Eugenol Reduced ΜPO, CD45 and HMGB1 Expression and Attenuated the Expression of Leukocyte Infiltration Markers in the Intestinal Tissue in Biliopancreatic Duct Ligation-Induced Pancreatitis in Rats.

Background and Objectives: Inflammation and dysregulation in the intestinal barrier function in acute pancreatitis (AP) trigger pancreatic lesions, systemic inflammatory response, and multiple organ dysfunction. Eugenol, as the main component of clove (Syzygium aromaticum), is known for its antioxidant and anti-inflammatory properties. We studied the potentially beneficial effect of eugenol in a rodent model of biliopancreatic duct ligation-induced AP. Materials and Methods: Rats were randomly divided into three groups: Sham, AP, and AP + eugenol (15 mg/kg/day). Serum TNFα, IL-6, IL-18, and resistin levels, as well as IL-6, TNFα, MPO, HMGB1, and CD45 tissue expression, were determined at various timepoints after the induction of AP. Results: Eugenol attenuated hyperemia and inflammatory cell infiltration in the intestinal mucosal, submucosal, and muscular layers. IL-6 and resistin serum levels were significantly reduced in the AP + eugenol group, while serum TNFα and IL-18 levels remained unaffected overall. TNFα pancreatic and intestinal expression was attenuated by eugenol at 72 h, while IL-6 expression was affected only in the pancreas. MPO, CD45, and HMGB1 intestinal expression was significantly reduced in eugenol-treated rats. Conclusions: Eugenol managed to attenuate the inflammatory response in the intestine in duct ligation-induced AP in rats.

Soluble E-cadherin as a diagnostic and prognostic marker in gastric carcinoma.

OBJECTIVE: Modifications in E-cadherin (E-Cad) expression are associated with dedifferentiation, progression, metastases and poor prognosis in many types of tumors. The aim of the present study was to identify a potential association of the pre- and post-operative soluble E-Cad levels (sE-Cad) with the clinicopathological parameters of patients with gastric cancer. PATIENTS AND METHODS: Serum sE-Cad levels were determined in 99 gastric cancer patients and 78 healthy volunteers using ELISA. RESULTS: Levels of sE-Cad were significantly increased in gastric cancer patients compared with these levels in healthy controls (p < 0.001). For the evaluation of the diagnostic significance of sE-Cad the area under the receiver operating characteristic (ROC) curve (AUC) was 0.835, while the optimal cut-off point of 9.9 microg/mL was determined to classify gastric cancer patients, which yielded sensitivity of 72.7%, specificity of 80.8% and accuracy of 76.3%. Poor differentiation (p = 0.009) and the presence of distant metastases (p < 0.001) were the two significant independent prognostic determinants for high sE-Cad levels in multivariate linear regression analysis. The preoperative levels of sE-Cad also proved helpful in classifying patients according to the choice treatment (curative versus palliative) (AUC, 0.656); when the optimal cut-off point was set at 17.60 microg/mL, the sensitivity was 57%, the specificity was 83% and accuracy was 75%. Survival was shorter in patients with increased sE-Cad (median, 7 months vs 39 months, p = 0.0002), although multivariate Cox regression analysis demonstrated a marginal prognostic significance of sE-Cad for survival (adjusted HR = 1.68, 95% CI = 0.93 to 3.02, p = 0.072). CONCLUSIONS: Serum sE-Cad levels could be considered as a diagnostic and prognostic marker in gastric cancer patients as well as a tool to select a treatment approach. The prognostic value of sE-Cad on overall survival requires further study.

Differential effects of cisplatin combined with the flavonoid apigenin on HepG2, Hep3B, and Huh7 liver cancer cell lines.

