chow diet containing 5% fat. Crocin was initiated at the 16th week of age and continued for 16 additional weeks. At 32 weeks of age, after blood sampling for plasma lipid determination and euthanasia, proximal aorta was removed and 3 µm sections were used to measure the atherosclerotic area and determine the expression of eNOS and HIF-1α by immunohistochemistry. RESULTS: Each group consisted of 31 animals (17 males and 14 females in each group). Crocin significantly reduced the atherosclerotic area (mm2 ± SEM) in treated mice compared to controls, both in males (0.0798 ± 0.017 vs. 0.1918 ± 0.028, P < 0.002, respectively) and females (0.0986 ± 0.023 vs. 0.1765 ± 0.025, P < 0.03, respectively). eNOS expression was significantly increased in crocin-treated mice compared to controls, both in males (2.77 ± 0.24 vs. 1.50 ± 0.34, P=0.004, respectively) and females (3.41 ± 0.37 vs. 1.16 ± 0.44, P=0.003, respectively). HIF-1α expression was significantly decreased in crocin-treated mice compared to controls, both in males (21.25 ± 2.14 vs. 156.5 ± 6.67, P < 0.001, respectively) and females (35.3 ± 7.20 vs. 113.3 ± 9.0, P < 0.01, respectively). No difference was noticed in total, low- and high-density lipoprotein cholesterol between treated and control mice. CONCLUSION: Crocin reduces atherosclerosis possibly by modulation of eNOS and HIF-1α expression in ApoE-/- mice without affecting plasma cholesterol.

Altered activity of xenobiotic detoxifying enzymes at menopause - A cross-sectional study.

Xenobiotic metabolism at menopause is an under-investigated topic, albeit women spend one-third of their life in the postmenopausal period. The present study examined the effect of menopause on the in vivo activities of CYP1A2, CYP2A6, xanthine oxidase (XO) and N-acetyltransferase-2 (NAT2) xenobiotic metabolizing enzymes. Enzyme activity was determined in 152 non-smoking volunteers following oral intake of a single dose of 200 mg caffeine and subsequent determination of caffeine metabolite ratios (CMRs) in a 6-h urine sample as follows: CYP1A2: (AFMU+1U+1X)/17U, CYP2A6: 17U/(17U + 17X), XO: 1U/(1U+1X) and NAT2: AFMU/(AFMU+1U+1X). CMRs among groups were analyzed using one-way ANOVA. Significantly lower CYP1A2 and higher CYP2A6 CMRs were observed in postmenopausal compared to premenopausal women and age-matched men. These changes could be attributed to menopause rather than chronological aging since an age-related effect was not observed in premenopausal women or men of any age group. XO CMRs were higher in postmenopausal women and men>50 compared to premenopausal women and men<50, respectively, suggesting an age-related increase in XO activity. No significant alterations were discerned in NAT2 CMRs, in either slow- or rapid-acetylators, indicating that menopause exerts minimal modulation of xenobiotics metabolized by this enzyme. This study provides evidence that the transition to menopause induces significant alterations in xenobiotic-metabolizing enzymes independent of chronological aging suggesting altered metabolism of pharmaceutical and environmental agents.

Effects of short-term saffron (Crocus sativus L.) intake on the in vivo activities of xenobiotic metabolizing enzymes in healthy volunteers.

Crocus sativus L., a perennial plant grown mainly around the Mediterranean and Iran, has many medicinal properties including anti-inflammatory, anti-depressive and cancer preventing properties. Aqueous herbal extracts may affect the activity of Phase I and II enzymes involved in xenobiotic metabolism. The present study was designed to determine whether C. sativus infusion alters the activity of CYP1A2, CYP2A6, XO and NAT2 enzymes in humans. Thirty-four healthy volunteers consumed infusion prepared from C. sativus stigmata for six days. Enzyme phenotyping was assessed in saliva and urine using caffeine metabolite ratios as follows: CYP1A2: 17X/137Χ (saliva) and CYP1A2: (AFMU+1U+1X)/17U, CYP2A6: 17U/(17U + 17X), XO: 1U/(1U+1X) and NAT2: AFMU/(AFMU+1U+1X) (urine). Following C. sativus intake, CYP1A2 index was reduced by ∼13.7% in saliva (before: 0.51 ±â€¯0.22, after: 0.44 ±â€¯0.14; p = 0.002) and ∼6.0% in urine (before: 3.81 ±â€¯1.20, after: 3.58 ±â€¯0.92; p = 0.054). CYP1A2 index was significantly reduced only in males (saliva, before: 0.65 ±â€¯0.22, after: 0.51 ±â€¯0.16; p = 0.0001; urine, before: 4.53 ±â€¯1.19, after: 4.03 ±â€¯0.87; p = 0.017) suggesting sexual dimorphism in CYP1A2 inhibition. There was no effect of C. sativus intake on CYP2A6, XO or NAT2 indices. Short-term consumption of C. sativus infusion is unlikely to result in significant herb-drug interactions involving the enzymes studied, with the exception of potential herb-CYP1A2 substrate interaction in males.

