Alteration of unfolded protein responses and autophagy signaling represented the molecular basis underlying saccharin toxicity to Drosophila (Diptera: Drosophilidae).

The purpose of this study was to develop a new control method for Drosophila using saccharin sodium dihydrate (saccharin), an artificial sweetener that is safe for humans and the environment, and to elucidate its mode of action. In this study, we confirmed that saccharin can dose-dependently inhibit the development of or kill vinegar flies (VFs) and spotted wing Drosophila (SWDs). In addition, we found that low concentrations of saccharin induced a similar effect as starvation in Drosophila, whereas high concentrations of saccharin induced changes in the unfolded protein response (UPR) and autophagy signaling that were unlike starvation and inhibited development or killed the VF and the SWD by performing real-time quantitative polymerase chain reaction analyses. Spinosad is a widely used plant protection agent for SWD control. When saccharin was cotreated with 0.25-1.0 ppm spinosad, an additive insecticidal activity was observed only at high concentrations of saccharin. However, when saccharin was cotreated with 2.0 ppm spinosad, an additive insecticidal activity was observed at low concentrations of saccharin. Taken together, alteration of UPR and autophagy signaling represented the molecular basis underlying saccharin toxicity to Drosophila and concurrent spraying of an insecticide with saccharin could enhance the insecticidal activities.

Phenotyping Aquatic Neurotoxicity Induced by the Artificial Sweetener Saccharin at Sublethal Concentration Levels.

Artificial sweeteners (ASs) have generally been applied as food additives to improve the taste of sweetness. Thus, their potential toxic effects have received extensive attention. Saccharin (SAC), discovered more than a century ago, has been used as the first noncaloric AS in foods and beverages for over 100 years. Although the toxicological effects such as carcinogenicity of SAC have been controversial for a long time, there is a paucity of knowledge covering its potential behavioral toxicity and neurotoxicity. Methodologically, in current research, adult zebrafish neurobehavioral phenotypic screening approaches were introduced to systematically delineate the potential behavioral and neural toxicity of SAC by phenotyping the comprehensive neuro-behavioral profiles of adult zebrafish, which were chronically (2 months) subject to SAC (0, 1, 10, and 50 mg/L) exposure. Subsequently, a cohort of standard neurobehavioral tests including the light/dark preference (LDP) test, novel tank diving (NTD) test, novel object recognition (NOR) test, social interaction test (SIT), color-associated learning and memory test, and conditional place preference test were applied to delineate the general adverse effect of SAC. Specifically, in a concentration-dependent manner, SAC significantly increased the preference toward the dark side in the LDP test, inhibited exploratory behavior to the top arena in the NTD test, dampened the motivation to explore the novel object in the NOR test, weakened social preference in the SIT, and interfered in the color-based associative learning and memory ability. For example, in the LDP test, SAC remarkably increased the swimming distance of zebrafish in the dark part from 222 ± 34.6 (control group) to 675 ± 35.0 (50 mg/L group). Finally, the quantity of certain key neurotransmitters was further measured to determine the alteration induced by SAC on the brain chemistry. In total, the current research would provide a versatile neurobehavioral phenomics-based strategy to phenotypically screen the neurotoxicity of food additives at the overall animal level and provide a reference for further neurotoxicity exploration at the tissue and molecular level.

Development of a novel flow cytometry-based approach for reticulocytes micronucleus test in rat peripheral blood.

