Caterina Loredana Mammola

Chelidonium majus is not hepatotoxic in Wistar rats, in a 4 weeks feeding experiment.

AIM OF THE STUDY Aerial parts of Chelidonium majus L. (Papaveraceae family) are traditionally used in the treatment of gallstones and dyspepsia, however several cases of hepatotoxicity are reported. In this work we evaluated the effects on liver function of a C. majus extract, obtained from the herbal material responsible for one case of hepatotoxicity. MATERIALS AND METHODS Experiments were performed in Wistar rats, after oral administration of doses corresponding to 1.5 and 3g/(kg day) of herbal drug, for 2 or 4 weeks. Blood samples were collected to perform biochemical analysis, whereas liver samples were used for histomorphological and immunohistochemical examination along with the determination of oxidative stress parameters. RESULTS No significant modification in animal body weight, food consumption, enzyme activities, hepatic histomorphology and MDA formation, at either time or dosage level. Conversely, C. majus induced a slight but significant decrease of GSH levels and SOD activity, especially at the high dose. CONCLUSIONS Our study suggests that C. majus, at doses about 50 and 100 times higher than those generally used in humans, does not alter hepatic function. However, the reduction in GSH levels and SOD activity suggests particular attention in use of C. majus or its preparations in situations (pharmacological treatments, physio-pathological conditions, etc.) that can compromise liver function.

Effects of Cimicifuga racemosa extract on liver morphology and hepatic function indices.

UNLABELLED Cimicifuga racemosa (black cohosh) is a herbaceous perennial plant, that has been traditionally used for a variety of ailments (dyspepsia, climacteric complaints, muscular rheumatisms, menstrual cramps). From laboratory and clinical studies, black cohosh seems to have a relatively good safety profile, even if a number of case reports of hepatotoxicity were a matter of recent concern. AIM A number of case reports indicated that C. racemosa could induce hepatotoxicity. We evaluated the effects of black cohosh extract on liver morphology, and on levels of various hepatic function indices in rats. METHODS Wistar rats received 300mg/kg/day of C. racemosa extract by gavage, for 30 days. Biochemical analysis of serum was conducted by an automated, random-access clinical chemistry analyzer. Liver samples were used for hystomorphological and immunohistochemical examination, for the detection of apoptosis (TUNEL assay), and for the determination of GSH level (spectrophotometrical analysis). RESULTS C. racemosa extract does not affect liver morphology and hepatic function indices, in rats. CONCLUSIONS On the basis of experimental data, the use of 300mg/kg/day of black cohosh appears quite safe in rats. Nevertheless, in humans the safety of C. racemosa should be further monitored, in terms of patient-related factors.

1H NMR-based urinary metabolic profiling reveals changes in nicotinamide pathway intermediates due to postnatal stress model in rat

The maternal separation protocol in rodents is a widely recognized model of early life stress allowing acute and chronic physiological consequences to be studied. An (1)H NMR-based metabolomic approach was applied to urines to evaluate the systemic metabolic consequences of maternal separation stress in female rats after the beginning of weaning and 4 weeks later when the rats were reaching adulthood. Furthermore, because maternal separation is considered as a model mimicking the inflammatory bowel syndrome, the lactulose/mannitol test was used to evaluate the influence of postnatal maternal separation on gut permeability and mucosal barrier function by (1)H NMR spectroscopy analysis of urine. The results showed no statistical differences in gut permeability due to maternal separation. The application of ANOVA simultaneous component analysis allowed the contributions of physiological adaptations to the animals development to be separated from the metabolic consequences due to postnatal stress. Systemic metabolic differences in the maternally separated pups were mainly due to the tryptophan/NAD pathway intermediate levels and to the methyladenosine level. Urinary NMR-based metabolic profiling allowed us to disentangle the metabolic adaptive response of the rats to postnatal stress during the animals growth, highlighting the metabolic changes induced by weaning, gut closure, and maturity.

Cassia angustifolia extract is not hepatotoxic in an in vitro and in vivo study.

Background:Cassia angustifolia L. (senna) is traditionally used as a laxative. Its major components are sennosides that are responsible for the laxative effect. Senna is recommended for the short-term treatment of acute constipation. Nevertheless people use its preparations as self-medication, often for long periods, to treat chronic constipation thus exposing themselves to adverse reactions. Most reactions were associated with hepatotoxicity. Aims: The present study was aimed to evaluate the toxicity of a C. angustifolia leafextract (standardized at 60% of sennosides) on rat liver cells and the long-term effects on liver functions, in Wistar rats. Methods: Cytotoxicity was assessed in a buffalo normal rat liver cell line (BRL-3A) by the trypan blue assay and the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide reduction test. In vivo effects were observed after oral administration of the extract for 4 or 8 weeks at doses of 12 and 58 mg/kg/day. At the end of treatment, animals were sacrificed, the postmortem examination was performed and serum was used for biochemical analysis. Liver samples were used for histomorphological and immunohistochemical examination along with the determination of oxidative stress parameters. Results and Conclusion: In BRL-3A cells, the extract was cytotoxic at concentrations that appear largely higher than those attainable in humans. In Wistar rats, the extract did not induce any significant change in all of the parameters tested. In summary, the present study indicates a lack of hepatotoxicity of senna at doses higher than those generally used in humans.

