In vitro toxicological assessment of PhSeZnCl in human liver cells.

Phenylselenenylzinc chloride (PhSeZnCl) is an air-stable selenolate, easily synthesizable through oxidative insertion of elemental zinc into the Se-halogen bond of the commercially available phenylselenyl chloride. PhSeZnCl was shown to possess a marked GPx-like activity both in NMR and in vitro tests, and to effectively react with cellular thiols, and was supposed for a potential use in the chemotherapy of drug-resistant cancers. However, activity of PhSeZnCl in hepatic cells has never been tested before now. In this in vitro approach, we evaluated the cytotoxic, genotoxic, and apoptotic activities, as well as the effects on cell cycle of PhSeZnCl in two preclinical hepatic models, namely HepG2 and HepaRG cells. Results showed that cell viability of HepG2 and HepaRG cells decreased in a dose-dependent manner, with a more marked effect in HepG2 tumour cells. Moreover, treatment with 50 µg/mL PhSeZnCl caused an increase of primary DNA damage (4 h) and a statistically significant increase of HepG2 cells arrested in G2/M phase. In addition, it altered mitochondrial membrane potential and induced chromosomal DNA fragmentation (24 h). In HepaRG cells, PhSeZnCl was able to determine a cell cycle-independent induction of apoptosis. Particularly, 50 µg/mL induced mitochondrial membrane depolarization after 24 h and apoptosis after 4 h treatment. Futhermore, all PhSeZnCl concentrations tested determined a significant increase of apoptotic cells after 24 h. Apoptosis was also highlighted by the detection of active Caspase-3 by Western Blot analysis after 24 h exposure. In conclusion, this first toxicological assessment provides new insights into the biological activity of PhSeZnCl in preclinical hepatic models that will be useful in future safety assessment investigation of this compound as a potential pharmaceutical. Supplementary Information: The online version contains supplementary material available at 10.1007/s43188-022-00148-y.

Cytotoxicity and Genotoxicity of Senecio vulgaris L. Extracts: An In Vitro Assessment in HepG2 Liver Cells.

Senecio vulgaris L. is a herbaceous species found worldwide. The demonstrated occurrence of pyrrolizidine alkaloids in this species and its ability to invade a great variety of habitats result in a serious risk of contamination of plant material batches addressed to the herbal teas market; this presents a potential health risk for consumers. In light of the above, this work aimed to assess the cytotoxic and genotoxic activity of S. vulgaris extracts in HepG2 cells. Dried plants were ground and extracted using two different methods, namely an organic solvent-based procedure (using methanol and chloroform), and an environmentally friendly extraction procedure (i.e., aqueous extraction), which mimicked the domestic preparation of herbal teas (5, 15, and 30 min of infusion). Extracts were then tested in HepG2 cells for their cytotoxic and genotoxic potentialities. Results were almost superimposable in both extracts, showing a slight loss in cell viability at the highest concentration tested, and a marked dose-dependent genotoxicity exerted by non-cytotoxic concentrations. It was found that the genotoxic effect is even more pronounced in aqueous extracts, which induced primary DNA damage after five minutes of infusion even at the lowest concentration tested. Given the broad intake of herbal infusions worldwide, this experimental approach might be proposed as a screening tool in the analysis of plant material lots addressed to the herbal infusion market.

Pro-Apoptotic Activity of Artichoke Leaf Extracts in Human HT-29 and RKO Colon Cancer Cells.

(1) Background: Cynara cardunculus L. subsp. scolymus (L.) Hegi, popularly known as artichoke, is an herbaceous plant belonging to the Asteraceae family. Artichoke leaf extracts (ALEs) have been widely used in traditional medicine because of their hepatoprotective, cholagogic, hypoglycaemic, hypolipemic and antibacterial properties. ALEs are also recognized for their antioxidative and anti-inflammatory activities. In this study, we evaluated the cytotoxic, genotoxic, and apoptotic activities, as well as effect on cell growth of ALEs on human colon cancer HT-29 and RKO cells. HT-29 and RKO cells exhibit a different p53 status: RKO cells express the wild-type protein, whereas HT-29 cells express a p53-R273H contact mutant. (2) Methods: Four different ALEs were obtained by sequential extraction of dried artichoke leaves; ALEs were characterized for their content in chlorogenic acid, cynaropicrin, and caffeoylquinic acids. HT-29 and RKO cells were used for in vitro testing (i.e., cytotoxicity and genotoxicity assessment, cell cycle analysis, apoptosis induction). (3) Results: Two out of the four tested ALEs showed marked effects on cell vitality toward HT-29 and RKO tumour cells. The effect was accompanied by a genotoxic activity exerted at a non-cytotoxic concentrations, by a significant perturbation of cell cycle (i.e., with increase of cells in the sub-G1 phase), and by the induction of apoptosis. (4) Conclusions: ALEs rich in cynaropicrin, caffeoylquinic acids, and chlorogenic acid showed to be capable of affecting HT-29 and RKO colon cancer cells by inducing favourable biological effects: cell cycle perturbation, activation of mitochondrial dependent pathway of apoptosis, and the induction of genotoxic effects probably mediated by the induction of apoptosis. Taken together, these results weigh in favour of a potential cancer chemotherapeutic activity of ALEs.