The micronucleus test (MNT) is the most widely applied short-term assay to detect clastogens or spindle disruptors. The use of flow cytometry (FCM) has been reported for micronucleated erythrocytes scoring in peripheral blood. The aim of this study was to develop a novel and practical protocol for MNT in rat peripheral blood by FCM, with the method validation. CD71-fluorescein isothiocyanate and DRAQ5 were adopted for the fluorescent staining of proteins and DNA, respectively, to detect micronuclei. To validate the method, groups of male Sprague-Dawley rats (five per group) received two oral gavage doses at 0 and 24 h of six chemicals (four positive mutagens: ethyl methanesulphonate [EMS], cyclophosphamide [CP], colchicine [COL], and ethyl nitrosourea [ENU]; two nongenotoxic chemicals: sodium saccharin and eugenol). Blood samples were collected from the tail vein before and on the five continuous days after treatments; all of which were analyzed for micronuclei presence by both the manual (Giemsa staining) and FCM methods. The FCM-based method consistently demonstrated highly sensitive responses for micronucleus detection at all concentrations and all time points for EMS, CP, COL, and ENU. Sodium saccharin and eugenol could be identified as negative in this protocol. Results obtained with the FCM-based method correlated well with the micronucleus frequencies (r = 0.659-0.952), and the proportion of immature erythrocytes (r = 0.915-0.981) tested by Giemsa staining. The method reported here, with easy operation, low background, and requirement for a regular FCM, could be an efficient system for micronucleus scoring.

A novel screening test to predict the developmental toxicity of drugs using human induced pluripotent stem cells.

In vitro human induced pluripotent stem (iPS) cells testing (iPST) to assess developmental toxicity, e.g., the induction of malformation or dysfunction, was developed by modifying a mouse embryonic stem cell test (EST), a promising animal-free approach. The iPST evaluates the potential risks and types of drugs-induced developmental toxicity in humans by assessing three endpoints: the inhibitory effects of the drug on the cardiac differentiation of iPS cells and on the proliferation/survival of iPS cells and human fibroblasts. In the present study, the potential developmental toxicity of drugs was divided into three classes (1: non-developmentally toxic, 2: weakly developmentally toxic and 3: strongly developmentally toxic) according to the EST criteria. In addition, the type of developmental toxicity of drugs was grouped into three types (1: non-effective, 2: embryotoxic [inducing growth retardation/dysfunction]/deadly or 3: teratogenic [inducing malformation]/deadly) by comparing the three endpoints. The present study was intended to validate the clinical predictability of the iPST. The traditionally developmentally toxic drugs of aminopterin, methotrexate, all-trans-retinoic acid, thalidomide, tetracycline, lithium, phenytoin, 5-fluorouracil, warfarin and valproate were designated as class 2 or 3 according to the EST criteria, and their developmental toxicity was type 3. The non-developmentally toxic drugs of ascorbic acid, saccharin, isoniazid and penicillin G were designated as class 1, and ascorbic acid, saccharin and isoniazid were grouped as type 1 while penicillin G was type 2 but not teratogenic. These results suggest that the iPST is useful for predicting the human developmental toxicity of drug candidates in a preclinical setting.

Synthesis, hepatotoxic evaluation and antipyretic activity of nitrate ester analogs of the acetaminophen derivative SCP-1.

A series of nitrate ester analogues of the acetaminophen derivative SCP-1 were prepared by triflic acid catalyzed O-acylation of SCP-1 with chloroalkanoyl chlorides followed by nitration with silver nitrate. The chloroesters and corresponding nitrate esters were obtained in high yields. Preliminary hepatotoxicity studies revealed nitrate esters 5b (MD-38) and 5c (MD-39) to be well tolerated by human hepatocytes and had little effect on the three cytochrome P450 enzymes tested (CYP3A4, CYP2E1 and CYP2D6). In addition, the nitrate ester 5c (MD-39) exhibited antipyretic activity similar to acetaminophen.

Evaluation of long-term effects of artificial sweeteners on rat brain: a biochemical, behavioral, and histological study.