Dietary effects of Sideritis scardica "mountain tea" on human in vivo activities of xenobiotic metabolizing enzymes in healthy subjects.

Sideritis scardica(S. scardica) is an endemic plant of the Balkan Peninsula traditionally used as herbal tea for inflammation and gastric disorders. Aqueous herbal extracts may affect the activity of Phase I and II enzymes involved in xenobiotic metabolism. The purpose of the present study was to determine whether S. scardica decoction alters the activity of CYP1A2, CYP2A6, XO, NAT2 and UGT1A1/1A6 enzymes in humans. Fourteen healthy subjects consumed S. scardica decoction for six days. Enzyme phenotyping was assessed in saliva and urine using caffeine and paracetamol metabolite ratios as follows: CYP1A2: 17X/137X (saliva) and (AFMU+1U+1X)/17U, CYP2A6: 17U/(17U + 17X), XO: 1U/(1U+1X), NAT2: AFMU/(AFMU+1U+1X) and UGT1A1/1A6: glucuronidated/total paracetamol (urine). After S. scardica intake, CYP1A2 index was reduced by ∼16% and ∼8% in saliva (before: 0.54 ±â€¯0.18, after: 0.46 ±â€¯0.09; p = 0.08) and urine (before: 3.59 ±â€¯0.52, after: 3.67 ±â€¯0.78; p = 0.12), respectively. CYP2A6 index was significantly reduced only in males (before: 0.76 ±â€¯0.08, after: 0.67 ±â€¯0.07; p = 0.004), suggesting sexual dimorphism in CYP2A6 inhibition. There was no effect of Sideritis scardica treatment on XO, NAT2 or UGT1A1/1A6 indices. Usual consumption of the aerial parts of S. scardica decoction is unlikely to result in herb-drug interactions involving the enzymes studied, with the exception of potential herb-CYP2A6 substrate interaction in males.

Development and validation of a reversed-phase HPLC method for licarbazepine monitoring in serum of patients under oxcarbazepine treatment.

Licarbazepine is the pharmacologically active metabolite of oxcarbazepine, a drug indicated for the treatment of partial seizures and bipolar disorders. Several HPLC methods have been developed thus far but there is lack of control for interferences from antipsychotic drugs. The aim of the present study was to develop a simple, low-cost and reliable HPLC-UV method for the determination of licarbazepine in human serum in the presence of co-administered antiepileptic, antipsychotic and commonly prescribed drugs. Sample preparation consisted of a single protein precipitation step with methanol. Separation lasted ~9 min on a reversed-phase C18 column using a mobile phase composed of 50 mm sodium-dihydrogen-phosphate-monohydrate/acetonitrile (70:30, v/v) delivered isocratically at 0.9 mL/min and 30°C. Wavelength was 210 nm and calibration curve was linear with r2 0.998 over the range 0.2-50.0 µg/mL. Coefficient of variation was <5.03% and bias <-4.92%. Recovery ranged from 99.49 to 104.52% and the limit of detection was 0.0182 µg/mL. No interferences from the matrix or from antiepileptic, antipsychotic and commonly prescribed drugs were observed. The method was applied to serum samples of patients under oxcarbazepine treatment and proved to be a useful tool for the therapeutic drug monitoring of licarbazepine during monotherapy or adjunctive treatment of seizures or affective disorders.

Effects of peppermint tea consumption on the activities of CYP1A2, CYP2A6, Xanthine Oxidase, N-acetyltranferase-2 and UDP-glucuronosyltransferases-1A1/1A6 in healthy volunteers.