The potential of apigenin (APG) to enhance cisplatin's (CDDP) chemotherapeutic efficacy was investigated in HepG2, Hep3B, and Huh7 liver cancer cell lines. The presence of 20 µM APG sensitized all cell lines to CDDP treatment (degree of sensitization based on the MTT assay: HepG2>Huh7>Hep3B). As reflected by sister chromatid exchange levels, the degree of genetic instability as well as DNA repair by homologous recombination differed among cell lines. CDDP and 20 µM APG cotreatment exhibited a synergistic genotoxic effect on Hep3B cells and a less than additive effect on HepG2 and Huh7 cells. Cell cycle delays were noticed during the first mitotic division in Hep3B and Huh7 cells and the second mitotic division in HepG2 cells. CDDP and CDDP + APG treatments reduced the clonogenic capacity of all cell lines; however, there was a discordance in drug sensitivity compared with the MMT assay. Furthermore, a senescence-like phenotype was induced, especially in Hep3B and Huh7 cells. Unlike CDDP monotherapy, the combined treatment exhibited a significant anti-invasive and anti-migratory action in all cancer cell lines. The fact that the three liver cancer cell lines responded differently, yet positively, to CDDP + APG cotreatment could be attributed to variations they present in gene expression. Complex mechanisms seem to influence cellular responses and cell fate.

Bacterial translocation in a rat model of large volume hepatic radiofrequency ablation.

BACKGROUND: In this experimental study, we investigated the possibility of bacterial translocation, constituting a potential cause of infectious complications, after performing large volume hepatic radiofrequency ablation (RFA). MATERIALS AND METHODS: Wistar rats were subjected to RFA of the left median liver lobe (approximately 28.5% of the liver volume) after midline laparotomy. At 30 min, 24 h, 48 h, 72 h or 1 wk postoperatively, (1) blood samples were collected from the portal and systemic circulation for assessment of endotoxin concentration, (2) tissue specimens were excised from mesenteric lymph nodes, non-ablated liver, pancreas, spleen, kidneys, and lungs for bacterial culture, and (3) segments of terminal ileum were excised for histopathologic examination, morphometric analysis, and apoptotic and mitotic rate estimation. At 1 and 48 h, ileal mucosa was collected for oxidative state assessment on the basis of glutathione to glutathione disulfate (GSH/GSSG) ratio. RESULTS: Endotoxin levels were increased in both the portal and systemic circulation. Intestinal bacteria were isolated from all the organs at all time points. Ileal mucosa became gradually atrophic, with a decrease in villous height and density. There was an increase of crypt apoptotic rate, a decrease of GSH/GSSG ratio, while there were only mild signs of inflammation. CONCLUSIONS: Large volume liver RFA in the rat resulted to endotoxemia and translocation of intestinal bacteria to proximal and distal to the intestine organs at both the early and late post-RFA periods. The intestinal mucosa barrier was disrupted as suggested by ileal mucosal atrophy, increased crypt apoptosis, and induction of oxidative stress.

The Use of Pistacia Lentiscus Chia Resin Versus Omeprazole in Protecting Male Rats Peptic Mucosa Against Cold Restraint Stress.

INTRODUCTION: Peptic mucosal damage induced by acute stress is a serious cause of morbidity and mortality in critically ill patients. The study aimed to investigate the protective, antioxidant and anti-inflammatory effects of pretreatment with Chios mastic gum (CMG), a traditionally consumed herbal resin naturally deriving from the trunk of Pistacia Lentiscus var. Chia compared to Omeprazole, a standard medication used in the prevention and treatment of gastritis, against the effects of cold restraint stress (CRS) in rat gastric and colonic mucosa. METHODS: Twenty-one male Wistar rats were randomly assigned to three groups: Control (C), Omeprazole (O), and CMG (M), according to the pre-treatment regime, and were subjected to CRS at 40C for 3 hours. The gastric and colonic mucosal lesions were histologically assessed. ELISA measured blood concentrations of TNF-α, IL-1ß, peroxidase, superoxide dismutase (SOD) and total antioxidant capacity (TEAC). RESULTS: In both groups, O and M, gastric mucosal hyperemia, haemorrhagic infiltration and mucosal oedema, as well as colonic mucosal hyperaemia and haemorrhagic infiltration were significantly reduced compared to the controls (p<0.05). No significant differences were observed between Groups O and M. TNF-α levels were significantly lower in group M compared to Group O (p=0.013). IL-1ß levels were significantly depressed in groups M and O compared to control (p≤ 0.001). The activity of both peroxidase and SOD enzymes decreased in group M compared to group O (p= 0.043 and p=0.047 respectively) and the control (p=0.018 and p< 0.001 respectively). CONCLUSIONS: The natural Chios mastic gum is a promising nutritional supplement with protective properties to the peptic mucosa against CRS, exerting anti-inflammatory and antioxidant effects.

