Davide Degli Esposti

First Experimental Demonstration of the Multipotential Carcinogenic Effects of Aspartame Administered in the Feed to Sprague-Dawley Rats

The Cesare Maltoni Cancer Research Center of the European Ramazzini Foundation has conducted a long-term bioassay on aspartame (APM), a widely used artificial sweetener. APM was administered with feed to 8-week-old Sprague-Dawley rats (100–150/sex/group), at concentrations of 100,000, 50,000, 10,000, 2,000, 400, 80, or 0 ppm. The treatment lasted until natural death, at which time all deceased animals underwent complete necropsy. Histopathologic evaluation of all pathologic lesions and of all organs and tissues collected was routinely performed on each animal of all experimental groups. The results of the study show for the first time that APM, in our experimental conditions, causes a) an increased incidence of malignant-tumor–bearing animals with a positive significant trend in males (p ≤ 0.05) and in females (p ≤ 0.01), in particular those females treated at 50,000 ppm (p ≤ 0.01); b) an increase in lymphomas and leukemias with a positive significant trend in both males (p ≤ 0.05) and females (p ≤ 0.01), in particular in females treated at doses of 100,000 (p ≤ 0.01), 50,000 (p ≤ 0.01), 10,000 (p ≤ 0.05), 2,000 (p ≤ 0.05), or 400 ppm (p ≤ 0.01); c) a statistically significant increased incidence, with a positive significant trend (p ≤ 0.01), of transitional cell carcinomas of the renal pelvis and ureter and their precursors (dysplasias) in females treated at 100,000 (p ≤ 0.01), 50,000 (p ≤ 0.01), 10,000 (p ≤ 0.01), 2,000 (p ≤ 0.05), or 400 ppm (p ≤ 0.05); and d) an increased incidence of malignant schwannomas of peripheral nerves with a positive trend (p ≤ 0.05) in males. The results of this mega-experiment indicate that APM is a multipotential carcinogenic agent, even at a daily dose of 20 mg/kg body weight, much less than the current acceptable daily intake. On the basis of these results, a reevaluation of the present guidelines on the use and consumption of APM is urgent and cannot be delayed.

Life-Span Exposure to Low Doses of Aspartame Beginning during Prenatal Life Increases Cancer Effects in Rats

Background In a previous study conducted at the Cesare Maltoni Cancer Research Center of the European Ramazzini Foundation (CMCRC/ERF), we demonstrated for the first time that aspartame (APM) is a multipotent carcinogenic agent when various doses are administered with feed to Sprague-Dawley rats from 8 weeks of age throughout the life span. Objective The aim of this second study is to better quantify the carcinogenic risk of APM, beginning treatment during fetal life. Methods We studied groups of 70–95 male and female Sprague-Dawley rats administered APM (2,000, 400, or 0 ppm) with feed from the 12th day of fetal life until natural death. Results Our results show a) a significant dose-related increase of malignant tumor–bearing animals in males (p < 0.01), particularly in the group treated with 2,000 ppm APM (p < 0.01); b) a significant increase in incidence of lymphomas/leukemias in males treated with 2,000 ppm (p < 0.05) and a significant dose-related increase in incidence of lymphomas/leukemias in females (p < 0.01), particularly in the 2,000-ppm group (p < 0.01); and c) a significant dose-related increase in incidence of mammary cancer in females (p < 0.05), particularly in the 2,000-ppm group (p < 0.05). Conclusions The results of this carcinogenicity bioassay confirm and reinforce the first experimental demonstration of APM’s multipotential carcinogenicity at a dose level close to the acceptable daily intake for humans. Furthermore, the study demonstrates that when life-span exposure to APM begins during fetal life, its carcinogenic effects are increased.

Consequences of Exposure to Carcinogens Beginning During Developmental Life

The increased incidence of cancer over the last 50-60 years may be largely attributed to two factors: the ageing of the population and the diffusion of agents and situations presenting carcinogenic risks. Today, we have entered into a new era in which populations are ever-increasingly exposed to diffuse carcinogenic risks, present not only in the occupational, but also in the general environment. We must now also consider an additional factor in the carcinogenic process, that is, the age in which exposure to carcinogenic risks begins. Apart from the paradigmatic cases of diethylstilboestrol and ionizing radiation, the available epidemiological data concerning the adult consequences of developmental exposure to carcinogens is very limited. However, important data have been provided by long-term experimental carcinogenicity bioassays conducted using rodents. This paper reports a selection of studies conducted in the laboratories of the Cesare Maltoni Cancer Research Center of the European Ramazzini Foundation in which exposure to the chemical agents vinyl acetate monomer, ethyl alcohol and aspartame was started during developmental life and continued into adulthood. The results of these studies provide supporting evidence that lifespan exposure to carcinogenic agents beginning during developmental life produces an overall increase in the carcinogenic effects observed. Moreover, when comparing prenatal and postnatal exposure, the data demonstrate that the development of cancers may appear earlier in life.

