Toxicological assessment of Opuntia dillenii (Ker Gawl.) Haw. cladode methanol extract, fractions and its alpha pyrones: Opuntiol and opuntioside.

ETHNOPHARMACOLOGICAL RELEVANCE: The edible plant Opuntia dillenii (Ker Gawl.) Haw. commonly known as Nagphana, belongs to the Cactaceae family. It is traditionally used to treat various ailments including inflammation, gastric ulcers, diabetes, hepatitis, asthma, whooping cough and intestinal spasm. AIM OF THE STUDY: Despite its traditional use in various countries, detailed toxicological studies of O. dillenii cladode are few. Thus in the current study, toxicity of O. dillenii cladode derived methanol extract, fractions and its α-pyrones: opuntiol and opuntioside have been addressed. METHODS: The test agents were assessed using both in vitro and in vivo toxicity assays. MTT on human embryonic kidney cell line (HEK-293), tryphan blue exclusion in rat neutrophils, Cytokinesis-B block micronucleus (CBMN) in human lymphocytes and genomic DNA fragmentation using agarose gel electrophoresis were performed. In acute toxicity test, mice orally received extract (5 g/kg) for 7 days followed by measurements of relative organ weight, biochemical (blood profile, liver and kidney function test) and histological studies (liver and kidney) were carried out. Rat bone marrow micronucleus genotoxicity assay was also conducted. RESULTS: O. dillenii derived test agents were non-cytotoxic and had no effect on the integrity of DNA. Methanol extract (5 g/kg) orally administered in mice did not cause any significant change in relative organ weights, biochemical parameters and liver and kidney histology as compared to vehicle control. In parallel, extract did not stimulate micronuclei formation in rat bone marrow polychromatic erythrocytes. CONCLUSION: These results led to conclude that edible O. dillenii extract is non-toxic via the oral route and appears to be non-cyto-, hepato-, nephro- or genotoxic, thereby supporting its safe traditional use against various ailments. Therefore, opuntiol and opuntioside may serve as lead compounds in designing new drug(s) derived from edible plants.

Functional cardiotoxicity assessment of cosmetic compounds using human-induced pluripotent stem cell-derived cardiomyocytes.

There is a large demand of a human relevant in vitro test system suitable for assessing the cardiotoxic potential of cosmetic ingredients and other chemicals. Using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), we have already established an in vitro cardiotoxicity assay and identified genomic biomarkers of anthracycline-induced cardiotoxicity in our previous work. Here, five cosmetic ingredients were studied by the new hiPSC-CMs test; kojic acid (KJA), triclosan (TS), triclocarban (TCC), 2,7-naphthalenediol (NPT), and basic red 51 (BR51) based on cytotoxicity as well as ATP assays, beating rate, and genomic biomarkers to determine the lowest observed effect concentration (LOEC) and no observed effect concentration (NOEC). The LOEC for beating rate were 400, 10, 3, >400, and 3 µM for KJA, TS, TCC, NPT, and BR51, respectively. The corresponding concentrations for cytotoxicity or ATP depletion were similar, with the exception of TS and TCC, where the cardiomyocyte-beating assay showed positive results at non-cytotoxic concentrations. Functional analysis also showed that the individual compounds caused different effects on hiPSC-CMs. While exposure to KJA, TS, TCC, and BR51 induced significant arrhythmic beating, NPT slightly decreased cell viability, but did not influence beating. Gene expression studies showed that TS and NPT caused down-regulation of cytoskeletal and cardiac ion homeostasis genes. Moreover, TS and NPT deregulated genomic biomarkers known to be affected also by anthracyclines. The present study demonstrates that hiPSC-CMs can be used to determine LOECs and NOECs in vitro, which can be compared to human blood concentrations to determine margins of exposure. Our in vitro assay, which so far has been tested with several anthracyclines and cosmetics, still requires validation by larger numbers of positive and negative controls, before it can be recommended for routine analysis.

Structure- and concentration-specific assessment of the physiological reactivity of α-dicarbonyl glucose degradation products in peritoneal dialysis fluids.