Green Tea Consumption and Risk of Breast Cancer and Recurrence-A Systematic Review and Meta-Analysis of Observational Studies.

Breast cancer (BC) is the most common cancer in women and several factors are involved in its onset. Green tea (GT) has been shown to have potential beneficial effects on different types of cancer. The aim of this review was to evaluate the association between GT regular consumption and risk of BC in women. The risk of BC recurrence and risk of BC in relation to menopausal status were also evaluated. A literature search of PubMed, Scopus, and Web of Science was conducted. Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were followed to perform the systematic review and meta-analysis. Full texts were downloaded for 40 studies; however, only 13 records were included in the meta-analysis. Eight were cohort studies and five were case-control studies. The pooled sample consisted of 163,810 people. An inverse statistically significant relationship between GT and BC risk, with an Odds Ratio (OR) = 0.85 ((95% CI = 0.80⁻0.92), p = 0.000)), was found. Egger's linear regression test did not show a potential publication bias (intercept 0.33, t = 0.40, p = 0.695), which was also confirmed by the symmetry of the funnel plot. Moreover, no high statistical heterogeneity (Chi² = 31.55, df = 13, I² = 58.79%, p = 0.003) was found. The results of this meta-analysis showed a potential protective effect of GT consumption on BC, especially for BC recurrence.

In vitro toxicity evaluation of estragole-containing preparations derived from Foeniculum vulgare Mill. (fennel) on HepG2 cells.

Estragole, a common component of herbs and spices, is a wellknown genotoxic hepatocarcinogen in rodents, whereas its potential toxic effect in humans is still debated. In the European contest, one of the major sources of human exposure to this phytochemical is Foeniculum vulgare Mill. (fennel). Therefore, the aim of this study was to evaluate the in vitro toxicity of estragole in the context of two complex phytochemical mixtures derived from fennel: fennel seed powder (FSPw) and fennel seed essential oil (FSEO). The estragole-containing preparations were analysed for their ability to cause cytotoxicity, genotoxicity, apoptosis and cell cycle perturbation in the human hepatoma (HepG2) cell line. None of the tested concentrations of FSPw induced DNA damage, nor apoptosis or cell cycle perturbation. Although FSEO did not induce any genetic damage as well, it exerted marked dose-dependent apoptotic effects on HepG2 cells with a concurrent cell cycle arrest in G2/M at the highest tested dose. Although prospective analyses are required to clarify the observed toxic effects of FSEO, our results support the hypothesis that the genotoxicity of estragole may be significantly reduced or null in the context of botanical mixtures.

Sulforaphane and Epigallocatechin Gallate Restore Estrogen Receptor Expression by Modulating Epigenetic Events in the Breast Cancer Cell Line MDA-MB-231: A Systematic Review and Meta-Analysis.

BACKGROUND/AIMS: Epigenetics refers to modifications in gene activity and expression without alteration at the DNA sequence. Environment and diet could influence gene expression. Diet modifications may be meaningful in preventing and treating chronic diseases, cancer included. Dietary bioactive compounds, such as polyphenols (e.g., curcumin, resveratrol, or epigallocatechin gallate [EGCG]) or isothiocyanate (e.g., sulforaphane [SFN]), can regulate histone acetylation. The aim of this systematic review and meta-analysis was to evaluate the effect of SFN and EGCG on breast cancer (BC) cells cultured in vitro. METHODS: Due to the enormous variability observed in study protocols and the innumerable genes involved, only studies analyzing the number of apoptotic cells in the MDA-MB-231 cell line were evaluated. The effect size (ES) was computed as the ratio of means. RESULTS: We identified 7 studies, 4 regarding the effect of 10 µM SFN on MDA-MB-231 cells (ES = 4.59, 95% confidence interval 4.05-5.20) and 3 focusing on the impact of 20 µM EGCG (ES = 2.84, 95% confidence interval 2.60-3.10). CONCLUSION: The findings suggest beneficial effects of dietary bioactive compounds such as SFN and EGCG and their effect on BC cells by restoring estrogen receptor gene expression, modulating epigenetic changes and events, and interfering with tumor growth rate. Publication bias limits the generalizability of the conclusions. High-quality studies are needed.