The aim of the present study was to compare the effects of artificial sweeteners (aspartame, saccharin, and sucralose) on rat brain. Twenty-four adult male Sprague-Dawley rats were included in the study. The control group (n = 6) received regular tap water, whereas other groups received aspartame (3 mg/kg/day, n = 6,) or saccharin (3 mg/kg/day, n = 6) or sucralose (1.5 mg/kg/day, n = 6) in the drinking water. Following 6 weeks, the passive avoidance learning (PAL) test was performed to evaluate the neurobehavioral effects of sweeteners. The brains were assessed for lipid peroxides, neuron count, and Glial fibrillary acidic protein (GFAP) immunohistochemistry. Our results demonstrated that chronic intake of sweeteners significantly impaired PAL performance in all groups. Hippocampal CA1-CA3 areas revealed significantly lower neuronal count in aspartame and increased GFAP expression in all groups. Brain lipid peroxides were significantly higher in all groups. Our findings suggest that long-term consumption of artificial sweeteners may have harmful effects on cognition and hippocampal integrity in rats.

Effects of daily exposure to saccharin sodium and rebaudioside A on the ovarian cycle and steroidogenesis in rats.

Saccharin sodium and rebaudioside A are widely used as non-caloric sweeteners in our daily life; however, the impacts and regulatory mechanisms of such sweeteners on reproduction remain unclear. In the present study, we used rats as animal models to evaluate the effects of daily exposure to saccharin sodium and rebaudioside A on ovarian biologic functions. Weanling rats were distributed into five experimental groups receiving normal water, 1.5 or 7.5 mM saccharin sodium solution, or 0.5 or 2.5 mM rebaudioside A solution for 48 days of exposure. The results showed an increased percentage of abnormal estrous cycles, augmented number of ovarian cysts, elevated serum progesterone levels, and increased expression of steroidogenesis-related factors in saccharin sodium-treated groups. Conversely, rebaudioside A-treated groups showed decreased serum progesterone levels. Our findings suggest that saccharin sodium exerts adverse biologic effects on ovaries, and rebaudioside A is a potential steroidogenic disruptor in female rats.

The effect of five artificial sweeteners on Caco-2, HT-29 and HEK-293 cells.

CONTEXT: Artificial sweeteners (AS) have been associated with tumor development (including colon cancer) in both animals and humans although evidence has been conflicting. OBJECTIVES: Additional research was thus conducted by studying the effects of 5 AS on the morphology, cell proliferation and DNA in cells by utilizing Caco-2, HT-29 (colon) and HEK-293 (kidney) cell lines. MATERIALS AND METHODS: Cells were exposed to sodium cyclamate, sodium saccharin, sucralose and acesulfame-K (0-50 mM) and aspartame (0-35 mM) over 24, 48 and 72 hours. Morphological changes were presented photographically and % cell viability was determined by using the MTT cell viability assay. Possible DNA damage (comet assay) induced by the AS (0.1, 1 and 10 mM, treated for 24, 48 and 72 hours) was studied. The appearance of "comets" was scored from no damage to severe damage (0-4). RESULTS: Cells became flatter and less well defined at higher AS concentrations (>10 mM). At concentrations >10 mM, decreased cell viability was noted with both increasing concentration and increasing incubation time for all cell lines tested. In general, HEK-293 cells seemed to be less affected then the colon cancer cells. Sucralose and sodium saccharin seemed to elicit the greatest degree of DNA fragmentation of all the sweeteners tested in all the cell lines used. DISCUSSION: Morphological cell alterations, cell viability and DNA fragmentation seemed to be more in the colon cancer cells. CONCLUSIONS: Further studies have to be performed to clarify mechanisms involved causing these alterations in mammalian cells.

Modified high-density lipoproteins by artificial sweetener, aspartame, and saccharin, showed loss of anti-atherosclerotic activity and toxicity in zebrafish.

Safety concerns have been raised regarding the association of chronic consumption of artificial sweeteners (ASs) with metabolic disorders, especially in the heart and brain. There has been no information on the in vivo physiological effects of AS consumption in lipoprotein metabolism. High-dosage treatment (final 25, 50, and 100 mM) with AS (aspartame, acesulfame K, and saccharin) to human high-density lipoprotein (HDL) induced loss of antioxidant ability along with elevated atherogenic effects. Aspartame-treated HDL3 (final 100 mM) almost all disappeared due to putative proteolytic degradation. Aspartame- and saccharin-treated HDL3 showed more enhanced cholesteryl ester transfer activity, while their antioxidant ability was disappeared. Microinjection of the modified HDL3 exacerbated the inflammatory death in zebrafish embryos in the presence of oxLDL. These results show that AS treatment impaired the beneficial functions of HDL, resulting in loss of antioxidant and anti-atherogenic activities. These results suggest that aspartame and saccharin could be toxic to the human circulation system as well as embryonic development via impairment of lipoprotein function.