Chelidonium majus L. does not potentiate the hepatic effect of acetaminophen

AIM The present study assessed the ability of Chelidonium majus to potentiate the hepatic effect of a sub-toxic dose of acetaminophen, in rats. RESULTS C. majus, when administered alone, did not alter the liver function parameters in male, whereas an increase in fibrinogen level was found in female rats. Moreover, it did not affect the hepatic histomorphology in both male and female rats. The sub-toxic dose of acetaminophen induced: a significant increase in activated partial thromboplastin time in both genders, a focal hepatocellular necrosis with minor lymphocytes infiltrate and a slight but significant increase in total bilirubin, AST, and ALT in male rats, and in prothrombin time in female rats. The co-administration of C. majus did not increase the effects induced by acetaminophen, in both genders. CONCLUSIONS C. majus does not modify the hepatic effects of acetaminophen in our in vivo experimental model.

Differential Redox State Contributes to Sex Disparities in the Response to Influenza Virus Infection in Male and Female Mice

Influenza virus replicates intracellularly exploiting several pathways involved in the regulation of host responses. The outcome and the severity of the infection are thus strongly conditioned by multiple host factors, including age, sex, metabolic, and redox conditions of the target cells. Hormones are also important determinants of host immune responses to influenza and are recently proposed in the prophylaxis and treatment. This study shows that female mice are less susceptible than males to mouse-adapted influenza virus (A/PR8/H1N1). Compared with males, PR8-infected females display higher survival rate (+36%), milder clinical disease, and less weight loss. They also have milder histopathological signs, especially free alveolar area is higher than that in males, even if pro-inflammatory cytokine production shows slight differences between sexes; hormone levels, moreover, do not vary significantly with infection in our model. Importantly, viral loads (both in terms of viral M1 RNA copies and tissue culture infectious dose 50%) are lower in PR8-infected females. An analysis of the mechanisms contributing to sex disparities observed during infection reveals that the female animals have higher total antioxidant power in serum and their lungs are characterized by increase in (i) the content and biosynthesis of glutathione, (ii) the expression and activity of antioxidant enzymes (peroxiredoxin 1, catalase, and glutathione peroxidase), and (iii) the expression of the anti-apoptotic protein Bcl-2. By contrast, infected males are characterized by high expression of NADPH oxidase 4 oxidase and phosphorylation of p38 MAPK, both enzymes promoting viral replication. All these factors are critical for cell homeostasis and susceptibility to infection. Reappraisal of the importance of the host cell redox state and sex-related effects may be useful in the attempt to develop more tailored therapeutic interventions in the fight against influenza.

Multifaceted Roles of GSK-3 in Cancer and Autophagy-Related Diseases

GSK-3 is a ubiquitously expressed serine/threonine kinase existing as GSK-3α and GSK-3β isoforms, both active under basal conditions and inactivated upon phosphorylation by different upstream kinases. Initially discovered as a regulator of glycogen synthesis, GSK-3 is also involved in several signaling pathways controlling many different key functions. Here, we discuss recent advances regarding (i) GSK-3 structure, function, regulation, and involvement in several cancers, including hepatocarcinoma, cholangiocarcinoma, breast cancer, prostate cancer, leukemia, and melanoma (active GSK-3 has been shown to induce apoptosis in some cases or inhibit apoptosis in other cases and to induce cancer progression or inhibit tumor cell proliferation, suggesting that different GSK-3 modulators may address different specific targets); (ii) GSK-3 involvement in autophagy modulation, reviewing signaling pathways involved in neurodegenerative and liver diseases; (iii) GSK-3 role in oxidative stress and autophagic cell death, focusing on liver injury; (iv) GSK-3 as a possible therapeutic target of natural substances and synthetic inhibitors in many diseases; and (v) GSK-3 role as modulator of mammalian aging, related to metabolic alterations characterizing senescent cells and age-related diseases. Studies summarized here underline the GSK-3 multifaceted role and indicate such kinase as a molecular target in different pathologies, including diseases associated with autophagy dysregulation.

