Toxic essential oils, part VI: Acute oral toxicity of lemon balm (Melissa officinalis L.) essential oil in BALB/c mice.

Despite being renowned for its volatiles, the data on the toxicity of the essential oil of lemon balm (Melissa officinalis L., Lamiaceae) is rather limited compared to its solvent/water-soluble extractibles. In this study, the aerial parts essential oil of M. officinalis, with over 130 constituents identified herein, 26 of which detected for the first time, was investigated for acute oral toxicity in BALB/c mice. The oil, composed of predominantly monoterpene aldehydes, citronellal (21.2-21.8%), neral (17.8-18.4%), and geranial (22.9-23.5%), which were assayed in parallel with the oil in some tests, induced significant changes in animal behavior, as well as altered biochemical parameters reflecting liver and kidney functions. Different pathological changes in the stomach, duodenum, liver, and kidneys were detected when the oil was administered in doses higher than 1 g kg-1. A depletion in the liver/kidney antioxidant capacities and an increased rate of lipid peroxidation was noted for animals treated with lemon balm oil. The calculated value of the oral LD50 in BALB/c mice (2.57 g kg-1) infers that the essential oil is only moderately toxic.

The Quantitative ER Immunohistochemical Analysis in Breast Cancer: Detecting the 3 + 0, 4 + 0, and 5 + 0 Allred Score Cases.

The currently used immunohistochemical approach in determining the estrogen receptor (ER) positivity of breast cancers (BCs) is inherently subjective and additionally limited by its semi-quantitative nature. The application of software in the analysis of digitized slide images may overcome some of these limitations. However, the utilization of such an approach requires that the entire staining procedure is standardized. BACKGROUND AND OBJECTIVES: We aimed to establish a procedure for the photometric and morphometric analysis of BC immunohistochemical parameters that can possibly be used for a diagnostic purpose that is in line with the current semi-quantitative scoring system. MATERIALS AND METHODS: Semi-quantitative analysis of ER-stained tissue sections was performed following the Allred scoring system guidelines. The quantitative analysis was performed in ImageJ software after color deconvolution. The quantitative analysis of 66 cases of invasive lobular BC included: Percent of ER-positive cells, average nuclear coloration intensity, and the quantitative ER score. The percent of ER-positive tumor cells was counted using a standard grid overlay, while optical density (0.0-1.0) was measured within each nucleus at the grid points. RESULTS: A statistical analysis revealed a significant positive correlation (r = 0.886, p < 0.001) between the subjective semi-quantitative and quantitative ER scores, with a large effect size (d = 3.8215). We observed strong statistically significant correlations between individual parameters of the total ER score, percentage of ER-positive nuclei, and color intensity, obtained by the two independent methods. CONCLUSIONS: Additionally, besides excluding subjectivity, the up to now unreported cases of 3 + 0, 4 + 0, and 5 + 0 Allred scores were detected only by the application of the proposed quantitative approach.

Differences in Angular Photogrammetric Soft-Tissue Facial Characteristics among Parents and Their Offspring.

Background and objectives: The objective of this study was to determine if the angular photogrammetric analysis of soft-tissue characteristics can determine similarities between parents and their offspring in the Serbian population. Materials and Methods: A total of 15 families (52 participants) met the participation criteria of this study and their facial profile images were analyzed using the ImageJ software. Subjects were divided into groups of mothers and fathers and four groups of children (divided according to their age and gender). In total, twelve angular measurements were made on the standardized digital images of the profiles of the participants and the obtained data were compared using one-way ANOVA. Results: The obtained results showed that there were statistically significant differences in the values of the nasal and cervicomental angles, as well as the angle of the total facial convexity, between the group of fathers, on one side, and groups of male/female children, on the other. Conclusions: This work represents the first photogrammetric analysis of facial soft-tissue characteristics of children and adults in the Serbian population. The data suggest that there are much more similarities between the facial soft-tissue angles of fathers and their male offspring. Furthermore, mothers tend to have statistically insignificant differences in angle sizes, compared to both male and female offspring.

Two different melatonin treatment regimens prevent an increase in kidney injury marker-1 induced by carbon tetrachloride in rat kidneys.