Etiology of inflammatory bowel disease: a unified hypothesis.

Inflammatory bowel disease (IBD), including both ulcerative colitis (UC) and Crohn's disease (CD), emerged and dramatically increased for about a century. Despite extensive research, its cause remains regarded as unknown. About a decade ago, a series of findings made me suspect that saccharin may be a key causative factor for IBD, through its inhibition on gut bacteria and the resultant impaired inactivation of digestive proteases and over digestion of the mucus layer and gut barrier (the Bacteria-Protease-Mucus-Barrier hypothesis). It explained many puzzles in IBD such as its emergence and temporal changes in last century. Recently I further found evidence suggesting sucralose may be also linked to IBD through a similar mechanism as saccharin and have contributed to the recent worldwide increase of IBD. This new hypothesis suggests that UC and CD are just two symptoms of the same morbidity, rather than two different diseases. They are both caused by a weakening in gut barrier and only differ in that UC is mainly due to increased infiltration of gut bacteria and the resultant recruitment of neutrophils and formation of crypt abscess, while CD is mainly due to increased infiltration of antigens and particles from gut lumen and the resultant recruitment of macrophages and formation of granulomas. It explained the delayed appearance but accelerated increase of CD over UC and many other phenomena. This paper aims to provide a detailed description of a unified hypothesis regarding the etiology of IBD, including the cause and mechanism of IBD, as well as the relationship between UC and CD.

Aspartame-fed zebrafish exhibit acute deaths with swimming defects and saccharin-fed zebrafish have elevation of cholesteryl ester transfer protein activity in hypercholesterolemia.

Although many artificial sweeteners (AS) have safety issues, the AS have been widely used in industry. To determine the physiologic effect of AS in the presence of hyperlipidemia, zebrafish were fed aspartame or saccharin with a high-cholesterol diet (HCD). After 12 days, 30% of zebrafish, which consumed aspartame and HCD, died with exhibiting swimming defects. The aspartame group had 65% survivability, while the control and saccharin groups had 100% survivability. Under HCD, the saccharin-fed groups had the highest increase in the serum cholesterol level (599 mg/dL). Aspartame-fed group showed a remarkable increase in serum glucose (up to 125 mg/dL), which was 58% greater than the increase in the HCD alone group. The saccharin and HCD groups had the highest cholesteryl ester transfer protein (CETP) activity (52% CE-transfer), while the HCD alone group had 42% CE-transfer. Histologic analysis revealed that the aspartame and HCD groups showed more infiltration of inflammatory cells in the brain and liver sections. Conclusively, under presence of hyperlipidemia, aspartame-fed zebrafish exhibited acute swimming defects with an increase in brain inflammation. Saccharin-fed zebrafish had an increased atherogenic serum lipid profile with elevation of CETP activity.

Effect of saccharin on albino rats' blood indices and the therapeutic action of vitamins C and E.