Peppermint leaves are widely used for the symptomatic treatment of digestive disorders. Previous studies have shown significant effects of its natural products on human enzyme activity; however, there is no study available concerning the effects of peppermint tea on metabolizing enzymes in humans. Aim of the present study was to investigate the effect of peppermint tea on CYP1A2, CYP2A6, Xanthine Oxidase (XO), N-acetyltranferase-2 (NAT2) and UDP-glucuronosyltransferases-1A1/1A6 (UGT1A1/1A6) activities in healthy subjects. Four males and five females consumed peppermint tea (2 g of dry leaves/200 mL water, twice daily) for six days. CYP1A2, CYP2A6, XO, NAT2 and UGT1A1/1A6 activities were determined before and at the end of the study period, using the following caffeine and paracetamol metabolic ratios: CYP1A2: 17MX/137MX (saliva) and (AFMU+1MU+1MX)/17MU (urine); CYP2A6: 17MU/(17MU + 17MX), XO: 1MU/(1MU+1MX), NAT2, AFMU/(AFMU+1MU+1MX) and UGT1A1/1A6 glucuronidated/total paracetamol, all determined in urine. NAT2 metabolic ratio was significantly reduced following peppermint consumption (0.15 ± 0.13 vs 0.14 ± 0.13; p < 0.05). CYP1A2 urine and saliva indices were reduced, yet not significantly, following peppermint consumption (urine: 3.17 ± 1.08 vs 2.91 ± 0.76, saliva: 0.56 ± 0.12 vs 0.50 ± 0.12; p > 0.05). Peppermint had no influence on CYP2A6, XO and UGT1A1/1A6 indices. Daily ingestion of peppermint tea may alter pharmacokinetics of clinically administered drugs and promote cancer chemoprevention through NAT2 inhibition.

Development and validation of a reversed-phase HPLC method for CYP1A2 phenotyping by use of a caffeine metabolite ratio in saliva.

CYP1A2 is important for metabolizing various clinically used drugs. Phenotyping of CYP1A2 may prove helpful for drug individualization therapy. Several HPLC methods have been developed for quantification of caffeine metabolites in plasma and urine. Aim of the present study was to develop a valid and simple HPLC method for evaluating CYP1A2 activity during exposure in xenobiotics by the use of human saliva. Caffeine and paraxanthine were isolated from saliva by liquid-liquid extraction (chlorophorm/isopropanol 85/15v/v). Extracts were analyzed by reversed-phase HPLC on a C18 column with mobile phase 0.1% acetic acid/methanol/acetonitrile (80/20/2 v/v) and detected at 273nm. Caffeine and paraxanthine elution times were <13min with no interferences from impurities or caffeine metabolites. Detector response was linear (0.10-8.00µg/ml, R(2) >0.99), recovery was >93% and bias <4.47%. Intra- and inter-day precision was <5.14% (n=6). The limit of quantitation was 0.10µg/ml and the limit of detection was 0.018±0.002µg/mL for paraxanthine and 0.032±0.002µg/ml for caffeine. Paraxanthine/caffeine ratio of 34 healthy volunteers was significantly higher in smokers (p<0.001). Saliva paraxanthine/caffeine ratios and urine metabolite ratios were highly correlated (r=0.85, p<0.001). The method can be used for the monitoring of CYP1A2 activity in clinical practice and in studies relevant to exposure to environmental and pharmacological xenobiotics.

Myxoid liposarcoma with cartilaginous differentiation: A case study with fish analysis and review of the literature.

Cartilaginous differentiation is rarely encountered in myxoid liposarcomas. To date, a small number of such cases have been described, and molecular or cytogenetic analysis was performed only in few of them. In the present study, we describe a primary myxoid liposarcoma with cartilaginous differentiation which arised in the left thigh of a 37-year-old man. Miscroscopically, the tumor consisted of areas with typical myxoid liposarcoma morphology and areas of sharply demarcated hyaline cartilage nodules. Here, we present the results of Fluorescence In Situ Hybridization (FISH) analysis that revealed the presence of FUS and DDIT3 gene rearrangements in both the liposarcomatous and cartilaginous components of the tumor. These findings confirm the neoplastic nature of the cartilage component in this rare tumor.

Evaluation of current prognostic and predictive markers in breast cancer: a validation study of tissue microarrays.

BACKGROUND: Tissue microarrays (TMAs) are an attractive alternative to analysis of whole sections (WS). For breast carcinomas, the recent recommendations for cut-offs (i.e. Ki67, H-score) have necessitated the re-evaluation of TMAs. MATERIALS AND METHODS: TMA results of immunohistochemistry (IHC) and Fluorescence in situ hybridization (FISH) testing for Estrogen receptors (ER), Progesterone receptors (PgR), Ki67 and HER2 were compared against the results of WS for 88 breast carcinomas. RESULTS: We found excellent agreement between the two methods for ER and PgR IHC evaluation, using the H-score (Kappa coefficient 0.972 and 0.9, respectively). There was also excellent correlation for HER2 IHC (Kappa coefficient 1) and amplification (Kappa coefficient 0.933). Furthermore, scoring of Ki67 was highly-correlated between TMAs and WS (Kappa coefficient 0.954). The latter excellent correlation has not, to our knowledge, been previously reported. CONCLUSION: For breast cancer, TMAs are an efficient and reliable alternative to the use of WS, using the currently recommended markers, evaluation protocols and cut-off values.