Acute kidney injury is a frequent disorder that can be mimicked by the application of different nephrotoxic agents, including carbon tetrachloride (CCl4), where kidney injury marker-1 (KIM-1) has been recognized as a highly specific marker. Melatonin is one of the most powerful natural antioxidants and has numerous beneficial properties. We evaluated the nephroprotective potential of 2 melatonin treatment regimens (pre- and post-intoxication) in a CCl4-induced acute kidney injury model based on the standard serum parameters, kidney tissue antioxidative capacity, KIM-1 levels, and kidney tissue morphological changes. The two treatment regimens were found to preserve kidney function, as judged from the evaluated standard serum parameters. Only when administered after the intoxication, melatonin preserved total kidney antioxidant capacity; pre-treatment melatonin only preserved reduced glutathione levels. An increase in tissue KIM-1 level was found to be prevented by both treatment regimens, which correlated with the morphological changes seen in the kidney tissues of animals treated with melatonin and CCl4. The findings of our study are in agreement with the known actions of melatonin in relieving kidney tissue oxidative burden, but also contribute to the understanding of its action by preventing an increase in KIM-1.

Effects of melatonin on oxidative stress parameters and pathohistological changes in rat skeletal muscle tissue following carbon tetrachloride application.

Animal models demonstrating skeletal muscle (SM) disorders are rarely investigated, although these disorders accompany liver disorders and can occur during prolonged exercise/training. In cases of SM disorders exogenous antioxidants, such as melatonin, could help by generally improving tissues antioxidant capacities. We aimed to analyze the potential of melatonin in preventing biochemical and structural changes in rat biceps muscle (BM) occurring after an acute exposure to carbon tetrachloride (CCl4). Biceps muscles obtained from male Wistar rats belonging to different experimental groups were biochemically (determination of tissue MDA, total antioxidant capacity, GSH, CAT, SOD and GPx activities) and pathologically analyzed. Also, serum levels of potassium, LHD and CK were analyzed in all experimental animals. The obtained results were statically compared with those from vehicle-treated control group. The applied melatonin prevented potassium and intracellular enzyme leakage (CK and LDH) that was induced by CCl4, as well as an increase in tissue MDA. From a panel of determined oxidative stress parameters melatonin was able to statistically significantly prevent changes in total antioxidative capacity and in CAT, SOD and GPx activities induced by CCl4. Microscopic analysis of BM from the animals exposed to CCl4 revealed significant muscle fiber disorganization and massive inflammatory cell infiltration. All these changes were significantly ameliorated in the group that received melatonin prior to CCl4. Changes in serum and tissue biochemical parameters accompanied the observed pathological changes, which demonstrated a significant influence of melatonin in preventing skeletal muscle damage induced by CCl4.

Immunohistochemical features of multifocal and multicentric lobular breast carcinoma.

PURPOSE: Patient age, tumor size, axillary lymph node status, expression of estrogen, progesterone and HER2 receptors, as well as the histological grade, are widely accepted prognostic and predictive parameters in breast carcinoma. In invasive lobular carcinoma (ILC), the prognostic factors, such as nodal status and tumor size are believed to be the most valuable ones. The aim of this was set to determine the possible linkage between MFity/MCity of ILC and immunohistochemical predictive (ER, PR, HER2) and prognostic parameters (BRCA1, p53, E-cadherin, Ki-67). MATERIAL/METHODS: Out of 2486 cases diagnosed with BC from south-eastern Serbia, the presence ILC was noted in 334 cases. Immunohistochemical characterization of predictive and prognostic parameters in ILC was done. RESULTS: The occurrence of multifocal (MFC) and multicentric carcinoma (MCC) was observed in 18.9% of ILC. Bilateral (BL) BCs were found to be statistically significantly more frequent in younger women, compared to those with MFC, MCC or nonMFC/MCC/BL. No significant correlation was found between MFC/MCC and the presence of axillary lymph node metastases, and expression of immunohistochemical predictive and prognostic parameters. Based on literature data and the findings from the current work, MFity/MCtiy might represent negative morphologic prognostic parameters in ILC. CONCLUSIONS: In 334 cases analyzed, no statistically significant correlations were observed between MF/MC and the expression of immunohistochemical predictive and prognostic parameters.

Seven-year survey of classical and pleomorphic invasive lobular breast carcinomas in women from southeastern Serbia: Differences in clinicopathological and immunohistochemical features.