The present work aimed to study some blood indices of rats as affected by saccharin and the therapeutic action of vitamins C and E. The used adult female Rattus norvegicus albino rats in the present study were weighing 100-120 g. Administration of saccharin at a dose of 35 mg kg⁻¹ body weight (b.wt.) day⁻¹ for 35 days significantly decreased serum glucose, triglycerides, cholesterol, total protein and albumin values. These decrements were by 20.16%, 22.76%, 44.92%, 20.16% and 40.44%, respectively, compared to control level (p value < 0.01). But it increased levels of kidney function indices. The effect of saccharin was more pronounced on creatinine. Activities of Alanine aminotranferease (ALT), aspartate aminotransferase (AST) and Alkaline phosphatase (ALP) increased significantly following saccharin treatment to rats. Concerning hematoligical parameters, the more obvious changes were observed in the increment of white blood cell (WBC), mean corpuscular volume (MCV) and platelets (PLT) and the decrease in hematocrit, hemoglobin (Hb) and red blood cells (RBCs) count in response to the administration of saccharin. In general, vitamin C or E (150 mg kg⁻¹ b.wt. day⁻¹ for 35 days) was able to reduce the effects of saccharin intake. Both vitamins, however, generally have beneficial effects in reducing the changes in the studied parameters.

Genotoxicity testing of low-calorie sweeteners: aspartame, acesulfame-K, and saccharin.

Low-calorie sweeteners are chemicals that offer the sweetness of sugar without the calories. Consumers are increasingly concerned about the quality and safety of many products present in the diet, in particular, the use of low-calorie sweeteners, flavorings, colorings, preservatives, and dietary supplements. In the present study, we evaluated the mutagenicity of the three low-calorie sweeteners in the Ames/Salmonella/microsome test and their genotoxic potential by comet assay in the bone marrow cells of mice. Swiss albino mice, Mus musculus, were orally administered with different concentrations of aspartame (ASP; 7, 14, 28, and 35 mg/kg body weight), acesulfame-K (ASK; 150, 300, and 600 mg/kg body weight), and saccharin (50, 100, and 200 mg/kg body weight) individually. Concurrently negative and positive control sets were maintained. The animals were sacrificed and the bone marrow cells were processed for comet assay. The standard plate-incorporation assay was carried with the three sweeteners in Salmonella typhimurium TA 97a and TA 100 strains both in the absence and presence of the S9 mix. The comet parameters of DNA were increased in the bone marrow cells due to the sweetener-induced DNA strand breaks, as revealed by increased comet-tail extent and percent DNA in the tail. ASK and saccharin were found to induce greater DNA damage than ASP. However, none could act as a potential mutagen in the Ames/Salmonella /microsome test. These findings are important, since they represent a potential health risk associated with the exposure to these agents.

The potential toxicity of artificial sweeteners.

Since their discovery, the safety of artificial sweeteners has been controversial. Artificial sweeteners provide the sweetness of sugar without the calories. As public health attention has turned to reversing the obesity epidemic in the United States, more individuals of all ages are choosing to use these products. These choices may be beneficial for those who cannot tolerate sugar in their diets (e.g., diabetics). However, scientists disagree about the relationships between sweeteners and lymphomas, leukemias, cancers of the bladder and brain, chronic fatigue syndrome, Parkinson's disease, Alzheimer's disease, multiple sclerosis, autism, and systemic lupus. Recently these substances have received increased attention due to their effects on glucose regulation. Occupational health nurses need accurate and timely information to counsel individuals regarding the use of these substances. This article provides an overview of types of artificial sweeteners, sweetener history, chemical structure, biological fate, physiological effects, published animal and human studies, and current standards and regulations.

Categorizing mistaken false positives in regulation of human and environmental health.

One of the concerns often voiced by critics of the precautionary principle is that a widespread regulatory application of the principle will lead to a large number of false positives (i.e., over-regulation of minor risks and regulation of nonexisting risks). The present article proposes a general definition of a regulatory false positive, and seeks to identify case studies that can be considered authentic regulatory false positives. Through a comprehensive review of the science policy literature for proclaimed false positives and interviews with authorities on regulation and the precautionary principle we identified 88 cases. Following a detailed analysis of these cases, we found that few of the cases mentioned in the literature can be considered to be authentic false positives. As a result, we have developed a number of different categories for these cases of "mistaken false positives," including: real risks, "The jury is still out," nonregulated proclaimed risks, "Too narrow a definition of risk," and risk-risk tradeoffs. These categories are defined and examples are presented in order to illustrate their key characteristics. On the basis of our analysis, we were able to identify only four cases that could be defined as regulatory false positives in the light of today's knowledge and recognized uncertainty: the Southern Corn Leaf Blight, the Swine Flu, Saccharin, and Food Irradiation in relation to consumer health. We conclude that concerns about false positives do not represent a reasonable argument against future application of the precautionary principle.