Validated analysis of HIF-1alpha expression in cancer cells using a controlled and comparative immunoassay.

The immunoreactivity of hypoxia inducible factor 1alpha (HIF-1alpha) has been considered a reliable indicator of the HIF-1 pathway activation in tissue hypoxia. However, HIF-1alpha immunoreactivity has been evaluated with different antibodies and heterogeneous protocols. The need to interpret contradictory findings requires, among other things, a comparison of the antibodies. This could be accomplished by using identical, well characterized antigenic targets and by decreasing the influence of other variables. We applied most of the commercially available antibodies, and an antibody developed in our laboratories, to the human cervical cancer HeLa cell line and tissue sections from a renal cell carcinoma systematically, and to other tumors selectively. The expression of HIF-1alpha in HeLa cells was induced by the hypoxia-mimetic DFO. Non-induced HeLa cells were used as 'genuine' negative controls in addition to routine ones. HeLa cells (both induced and not induced) were also examined by immunofluorescence and Western blotting. We found that the antibodies showed immunostaining patterns with remarkable qualitative and quantitative differences, an observation not emphasized in previous literature. Certain antibodies require careful application to avoid specificity issues, and others to avoid low sensitivity problems. Pairing certain antibodies can optimize evaluation of HIF-1alpha expression. Most previous immunohistochemical studies of HIF-1alpha have attempted to map hypoxic neoplastic tissues or to demonstrate hypoxia in studies of neoangiogenesis, rather than 'measuring' HIF-1alpha expression or activation, because this requires a validated immunoassay. Our study thus allows for the development of a controlled and comparative HIF-1alpha immunoassay, which could be valuable if HIF-1alpha becomes a therapeutic target.

Increased HIF-1 alpha immunostaining in psoriasis compared to psoriasiform dermatitides.

BACKGROUND: Expression of hypoxia inducible factor 1 (HIF-1) alpha can be linked to inflammation through reciprocal interactions with several cytokines. This finding is in accordance with the previously noted HIF-1 alpha overexpression in psoriatic lesional keratinocytes. METHODS: In this study we have employed a carefully selected panel of antibodies and quantitative morphometric analysis to compare HIF-1 alpha immunoreactivity in psoriatic biopsy samples vs. that in samples obtained from psoriasiform dermatitides. RESULTS: We found statistically significant HIF-1 alpha overexpression in psoriasis. Furthermore, we observed a previously unreported preferential localization of immunoreactive keratinocytes in the immediate vicinity of inflamed and elongated papillae. This pattern of immunoreactivity may partially account for the observed increase of HIF-1 alpha immunostaining in psoriasis. CONCLUSIONS: Overall, our findings imply that possible novel therapeutic intervention(s) on the HIF-1 alpha pathway may offer another approach in controlling the inflammatory activity of psoriatic lesions.

INCENP (inner centromere protein) is overexpressed in high grade non-Hodgkin B-cell lymphomas.

Inner centromere protein (INCENP) is a member of the Chromosomal Passenger Complex (CPC), which is a four member protein complex essential for proper completion of mitosis and cell division (cytokinesis). Inappropriate chromosomal segregation and cytokinesis due to deregulated expression of chromosome passenger proteins may lead to aneuploidy and cancer including lymphomas. According to our knowledge this is the first study investigating immunohistochemical expression of INCENP in lymphoma cases and cancer tissues in general. Our purpose was to characterize the expression of INCENP in cases of non-Hodgkin B-cell lymphomas, to compare the immunoreactivity between low and high grades and to evaluate the correlation between INCENP and MIB-1 labeling indices. We examined INCENP and MIB-1 immunoreactivity in paraffin sections of 55 samples of non-Hodgkin B-cell lymphomas, obtained from 55 patients, 31 men and 24 women. Thirty were of high grade and 25 were of low grade. Our results showed significantly higher nuclear immunohistochemical expression of INCENP in high grade B-cell lymphomas versus low grade ones. Also INCENP expression was significantly correlated with MIB-1 labeling index. Taken together our results point to a possible association between increased INCENP immunostaining and B-cell lymphoma aggressiveness and also stress the need for further investigating the expression of INCENP and other mitotic regulatory proteins in lymphomas and other malignant neoplasms.