Acid sphingomyelinase as target of Lycium Chinense: promising new action for cell health.

BACKGROUND: Sphingomyelin plays very important roles in cell function under physiological and pathological conditions. Physical and chemical stimuli produce reactive oxygen species that stimulate acid sphingomyelinase to induce apoptosis. Antioxidant plants of the traditional Chinese Pharmacopoeia, such as Lycium Barbarum and Lycium Chinense, have become increasingly popular in Western countries. We investigated the effects of Lycium Chinense on acid sphingomyelinase and sphingomyelin species in relation to gene expression. METHODS: We prepared Lycium Chinense berry extracts and evaluated their antioxidant properties. Increasing amount of extracts was used to test cytotoxic and genotoxic effect on HepG2 cells. Gene expression, protein amount and enzyme activity of acid sphingomyelinase were tested by RT-PCR, immunoblotting and enzymatic activity assay, respectively. Sphingomyelin species were analyzed by UFLC MS/MS. A panel of 96 genes involved in oxidative stress, proliferation, apoptosis and cancer was used to test the effect of LC on gene expression. GLRX2, RNF7, and PTGS1 proteins were analyzed by immunoblotting. RESULTS: We showed that Lycium Chinense berries have high antioxidant properties, have an IC50value of 9.55 mg/mL, do not induce genotoxic effect and maintain high level of cell viability. The berry extracts inhibit acid sphingomyelinase activity and increase both very long fatty acid sphingomyelin species and unsaturated fatty acid sphingomyelin species. Among 96 genes, Lycium Chinense berries up-regulate Glutaredoxin 2 and Ring Finger Protein 7 genes and proteins, able to protect cells from apoptosis. Intrigantly, Lycium Chinense berries down-regulates Prostaglandin H synthase 1 gene but the protein is not expressed in HepG2 cells. CONCLUSION: The results identify acid sphingomyelinase as a novel target of Lycium Chinense berries to decrease saturated/unsaturated fatty acid sphingomyelin ratio, known to be useful for cell health. Consistent with these data, the berries regulate specifically gene expression to protect cells from apoptosis.

In Vitro Safety/Protection Assessment of Resveratrol and Pterostilbene in a Human Hepatoma Cell Line (HepG2).

The aim of this work was to evaluate in vitro the genotoxic and/or antigenotoxic effects of resveratrol (RESV) and pterostilbene (PTER) on HepG2 cells. Moreover, additional tests were performed to evaluate early and late apoptosis events induced by the tested stilbenes. RESV and PTER did not show any genotoxic activity. As regards antigenotoxicity testing, RESV and PTER showed a typical, U-shaped hormetic dose-response relationship characterized by a biphasic trend with small quantities having opposite effects to large ones. HepG2 cells treated with PTER exhibited a marked increase in early apoptosis (40.1%) at 250 microM; whereas, the highest concentration tested for both RESV and PTER significantly increased the proportion of HepG2 cells undergoing late apoptosis (32.5 and 51.2%, respectively). The observed pro-apoptotic activity could, at least in part, explain the hormetic response observed when the compounds were tested for antigenotoxicity (i.e., in the presence of induced DNA damage).

Protective effects of probiotic Lactobacillus rhamnosus IMC501 in mice treated with PhIP.

The aim of the present study was to investigate the antigenotoxic properties of the probiotic Lactobacillus rhamnosus IMC501; DNA damage was induced by one representative food mutagen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Mice were treated orally with suspensions of lactobacilli for 10 days before administration of food mutagen. During the treatment, the abundance of lactobacilli in feces, as assessed by qPCR analysis, increased, whereas ß-glucuronidase and N-acetyl-ß-glucosaminidase activities decreased. The extent of DNA damage was measured in colon and liver cells by comet assay. In colonocytes, diet supplementation with IMC501 resulted in a significant inhibition of DNA damage induced by PhIP. The results obtained in this in vitro study suggest that Lactobacillus rhamnosus IMC501 used as a dietary supplement can provide a useful integration of antimutagen food components of the normal diet, which are generally lower than the protective level.