Antineoplastic and cytogenetic effects of chlorpromazine on human lymphocytes in vitro and on Ehrlich ascites tumor cells in vivo.

The inhibitory effect of phenothiazines in tumor growth and cancer cell proliferation in vitro and in vivo has been established. These reports motivated us to investigate the genotoxic, cytotoxic, and cytostatic potential of chlorpromazine, alone or in combination with mitomycin C, in vitro and in vivo. Sister chromatid exchange levels were assessed providing a quantitative index of genotoxicity. In-vitro studies were performed on human lymphocyte cultures and in-vivo studies involved Ehrilch ascites tumor (EAT) cells. An antitumour study was also conducted on the survival time and the ascitic volume in EAT-bearing Balb/C mice. The combination of chlorpromazine plus caffeine and mitomycin C exerted cytostatic and cytotoxic actions in human lymphocytes. The combination of chlorpromazine plus mitomycin C exerted cytostatic and cytotoxic actions in EAT cells, significantly increased the survival span of the mice inoculated with EAT cells, and suppressed the expected tumor growth increase. The findings of this basic study illustrate that high chlorpromazine concentrations increase chemotherapeutic effectiveness of mitomycin C. Chlorpromazine concentrations within the observed human plasma concentration range need to be tested along with antineoplastic agents in vitro for its synergistic action so as to evaluate a potential clinical application. Further investigation including other phenothiazines, biological systems, and cancer models is required.

Supplementation of melatonin protects human lymphocytes in vitro from the genotoxic activity of melphalan.

Melatonin (MLT) is a natural oncostatic factor of the human body as well as an antioxidant thus protects the nuclear DNA from oxidative damage. It also has the ability to reduce the side effects of various drugs when used as a combination therapy. The anti-neoplastic agent melphalan (MEL), which encompasses a number of side effects, is a strong alkylating agent and a potent inducer of sister chromatid exchanges (SCEs). The aim of the current in vitro study was to investigate the ability of MLT to reduce the genotoxic effect of MEL on normal human cultured peripheral lymphocytes. Cells were treated with both agents at various concentrations (MLT 100, 200 and 400 microM and MEL 330, 490 and 650 nM) and incubated for 72 h prior harvesting. The levels of cytostaticity, cytotoxicity and genotoxicity were qualitatively evaluated using the proliferation rate index, the mitotic index and the SCE methodology, respectively. Our results demonstrated the protective effect of MLT on cells treated with MEL in vitro. The greatest protective effect of MLT at 100 and 400 microM was illustrated against 330 nM of MEL in comparison with all other doses of MEL. These observations imply that MLT may be proved useful in reducing some of the toxic effects associated with certain classes of chemotherapeutic agents and other chemical and physical mutagens and carcinogens, acting both as an antioxidant-radical scavenger and a protective mechanism against cellular damage due to exposure to free radical-producing agents. It is essential to investigate substances with protective properties which are normally produced from the human body.

Evidence of increased chromosomal instability in infertile males after exposure to mitomycin C and caffeine.

AIM: To evaluate the genetic instability of 11 fertile and 25 infertile men. METHODS: The methodology of sister chromatid exchanges (SCEs) was applied to cultures of peripheral blood lymphocytes, and the levels of SCEss were analyzed as a quantitative index of genotoxicity, along with the values of the mitotic index (MI) and the proliferation rate index (PRI) as qualitative indices of cytotoxicity and cytostaticity, respectively. The genotoxic and antineoplastic agent, mitomycin C (MMC), and caffeine (CAF)--both well-known inhibitors of DNA repair mechanism--were used in an attempt to induce chromosomal instability in infertile men, so as to more easily detect the probable underlying damage on DNA. RESULTS: Our experiments illustrated that infertile men, compared with fertile ones, demonstrated a statistically significant DNA instability in peripheral blood lymphocytes after being exposed simultaneously to MMC and CAF. CONCLUSION: The current study showed vividly that there was genetic instability in infertile men which probably contributes to the development of an impaired reproductive capacity.