Autograft ossiculoplasty in cholesteatoma surgery: a histological study

Abstract Conclusion: The results of the present study reject the hypothesis that epithelial inclusions into the ossicles could cause cholesteatoma recurrences, but strongly suggest the performance of a safe cleaning procedure for ossicular remnants to make them usable in ossiculoplasty in patients with partially or non-encapsulated cholesteatoma. Objective: The aim of the study was to define, before any sort of cleaning procedure, if there is any epithelial inclusion inside the ossicles of patients with cholesteatoma and if the findings could be correlated with surgical aspect of cholesteatoma. Methods: The specimens used for this study comprised 19 mallei and 15 incudes, which were obtained intraoperatively from 24 patients. Each ossicle was grouped on the basis of the intraoperative aspect of the cholesteatoma as follows. Grade 1: 10 ossicles obtained from encapsulated cholestatoma, non-invasive, easily cleavable. Grade 2: 14 ossicles obtained from partially encapsulated cholesteatoma, non-invasive, not easily cleavable. Grade 3: 10 ossicles obtained from non-encapsulated cholesteatoma, invasive, not cleavable. Two stapes and one malleus were taken from patients who underwent middle ear surgery for conductive hearing loss and were used as controls. The ossicles were examined histopathologically after removal. Results: Our results do not show any epithelial inclusion inside the ossicles independently from the macroscopic aspect or growing aggressiveness of cholesteatoma. In addition there was no infiltration of inflammatory cells in grade 1, but it was present in one incus (7.1%) of grade 2 and in five ossicles (50%) of grade 3. In ossicles of grade 3 up to four layers of epithelial cells were found on the surface of the ossicles.

β-caryophyllene and low-doses of doxorubicin against liver cancer cells: a “metronomic chemotherapy”

Cholangiocarcinoma and hepatocellular carcinoma are primary liver cancers, both representing a growing challenge due to their increasing morbidity and mortality. A “metronomic chemotherapy”, consisting of the repeated administration of low and/or continuous doses of anti-neoplastic drugs, represents an alternative approach to the standard chemotherapy [1]. Numerous natural substances exhibited in vitro chemosensitizing features: in particular, the natural sesquiterpene β-caryophyllene (CRY) has been proved to increase the cytotoxicity of doxorubicin (DOXO) in leukemic cells [2]. Hence, our aim has been to evaluate the ability of CRY to enhance the efficacy of low-dose DOXO in human liver cancer cells, by applying a metronomic protocol. To this end, human liver HepG2 and CCA cells have been used as models of hepatocellular carcinoma and cholangiocarcinoma. The metronomic protocol was based on a 2h low-time exposition to the test substances, followed by 72h incubation for restoring. This scheduling has been applied 3 times and cytotoxicity was measured by MTT assay. Both the substances alone (CRY 1-100 μg/ml; DOXO 1-500 μg/ml) and the combination of DOXO with a nontoxic concentration of CRY were assessed. We found that the repeated treatments with low concentrations produced a significant potentiation (about 30 %) of DOXO cytotoxicity in HepG2. The combination with CRY increased the DOXO activity, reaching a 70 % inhibition of cell viability at 50 μg/ml after 2 repeated treatments. Similar effects were found in CCA, although repeated treatments induced no additional potentiation. These results highlight a possible role of CRY as a chemosensitizing agent for DOXO-based chemotherapy of liver cancer.

Natural sesquiterpenes β-caryophyllene and β-caryophyllene oxide as chemopreventive agents for cholangiocarcinoma

Cholangiocarcinoma (CCA) represents a particular type of liver cancer originating from the epithelial cells lining the intrahepatic and extrahepatic biliary tree. It is often clinically silent until it becomes an advanced disease with obstructive symptoms and a poor prognosis for patients [1]. In this context, searching for alternative therapeutic strategies becomes a pivotal goal in the battle against liver cancer. In present research, β-caryophyllene (CRY) and β-caryophyllene oxide (CRYO), two natural sesquiterpenes with protective properties [2], have been investigated for their ability to inhibit the growth of the human Mz-ChA-1 cholangiocarcinoma cells from extrahepatic bile ducts and the H69 non-malignant cholagiocytes. The cells were exposed to the test compounds for 24 h, then the cytotoxicity was measured by MTT assay [3]; doxorubicin was included as a standard cytotoxic agents. Both sesquiterpenes reduced the cholangiocytes proliferation. Particularly, CRY produced a marked cytotoxicity in the Mz-ChA-1cells at concentrations lower than that required for CRYO. Surprisingly, the antiproliferative effects produced by both CRY and CRYO were higher than that of the standard doxoru- bicin. In the non-malignant cholangiocytes, in spite of a low cytotoxicity of CRYO, CRY reduced the cell proliferation already at lower concentrations, acting in a similar way to doxorubicin. Being CRYO an epoxide analogue of CRY, the different cytotoxicity found could be due to their structural features and bioavailability. Present results encourage further studies on CRY and CRYO as chemopreventive agents for cholangiocarcinoma. This work was supported by grants from Sapienza University Project 2014. Dr. Antonella Di Sotto was financed by “Enrico and Enrica Sovena” Foundation.