PURPOSE: The occurrence of different variants of invasive lobular carcinoma (ILC) of the breast is variable. For example, the pleomorphic variant of ILC has an incidence of around 5%; however, the number of analyzed cases of ILC is shadowed by the number of ductal type carcinoma (IDC). Thus, we aimed to analyze the classical and pleomorphic ILCs in women from southeastern Serbia. METHODS: Analyzed were 296 cases (11.91%) diagnosed with ILC, out of 2486 cases of all breast cancers (BCs), during a 7-year period (2005-2011) from southeastern Serbia. The differences in clinicopathological and immunohistochemical features (estrogen receptor/ER, progesterone receptor/ PR, HER-2, Ki-67, BRCA-1, p53 and E-cadherin) of these cases of ILCs were assessed and compared. RESULTS: Pleomorphic ILC occurred relatively rarely compared to other variants, however almost one fifth of the ILC cases were pleomorphic. No statistically significant correlation was found between patient age, tumor stage and the presence/absence of multifocality (MFC), multicentricity (MCC) and bilaterality (BL) on one side, and ILC variant on the other. Only the expression of two prognostic and predictive immunohistochemical markers, important for endocrine therapy, ER and PR, showed significant correlation with the ILC variant. CONCLUSIONS: Although higher tumor stage, incidence of multicentricity, overexpression of HER2 and higher p53 positivity were deemed to be characteristic of pleomorphic ILC, in our study that included a much larger number of cases than previous studies did, such correlations were not observed. Thus, it appears that the only two features of pleomorphic ILCs is absence of ER and PR positivity.

Evaluation of pathological parameters and morphometric data of desmoplastic lobular breast carcinoma.

BACKGROUND: Invasive lobular breast cancer (ILC) is the second most frequent form of breast cancer. While cancer cells are regularly investigated, tumor stroma represents a highly unexplored field. AIMS: The aim of this study is to perform a detailed investigation of clinical, immunohistochemical, and morphometric characteristics of desmoplastic (D) and nondesmoplastic (ND) ILC. MATERIALS AND METHODS: This study included twenty cases of ILC that were divided into two groups designated as D and ND groups. Medical histories and diagnosis data were obtained from the archives of the Center of Pathology, Clinical center Nis (Serbia). Morphometric analysis of hematoxylin and eosin stained slides was performed using ImageJ software, and the obtained data were further statistical processed. RESULTS: Statistical analyses of the data revealed that no significant differences between D and ND groups when patient age, estrogen receptor (ER), and progesterone receptor (PR) expressions and morphometrical parameters (such as the distance between groups of cancer cells and nucleocytoplasmic ratio) were compared. However, D and ND groups statistically, significantly differed in the occurrence of axillary lymph node metastasis, and when the ER and PR data were included, in certain nuclear parameters (cell/nucleus area, perimeter, Feret's diameter, and circularity). CONCLUSIONS: Desmoplastic stroma was observed more frequently in patients without axillary lymph node metastases, whereas the expression of ER and PR had no influence on its development. According to the measured morphometric parameters larger cells/nuclei belonged to ND group.

Effect of two esters of N-methylanthranilic acid from Rutaceae species on impaired kidney morphology and function in rats caused by CCl4.

AIMS: Herein we investigated the potential protective effects of methyl N-methylanthranilate (MA) and isopropyl N-methylanthranilate (IA), two naturally occurring plant constituents from Rutaceae taxa, in a rat model of acute intoxication with carbon tetrachloride (CCl4) by tracking changes in kidney tissue morphology and function. MAIN METHODS: The antioxidant capacity of IA and MA was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid radical cation (ABTS(+)) assays and superoxide-scavenging test. Wistar rats were treated daily with MA and IA for seven days in a dose of 200mg/kg. Twenty-four hours after a CCl4 injection, rats were sacrificed and blood samples were used for the evaluation of urea and creatinine. Kidney tissue specimens were stained with hematoxylin and eosin, periodic acid-Schiff and Jones stain and evaluated for morphological changes. Quantification of structural changes determined by histological analysis of kidney tissue was assessed by a morphometric analysis of glomeruli using ImageJ software. KEY FINDINGS: IA and MA applied in high doses on their own did not cause any significant damage to kidney tissue. A pretreatment with MA prior to the administration of CCl4 significantly prevented the increase of serum levels of decreased kidney function markers, while that of IA did not. Histopathological evaluation of the kidneys also revealed that MA reduced the incidence of kidney lesions. SIGNIFICANCE: Our experiments showed that methyl-, and not isopropyl-, N-methylanthranilate possesses a protective potential against CCl4-induced kidney damage in rats. The results are of interest due to the presence of natural or synthetic methyl N-methylanthranilate in the human diet and their potent analgesic properties.