Mycotoxins' activity at toxic and sub-toxic concentrations: differential cytotoxic and genotoxic effects of single and combined administration of sterigmatocystin, ochratoxin A and citrinin on the hepatocellular cancer cell line Hep3B.

Food safety organizations indicate the likelihood of constant human and animal exposure to mycotoxin mixtures as a possible negative public health impact. Risk assessment demonstrates that certain mycotoxins of Aspergillus and Penicillium spp. are toxic and hold a significant genotoxic efficacy at nanomolar concentrations. The aim of the current study was to investigate the potential cytogenetic effects of sterigmatocystin (STER), ochratoxin A (OTA) and citrinin (CTN) alone or in combination, at pM to µΜ concentrations, on the human hepatocellular cancer cell line Hep3B. MTT reduction, mitotic divisions, cell cycle delays and sister chromatid exchange rates (SCE) were determined as endpoints of metabolic activity, cytotoxicity, cytostaticity, and genotoxicity, respectively. All mycotoxin treatments induce SCE rates from 10-12 M, while their cytotoxic and cytostatic potential varies. In PRI and MI assays, but not at MTT, STER alone or in combination with OTA + CTN appeared cytostatic and cytotoxic, even at 10-12 M, while CTN alone and all other combinations displayed substantial cellular survival inhibition in doses ≥ 10-8 M. Co-administration of STER + OTA or STER + CTN in concentrations ≤ 10-1 M, increased the MI and MTT activity, while it did not affect the PRI. Mycotoxin co-treatments revealed in general similar-to-additive or antagonistic genotoxic and cytotoxic effects. Our results for the first time describe that STER alone or in combination with OTA and/or CTN share a cytotoxic and cytogenetic potential even at picoMolar concentrations on human hepatoma cells in vitro.

Serum profiles of M30, M65 and interleukin-17 compared with C-reactive protein in patients with mild and severe acute pancreatitis.

BACKGROUND: Several studies state that a test of severity early in the course of acute pancreatitis is still needed. In this prospective study, an assay of the biomarkers M30 and M65 as well as of interleukin 17 (IL-17) is investigated. METHODS: One hundred and fifty patients and 70 controls were evaluated. The prognostic value of M30, M65 and their ratio M30/M65 is assessed by ELISA. The same method is used for the study of IL-17. RESULTS: At 24 h after symptom onset, the concentrations of M30 and M65 as well as their ratio, differed significantly in severe compared to mild disease (P = 0.016). C-reactive protein (CRP) was significantly higher (P < 0.001) in severe pancreatitis on the same day. The sensitivity of M65 to show severe acute pancreatitis at 24 h was 100% for values above the cut-off point of 428.15 U/l. The sensitivity of CRP was 100% as well. Concerning IL-17, its concentrations were higher in patients than in the control group (P < 0.001) in the first 24 h. CONCLUSIONS: Plasma concentrations of M65 and the M30/M65 ratio can be useful in predicting the severity of acute pancreatitis as early as 24 h after the onset of symptoms. The rates of IL-17 early in the course of acute pancreatitis are indicative of the disease.

The effects of apigenin on the expression of Fas/FasL apoptotic pathway in warm liver ischemia-reperfusion injury in rats.