In peritoneal dialysis (PD), glucose degradation products (GDPs), which are formed during heat sterilization of dialysis fluids, lead to structural and functional changes in the peritoneal membrane, which eventually result in the loss of its ultrafiltration capacity. To determine the molecular mechanisms behind these processes, the present study tested the influence of the six major α-dicarbonyl GDPs in PD fluids, namely, glyoxal, methylglyoxal, 3-deoxyglucosone (3-DG), 3-deoxygalactosone (3-DGal), 3,4-dideoxyglucosone-3-ene (3,4-DGE), and glucosone with respect to their potential to impair the enzymatic activity of RNase A as well as their effects on cell viability. For comprehensive risk assessment, the α-dicarbonyl GDPs were applied separately and in concentrations as present in conventional PD fluids. Thus, it was shown that after 5 days, glucosone impaired RNase A activity most distinctly (58% remaining activity, p < 0.001 compared to that of the control), followed by 3,4-DGE (62%, p < 0.001), 3-DGal (66%, p < 0.001), and 3-DG (76%, p < 0.01). Methylglyoxal and glyoxal caused weaker inactivation with significant effects only after 10 days of incubation (79%, 81%, p < 0.001). Profiling of the advanced glycation end products formed during the incubation of RNase A with methylglyoxal revealed predominant formation of the arginine modifications imidazolinone, CEA/dihydroxyimidazoline, and tetrahydropyrimidine at Arg10, Arg33, Arg39, and Arg85. Particularly, modification at Arg39 may severely affect the active site of the enzyme. Additionally, structure- and concentration-specific assessment of the cytotoxicity of the α-dicarbonyl GDPs was performed. Although present at very low concentration, the cytotoxic effect of PD fluids after 2 days of incubation was exclusively caused by 3,4-DGE (14% cell viability, p < 0.001). After 4 days of incubation, 3-DGal (13% cell viability, p < 0.001), 3-DG (24%, p < 0.001), and, to a lower extent, glyoxal and methylglyoxal (both 57%, p < 0.01) also reduced cell viability significantly. In conclusion, 3,4-DGE, 3-DGal, and glucosone appear to be the most relevant parameters for the biocompatibility of PD fluids.

Final report of the safety assessment of Kojic acid as used in cosmetics.

Kojic acid functions as an antioxidant in cosmetic products. Kojic acid was not a toxicant in acute, chronic, reproductive, and genotoxicity studies. While some animal data suggested tumor promotion and weak carcinogenicity, kojic acid is slowly absorbed into the circulation from human skin and likely would not reach the threshold at which these effects were seen. The available human sensitization data supported the safety of kojic acid at a use concentration of 2% in leave-on cosmetics. Kojic acid depigmented black guinea pig skin at a concentration of 4%, but this effect was not seen at 1%. The Cosmetic Ingredient Review (CIR) Expert Panel concluded that the 2 end points of concern, dermal sensitization and skin lightening, would not be seen at use concentrations below 1%; therefore, this ingredient is safe for use in cosmetic products up to that level.

An assessment of the genotoxicity and human health risk of topical use of kojic acid [5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one].

Kojic acid (KA), a natural substance produced by fungi or bacteria, such as Aspergillus, Penicillium or Acetobacter spp, is contained in traditional Japanese fermented foods and is used as a dermatological skin-lightening agent. High concentrations of KA (>or=1000 microg/plate) were mutagenic in S. typhimurium strains TA 98, TA 100, TA 1535, TA102 and E. coli WP2uvrA, but not in TA 1537. An Ames test following the "treat and plate" protocol was negative. A chromosome aberration test in V79 cells following a robust protocol showed only a marginal increase in chromosome aberrations at cytotoxic concentrations after prolonged (>or=18 h) exposure. No genotoxic activity was observed for hprt mutations either in mouse lymphoma or V79 cells, or in in vitro micronucleus tests in human keratinocytes or hepatocytes. All in vivo genotoxicity studies on KA doses were negative, including mouse bone marrow micronucleus tests after single or multiple doses, an in vivo/in vitro unscheduled DNA synthesis (UDS) test, or a study in the liver of the transgenic Muta(TM) Mouse. On the basis of pharmacokinetic studies in rats and in vitro absorption studies in human skin, the systemic exposure of KA in man following its topical application is estimated to be in the range of 0.03-0.06 mg/kg/day. Comparing these values with the NOAEL in oral subchronic animal studies (250 mg/kg/day), the calculated margin of safety would be 4200- to 8900-fold. Comparing human exposure with the doses that were negative for micronuclei, UDS and gene mutations in vivo, the margins of safety are 16000 to 26000-fold. In conclusion, the topical use of KA as a skin lightening agent results in minimal exposure that poses no or negligible risk of genotoxicity or toxicity to the consumer.