Methyl and isopropyl N-methylanthranilates attenuate diclofenac- and ethanol-induced gastric lesions in rats.

AIMS: Two natural alkaloids, methyl (M) and isopropyl (I) N-methylanthranilates, with recently demonstrated significant pharmacological activities, were assayed for their possible overall effect on intact gastric mucosa and their protective properties towards the onset of gastric lesions induced by diclofenac (a non-steroidal anti-inflammatory drug, NSAID) or ethanol. MAIN METHODS: The influence of I and M on gastric mucosa integrity was assessed by oral administration in doses of 200mg/kg. The gastroprotective action of I and M in doses of 50, 100 and 200mg/kg was analyzed in the diclofenac and ethanol-induced gastric lesion models in rats. After the treatment, the stomachs of the animals were analyzed (captured by a digital camera). Ulcer scoring, morphometric and histopathological analyses of the stomachs were done. KEY FINDINGS: The oral application of these compounds on their own, even in quite high doses (200mg/kg) did not induce gastric lesions. Both alkaloids exerted a very strong antiulcer activity, even in low doses (50mg/kg), by decreasing the number of lesions caused by the application of either diclofenac or ethanol, eliminating them completely or reducing them to a form of mucosal hyperemia. SIGNIFICANCE: Their possible mechanism of action was discussed and due to their many positive properties including anxiolytic, antidepressant, antinociceptive, anti-inflammatory and gastroprotective activities, as well as a cheap and simple synthetic route for their preparation, methyl and isopropyl N-methylanthranilates, both alike, might represent a cost effective alternative sought for in the treatment of peptic ulcers and/or new safer NSAIDs for pain management.

Toxic essential oils. Part II: chemical, toxicological, pharmacological and microbiological profiles of Artemisia annua L. volatiles.

Botanical drugs based on Artemisia annua L. (Asteraceae) are important in the treatment of malaria. Alongside with artemisinin, this aromatic species produces high and variable amounts of other chemicals that have mostly unknown biological/pharmacological activities. Herein, we have studied the toxicological/pharmacological profile of volatile constituents of a Serbian population of A. annua. Fifty-eight components were identified, among them, artemisia ketone (35.7%), α-pinene (16.5%) and 1,8-cineole (5.5%) were the most abundant ones. Significant variability of A. annua volatile profile was confirmed by means of agglomerative hierarchical cluster analysis indicating the existence of several different A. annua chemotypes. In an attempt to connect the chemical profile of A. annua oil with its biological/toxicological effects, we have evaluated in vivo and/or in vitro toxicity (including hepato- and nephrotoxicity/protection), antinociceptive, antioxidant (DPPH, ABTS and superoxide radical scavenging activity assays), enzyme inhibiting (protein kinase A and α-amylase) and antimicrobial potential of A. annua oil and of its constituents. Our results revealed that the beneficial properties of A. annua botanical drugs are not limited only to their antimalarial properties. Taking into account its relatively low toxicity, the usage of A. annua volatiles (at least of the herein studied population) does not represent a health risk.

Exploitation of the antioxidant potential of Geranium macrorrhizum (Geraniaceae): hepatoprotective and antimicrobial activities.

In this study we evaluated in vitro (radical scavenging) and in vivo (hepatoprotective effect) antioxidant activities and antimicrobial properties of the extracts of the above- and underground parts of Geranium macrorrhizum L. (Geraniaceae), an ethnopharmacologically renowned plant species. The antioxidant activity and total phenol and flavonoid contents of four different solvent extracts were evaluated by seven different methods. The methanol extracts, administered i.p. to rats (120-480 mg/kg), were evaluated for hepatoprotective activity in a CCl4-induced hepatotoxicity model. The same extracts were tested for antimicrobial activity against seven bacterial and two fungal species. The administered methanol extracts with the highest antioxidant potential showed a significant dose-dependent hepatoprotective action against CCl4-induced liver damage in both decreasing the levels of liver transaminases and bilirubin and in reducing the extent of morphological malformations of the liver. The leaf methanol extract displayed a very strong antibacterial activity, especially against Staphylococcus aureus, with low minimal inhibitory and bactericidal concentrations. These results justify the frequent use of this plant species in folk medicine. Besides the known astringent effect, one can expect that the observed antimicrobial activity against several human pathogens contributes to the wound healing properties of this plant.