Induction of systemic protection against rust infection in broad bean by saccharin: effects on plant growth and development.

Here, we examine the effect of saccharin on the induction of systemic resistance in broad bean (Vicia faba) to the rust fungus Uromyces viciae-fabae. Saccharin was applied to beans at the three-leaf stage, either as a soil drench or by painting the solution on to first leaves. Plants were then challenge inoculated with the rust 1, 6, 10 and 14 d following saccharin treatment, after which they were harvested, assessed for the intensity of rust infection and plant growth measurements conducted. Foliar application of saccharin did not induce systemic protection to rust infection until 14 d after application and was less effective than saccharin applied as a soil drench. When saccharin was applied as a drench, systemic protection was not observed until 6 d after application, but was still apparent in plants 14 d after application of the drench. Irrespective of the method of application, saccharin had no significant effect on fresh and dry weights or leaf area of the plants. Saccharin applied as a drench did, however, reduce the number of leaflets produced.

Effects of a saccharin and cyclamate mixture on rat embryos.

Sodium saccharin (NaS) and calcium cyclamate (CaC) are artificial sweeteners widely used in food and drink. To evaluate their toxicological effects on preimplantation mammalian embryos, pregnant rats were gavaged with 1.65 mg NaS/kg bw + 3.85 mg CaC/kg bw (DI) or 6.6 mg NaS/kg bw + 15.4 mg CaC/kg bw (D2) on days 1, 2, 3 and 4 of pregnancy (positive vaginal smear = day 1). The female rats were killed on day 5 of the pregnancy (GD 5), maternal organs weighed, and the blastocysts collected, counted and evaluated for gross morphology, cell number and mitotic index. There was no alteration in maternal organ weights, but there was an increase of the cell number/embryo in the dams treated with that NaS + CaC mixtures (D1 = 37.20 +/- 7.96; D2 = 37.26 +/- 10.90) compared to control group (32.24 +/- 6.73). Embryos whose dams were exposed to NaS + CaC may have adapted for implantation into the uterus but more studies are needed to demonstrate this mechanism of action.

Development and implementation of the IPCS conceptual framework for evaluating mode of action of chemical carcinogens.

The framework developed by the International Programme on Chemical Safety (IPCS) for assessing the mode of action of tumour induction of chemicals in experimental animals has been illustrated with d-limonene, sodium saccharin, di(2-ethylhexyl)phthalate (DEHP) and sulfamethazine as examples. d-Limonene causes renal tumours only in male rats through a response associated with alpha(2u)-globulin. Sodium saccharin induces urinary bladder tumours only in male rats through formation of a urinary precipitate causing erosion of the bladder surface and extensive regenerative hyperplasia. DEHP causes liver tumours in rats and mice through activation of the receptor PPAR alpha leading to peroxisome proliferation and hepatocellular proliferation. Sulfamethazine induces thyroid follicular cell tumours in rats and mice through a mechanism involving altered thyroid hormone homeostasis.

Carcinogenicity of saccharin in laboratory animals and humans: letter to Dr. Harry Conacher of Health Canada.

We appreciate this opportunity to provide input to the Health Protection Branch's (HPB's) review of the artificial sweetener saccharin. Concerns with regard to the safety of saccharin are of great public health significance and of great interest to the public because saccharin is consumed by tens of millions of people, including children and fetuses. Any evidence of carcinogenesis--and there is ample such evidence--of such a widely used chemical should spur health officials to minimize human exposure to it. It is worth noting that on October 31, 1997, the Board of Scientific Counselors of the National Toxicology Program, a unit of the National Institute of Environmental Health Sciences (NIEHS), voted not to delist saccharin from its Report on Carcinogens.