Genotoxicity assessment of five tremorgenic mycotoxins (fumitremorgen B, paxilline, penitrem A, verruculogen, and verrucosidin) produced by molds isolated from fermented meats.

A number of toxinogenic fungal species, particularly producers of tremorgenic mycotoxins, have been isolated from traditional fermented meats. Tremorgenic mycotoxins are a group of fungal metabolites known to act on the central nervous system, causing sustained tremors, convulsions, and death in animals. However, the mode of action of these mycotoxins has not been elucidated in detail, and their genotoxic capacity has hardly been investigated. Because genotoxicity is one of the most prominent toxicological end points in food safety testing, we assessed the genotoxicity of five tremorgenic mycotoxins (fumitremorgen B, paxilline, penitrem A, verrucosidin, and verruculogen) associated with molds found in fermented meats. The mycotoxins were tested in two short-term in vitro assays with the use of different genotoxic end points in different phylogenetic systems (the Ames Salmonella/mammalian-microsome assay and the single-cell gel electrophoresis assay of human lymphocytes). According to the results obtained in this study, all of the investigated mycotoxins except penitrem A exhibited a certain degree of genotoxicity. Verrucosidin appeared to have the highest toxic potential, testing positive in both assays. Verruculogen tested positive in the Salmonella/mammalian-microsome assay, and paxilline and fumitremorgen B caused DNA damage in human lymphocytes. The use of fungal starter cultures to avoid tremorgen contamination in fermented meats is recommended.

In vitro response of human breast cancer cell lines to the growth-inhibitory effects of styrylpyrone derivative (SPD) and assessment of its antiestrogenicity.

Previous studies have shown that a styrylpyrone derivative (SPD) from a local tropical plant had antiprogestin and antiestrogenic effects in early pregnant mice models (Azimahtol et al. 1991). Antiprogestins and antiestrogens can be exploited as a therapeutic approach to breast cancer treatment and thus the antitumor activity of SPD was tested in three different human breast cancer cell lines that is: MCF- 7, T47D and MDA-MB-231, employing, the antiproliferative assay of Lin and Hwang (1991) slightly modified. SPD (10(-10) - 10(-6) M) exhibited strong antiproliferative activity in estrogen and progestin-dependent MCF-7 cells (EC50 = 2.24 x 10(-7) M) and in hormone insensitive MDA-MB-231 (EC50 = 5.62 x 10(-7) M), but caused only partial inhibition of the estrogen- insensitive T47D cells (EC50 = 1.58 x 10(-6) M). However, tamoxifen showed strong inhibition of MCF-7 cells (EC50 = 1.41 x 10(-6) M) and to a lesser extent the T47D cells (EC50 = 2.5 x 10(-6) M) but did not affect the MDA-MB-231 cells. SPD at 1 microM exerted a beffer antiestrogenic activity than 1 microM tamoxifen in suppressing the growth of MCF-7 cells stimulated by 1 nM estradiol. Combined treatment of both SPD and tamoxifen at 1 microM showed additional inhibition on the growth of MCF-7 cells in culture. The antiproliferative properties of SPD are effective on both receptor positive and receptor negative mammary cancer cells, and thus appear to be neither dependent on cellular receptor status nor cellular hormone responses. This enhances in vivo approaches as tumors are heterogenous masses with varying receptor status.