Antigenotoxic effect, composition and antioxidant activity of Dendrobium speciosum.

The chemical composition, the antiradical properties of Dendrobium speciosum (Orchidaceae) leaves and stem extracts have been studied. Furthermore, in view of the use of this orchid as "bush foods", the genotoxic/antigenotoxic effects of the extracts have been evaluated.

In vitro testing for genotoxicity of indigo naturalis assessed by micronucleus test.

In the field of cosmetic dyes, used for coloring the hair and skin, there is a clear tendency to replace the widely used synthetic dyes by natural colorants, such as henna and mixtures of henna with indigo. The aim of this study was to estimate the genotoxicity of water and DMSO solutions of indigo naturalis (prepared from Indigofera tinctoria leaves) using the cytokinesis-blocked micronucleus (CBMN) assay in the human metabolically active HepG2 cell line. The cytotoxic effects of indigo solutions were first assessed by propidium iodide and fluorescein-diacetate simultaneous staining. For both solutions, cytotoxicity was always under 10%. Data obtained in the CBMN assay (for all concentrations tested) indicated that the frequency of MN (micronuclei) in exposed cells was no higher than the control. Both the water and DMSO solutions showed the same behavior. These results indicate that indigo naturalis exhibits neither cytotoxicity, nor genotoxicity for all concentrations tested, which may justify excluding indigofera and its components from the list of carcinogenic agents.

Influence of iodide excess and interferon-gamma on human primary thyroid cell proliferation, thyroglobulin secretion, and intracellular adhesion molecule-1 and human leukocyte antigen-DR expression.

BACKGROUND: The effect of iodide on thyroid cell proliferation and function in vivo or in cultured thyroid cells has been previously reported and is still controversial. The aim of this study was to clarify these conflicting results by examining if prolonged high iodide exposition with or without interferon (IFN)-gamma has an effect on human primary thyroid cell proliferation, thyroglobulin (Tg) production, and intercellular adhesion molecule-1 (ICAM-1) and human leukocyte antigen (HLA)-DR expression. METHODS: Primary human thyroid cells were used. Cells were cultured in Coon's modified Ham's F-12 medium supplemented with 5% fetal calf serum in monolayer conditions to induce proliferation and were aggregated for molecular expression and Tg production analysis. HLA-DR and ICAM-1 expression were measured by flow cytometry and Tg by immunometric assay. RESULTS: Potassium iodide (KI) was more potent in arresting primary human thyroid cell proliferation as compared to sodium iodide and the effect was mediated by its action at G0/G1 and G2/M phases of the cell cycle. There were no signs of apoptosis or necrosis. An excess of KI alone did not change the expression of HLA-DR and Tg production, but gradually increased ICAM-1. Low-dose IFN-gamma and excess KI in combination transiently inhibited HLA-DR expression, while ICAM-1 was expressed at a higher level than with IFN-gamma alone. Tg production was moderately increased with low-dose IFN-gamma. However, a combination of high-dose KI with low-dose IFN-gamma significantly decreased Tg secretion, compared with IFN-gamma alone. CONCLUSIONS: Augmented ICAM-1 in the presence of iodide excess and low-dose IFN-gamma could induce secretion of proinflammatory cytokines and lymphocytic infiltration in the thyroid gland. Decreased Tg production in the presence of KI excess and IFN-gamma could explain the development of hypothyroidism after adding iodide in a diet of subjects that already have lymphocytic infiltration and/or mild inflammation in the thyroid gland.

Modulatory activity of a Lactobacillus casei strain on 1,2-dimethylhydrazine-induced genotoxicity in rats.