BACKGROUND: The aim of this experimental study was to investigate the role of apigenin in liver apoptosis, in an experimental model of hepatic ischemia-reperfusion in rats. MATERIALS AND METHODS: Forty-eight Wistar rats (apigenin and control groups), 14 to 16 weeks old and weighing 220 to 350 g, were used. They were all subjected to hepatic ischemia by occlusion of the hepatic artery and portal vein for 45 minutes and reperfusion was followed for 60, 120, and 240 minutes. Apigenin was administrated intraperitoneally. Liver tissues were used for the detection of apoptosis by TUNEL assay and caspase 3 antibodies. Expression analysis of Fas/FasL genes was evaluated by real time PCR. RESULTS: The expression analysis of Fas and FasL genes was increasing during reperfusion (significantly in the group of 240 minutes of reperfusion). It was in the same group that apigenin decreased Fas receptor levels and inhibited apoptosis as confirmed by TUNEL assay and caspase 3 antibodies. CONCLUSIONS: The effects of apigenin in the Fas/FasL mediated pathway of apoptosis, in the hepatic ischemia-reperfusion, seem to have a protective result on the hepatic cell.

Time course changes of anti- and pro-apoptotic proteins in apigenin-induced genotoxicity.

BACKGROUND: Apigenin (4',5,7-trihydroxyflavone, AP), an active component of many medicinal Chinese herbs, exhibits anticancer properties in vitro and in vivo. This study aims to investigate the genotoxic, cytostatic, and cytotoxic effects of AP and time course changes in the levels of anti- and pro-apoptotic proteins involved in the DNA damage response in HepG2 cells. METHODS: The genotoxic potential of AP was determined by sister chromatid exchanges (SCEs) and chromosomal aberrations (CAs) analysis. The levels of cytostaticity and cytotoxicity were evaluated by the proliferation rate and mitotic indices, respectively. MTT was used to study cytotoxicity, while the induction of apoptosis and the expression of apoptosis-related proteins were determined by ELISA. RESULTS: At concentrations greater than 10 µM, AP decreased cell survival in a dose- (48 h: 10 vs. 20 µΜ, P < 0.001 and 20 vs. 50 µΜ, P = 0.005; 72 h: 10 vs. 20 µΜ, P < 0.001 and 20 vs. 50 µΜ, P = 0.001) and time-dependent manner (20 µΜ: 24 vs. 48 h, P < 0.001 and 48 vs. 72 h, P = 0.003; 50 µΜ: 24 vs. 48 h, P < 0.001 and 48 vs. 72 h, P < 0.001; 100 µΜ: 24 vs. 48 h, P < 0.001 and 48 vs. 72 h, P < 0.001). SCEs rates, cell proliferation, and mitotic divisions were also affected in a dose-dependent manner (P < 0.001). There was no change in the frequency of aberrant cells (1 µΜ ΑP: P = 0.554; 10 µM AP: P = 0.337; 20 µΜ AP: P = 0.239). Bcl-2 levels were reduced 3 h after AP administration (P = 0.003) and remained reduced throughout the 48 h observation period (6 h, P = 0.044; 12 h, P = 0.001; 24 h, P = 0.042; 48 h, P = 0.012). Bax and soluble Fas exhibited a transient upregulation 24 h after AP treatment. The Bax/Bcl-2 ratio was also increased at 12 h and remained increased throughout the 48 h observation period. CONCLUSION: AP exhibited dose-dependent genotoxic potential in HepG2 cells. The protein levels of sFas, Bcl-2, and Bax were affected by AP to promote cell survival and cell death, respectively.

Corticotropin-releasing hormone receptors mediate opposing effects in cholestasis-induced liver cell apoptosis.

CRH receptors are expressed in human and rat liver. The current study investigated the biological role of the CRH system in the hepatocellular apoptotic process and aimed to reveal the responsible molecular mechanisms. Using a rat experimental model of common bile duct surgical ligation leading to obstructive jaundice and cholestasis, liver apoptosis was induced in the hepatic parenchyma as confirmed by the elevated expression of the early apoptotic neoepitope M30. This effect was reversed by administration of the nonselective CRH antagonist astressin but not by the selective CRH(2) antagonist astressin2B, suggesting that antagonism of the endogenous CRH(1) blocked the cholestasis-induced apoptotic mechanism. No effect was observed in the noncholestasis controls. In our experimental model, early and late apoptosis-preventing markers were induced in parallel to apoptosis; elevated gene transcript levels of the anti-apoptotic bcl-2 were found by real-time PCR in the first postoperative day and increased serum hepatocyte growth factor levels were measured by ELISA in the third postoperative day. Selective CRH(2) antagonism reversed the elevated expression of bcl-2 and hepatocyte growth factor, suggesting that this receptor type mediated antiapoptotic actions of the endogenous CRH system, opposing the preapoptotic ones mediated by CRH(1). In conclusion, the present study indicated that the CRH neuroendocrine system regulates cholestasis-induced apoptosis in the hepatic parenchyma via receptor-specific pathways. These data may contribute to better understanding of the CRH biology and its pathophysiological significance in the periphery.