The present study was designed to investigate the putative antigenotoxic effects of supplementing the diet of rats treated with the colon carcinogen 1,2-dimethylhydrazine hydrochloride (DMH) with a Lactobacillus casei strain using an in vivo approach. The antigenotoxic response was evaluated in colon and liver cells using the alkaline comet assay. Since the balance between the bioactivation and detoxification metabolic pathways is crucial for the formation of toxic and genotoxic metabolites, alterations in the level of some xenobiotic metabolizing enzymes (XME) were studied in liver preparations. In the challenge group (L. casei + DMH), lactobacilli-supplemented diet, there was a decrease in the extent of DMH-induced DNA damage, especially in colon cells. Compared with control rats, there was less basal DNA damage in colon cells of rats fed on a lactobacilli-supplemented diet. These findings are the first to give clear evidence of DNA-protective effects of lactobacilli against basal DNA damage. Moreover, the chemopreventive effects were accompanied by changes in the activities of several XME. The observed decrease in the concentration of nonenzymatic antioxidants (i.e. GSH) and the reduced activity of enzymatic antioxidants (i.e., GST, GPx, and SOD) in liver could reflect an overall reduction in the level of oxidative stress in rats on a diet supplemented with the L. casei suspension compared with control rats (basal state). Thus, the concentrations of GSH and the activities of GST, GPx, and SOD could be downregulated by supplementing the diet with L. casei as a response to an improved antioxidant status.

Evaluation of genotoxic and antigenotoxic effects of hydroalcoholic extracts of Zuccagnia punctata Cav.

Zuccagnia punctata Cav. (Fabaceae), a widely used plant species in Argentine folk medicine, has been shown to have a broad spectrum of antibacterial, antifungal, antioxidant and cytoprotective activities. In this study, the hydroalcoholic extract of Zuccagnia punctata and 2',4'-dihydroxychalcone isolated from it were investigated for genotoxicity/antigenotoxicity in the in vitro comet assay test on human hepatoma HepG2 cells. No acute toxicity of the extract could be determined. HepG2 cells were treated with three different concentrations (2.5, 5.0 and 10.0 microg/mL) or 2',4'-dihydroxychalcone (0.01, 0.10 and 1.00 microg/mL). To explore the potential mechanisms of action, two approaches were followed: co-treatment with 4-nitroquinoline-N-oxyde (4-NQO), a direct genotoxic compound, and a pre-treatment protocol with benzo[a]pyrene (B[a]P), an indirect genotoxic compound. The natural products neither affected cell viability nor induced DNA damage in the concentration range tested. Zuccagnia punctata tinctures were able to diminish the DNA damage induced in HepG2 cells by 4-NQO and B[a]P in 31% and 10%, respectively at 10 microg/mL. Pre-treatment of HepG2 cells with 2',4'-dihydroxychalcone was highly effective in decreasing B[a]P-induced DNA damage at a statistically significant level, with an almost clear dose-response relationship. The inhibition values were 28.2-43.9% for the tested concentrations of 0.01-1 microg/mL, respectively. The results clearly indicate that the phytoextract from Zuccagnia punctata, under the experimental conditions tested, is not genotoxic and that 2',4'-dihydroxychalcone contributes to a high degree to the antigenotoxic effects of Zuccagnia punctata tincture.

In vitro protective effects of Terminalia arjuna bark extracts against the 4-nitroquinoline-N-oxide genotoxicity.

We determined the antimutagenic potential of chloroform, acetone, methanol, methanol+HCl, diethyl ether, and ethyl acetate extracts of Terminalia arjuna bark against the model mutagen 4-nitroquinoline-N-oxide (4-NQO) using the Salmonella/microsome, comet, and micronucleus (MN) tests. Salmonella typhimurium TA100 strain and human peripheral white blood cells were coincubated with various concentrations (from 5 to 500 microg) of the six extracts and 4-NQO (from 0.05 to 2 microg). We found that the 4-NQO mutagenicity was inhibited by more than 70% in the Salmonella/microsome test at the highest nontoxic extract dose of ethyl acetate (50 microg/plate), chloroform (100 microg/plate), acetone, (100 microg/plate), and methanol (500 microg/plate). A less marked antimutagenicity activity (inhibition of about 40-45%) was observed for the acidic methanol and diethyl ether extracts. The comet assay showed that acetone extract (100 microg/mL) was more effective in reducing the DNA damage caused by 4-NQO (ca. 90%), whereas the chloroform, ethyl acetate, and diethyl ether extracts were cytotoxic. In the MN test, the decrease in 4-NQO clastogenicity was observed by testing the mutagen especially with chloroform and ethyl acetate extracts (inhibition about 40-45%). The acetone and methanol extracts showed a less marked activity (33% and 37%, respectively). The results of the present study suggest that T. arjuna bark contains some nonpolar as well as polar compounds with antimutagenic activity against 4-NQO. Several explanations can be suggested, but further investigations are necessary to definitely identify the active compounds.