DNA damage and chromosomal aberrations in various types of male factor infertility.

OBJECTIVE: To understand and delineate the nature and level of DNA damage in association to semen parameters in infertile men. DESIGN: A prospective experimental study. SETTING: Alexandroupolis University General Hospital. PATIENT(S): Eleven fertile and 27 infertile men with various types of infertility. INTERVENTION(S): DNA damage was induced by addition of mitomycin C and caffeine to lymphocyte cultures. MAIN OUTCOME MEASURE(S): Sister chromatid exchange (SCE) levels were assessed in cultures providing a quantitative index of genotoxicity and chromosomal analysis was performed using G-banding and C-banding techniques. RESULT(S): Karyotyping analysis indicated chromosomal fragility, trisomic lines, and marker chromosomes in some infertile men. Double minute chromosomes were noticed in 11 infertile men and were positively correlated with elevated SCE levels. Necrospermia and varicocele, irrespectively of the degree of severity, were positively correlated with elevated SCE levels. CONCLUSION(S): Infertile men are prone to have DNA damage; the nature and level of DNA damage varies and is associated with semen parameters. The presence of double minute chromosomes alone is associated with increased double-stranded breaks and abnormal sperm concentration. This study could provide the basis to establish whether and through which process double minute chromosomes could be related to poor semen parameters and regulation of DNA repair.

Induction of CYP1A and cyp2-mediated arachidonic acid epoxygenation and suppression of 20-hydroxyeicosatetraenoic acid by imidazole derivatives including the aromatase inhibitor vorozole.

Cytochrome P450 (P450) enzymes metabolize the membrane lipid arachidonic acid to stable biologically active epoxides [eicosatrienoic acids (EETs)] and 20-hydroxyeicosatetraenoic acid (20-HETE). These products have cardiovascular activity, primarily acting as vasodilators and vasoconstrictors, respectively. EET formation can be increased by the prototype CYP1A or CYP2 inducers, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or phenobarbital (PB), respectively. We report here that imidazole derivative drugs: the anthelminthics, albendazole and thiabendazole; the proton pump inhibitor, omeprazole; the thromboxane synthase inhibitor, benzylimidazole; and the aromatase (CYP19) inhibitor vorozole (R76713, racemate; and R83842, (+) enantiomer) increased hepatic microsomal EET formation in a chick embryo model. Albendazole increased EETs by transcriptional induction of CYP1A5 and the others by combined induction of CYP1A5 and CYP2H, the avian orthologs of mammalian CYP1A2 and CYP2B, respectively. All inducers increased formation of the four EET regioisomers, but TCDD and albendazole had preference for 5,6-EET and PB and omeprazole for 14,15-EET. Vorozole, benzylimidazole, and TCDD also suppressed 20-HETE formation. Vorozole was a remarkably effective and potent inducer of multiple hepatic P450s at a dose range which overlapped its inhibition of ovarian aromatase. Increased CYP1A activity in mouse Hepa 1-6 and human HepG2 cells by vorozole and other imidazole derivatives demonstrated applicability of the findings to mammalian cells. The findings suggest that changes in P450-dependent arachidonic acid metabolism may be a new source of side effects for drugs that induce CYP1A or CYP2. They demonstrate further that in vivo induction of multiple hepatic P450s produces additive increases in arachidonic acid epoxygenase activity and can occur concurrently with inhibition of ovarian aromatase activity.