Results of Long-Term Carcinogenicity Bioassay on Sprague-Dawley Rats Exposed to Aspartame Administered in Feed

Abstract:  Aspartame (APM) is one of the most widely used artificial sweeteners in the world. Its ever‐growing use in more than 6000 products, such as soft drinks, chewing gum, candy, desserts, etc., has been accompanied by rising consumer concerns regarding its safety, in particular its potential long‐term carcinogenic effects. In light of the inadequacy of the carcinogenicity bioassays performed in the 1970s and 1980s, a long‐term mega‐experiment on APM was undertaken at the Cesare Maltoni Cancer Research Center of the European Ramazzini Foundation on groups of male and female Sprague‐Dawley rats (100–150/sex/group), 8 weeks old at the start of the experiment. APM was administered in feed at concentrations of 100,000, 50,000, 10,000, 2,000, 400, 80, or 0 ppm. Treatment lasted until spontaneous death of the animals. The results of the study demonstrate that APM causes: (a) an increased incidence of malignant tumor‐bearing animals, with a positive significant trend in both sexes, and in particular in females treated at 50,000 ppm (P≤ 0.01) when compared to controls; (b) an increase in lymphomas–leukemias, with a positive significant trend in both sexes, and in particular in females treated at doses of 100,000 (P≤ 0.01), 50,000 (P≤ 0.01), 10,000 (P≤ 0.05), 2000 (P≤ 0.05), and 400 ppm (P≤ 0.01); (c) a statistically significant increased incidence, with a positive significant trend, of transitional cell carcinomas of the renal pelvis and ureter in females and particularly in those treated at 100,000 ppm (P≤ 0.05); and (d) an increased incidence of malignant schwannomas of the peripheral nerves, with a positive trend in males (P≤ 0.05). The results of this mega‐experiment indicate that APM, in the tested experimental conditions, is a multipotential carcinogenic agent.

Actein activates stress- and statin-associated responses and is bioavailable in Sprague-Dawley rats.

The purpose of this study was to assess in rats the pharmacological parameters and effects on gene expression in the liver of the triterpene glycoside actein. Actein, an active component from the herb black cohosh, has been shown to inhibit the proliferation of human breast cancer cells. To conduct our assessment, we determined the molecular effects of actein on livers from Sprague‐Dawley rats treated with actein at 35.7 mg/kg for 6 and 24 h. Chemogenomic analyses indicated that actein elicited stress and statin‐associated responses in the liver; actein altered expression of cholesterol and fatty acid biosynthetic genes, p53 pathway genes, CCND1 and ID3. Real‐time RT‐PCR validated that actein induced three time‐dependent patterns of gene expression in the liver: (i) a decrease followed by a significant increase of HMGCS1, HMGCR, HSD17B7, NQO1, S100A9; (ii) a progressive increase of BZRP and CYP7A1 and (iii) a significant increase followed by a decrease of CCND1 and ID3. Consistent with actein’s statin‐ and stress‐associated responses, actein reduced free fatty acid and cholesterol content in the liver by 0.6‐fold at 24 h and inhibited the growth of human HepG2 liver cancer cells. To determine the bioavailability of actein, we collected serum samples for pharmacokinetic analysis at various times up to 24 h. The serum level of actein peaked at 2.4 μg/mL at 6 h. Actein’s ability to alter pathways involved in lipid disorders and carcinogenesis may make it a new agent for preventing and treating these major disorders.

Results of a Long‐Term Carcinogenicity Bioassay on Sprague‐Dawley Rats Exposed to Sodium Arsenite Administered in Drinking Water

Abstract:  Arsenic (As) is a metal found in nature whose acute and chronic toxic effects have been known for decades. Hundreds of millions of people are at risk of exposure to As and its various chemical forms which can occur in the occupational and general environment in air, water, soil, food, and medicines. Several epidemiological studies have shown that prolonged exposure to As can induce various types of malignant tumors in humans, namely, skin, lung, liver, kidney, and bladder cancers. These effects have been observed particularly in geographic areas where people are exposed to well water with high concentrations of As. While the risks of As at high concentrations are well documented, there is still a great deal of uncertainty regarding the risk of exposure to As at very low levels. This uncertainty is due to the absence of adequate epidemiological data and the insufficiency of experimental data currently available. Given the limited evidence demonstrating the carcinogenic potential of As in animals, a long‐term carcinogenicity bioassay on sodium arsenite (NaAsO2) was performed at the Cesare Maltoni Cancer Research Center (CMCRC) of the European Ramazzini Foundation (ERF). NaAsO2 was administrated with drinking water at concentrations of 200, 100, 50, or 0 mg/L, for 104 weeks to Sprague‐Dawley rats (50/sex/group), 8 weeks old at the start of the study. The animals were monitored until spontaneous death at which time each animal underwent complete necropsy. Histopathological evaluation of all pathological lesions and of all organs and tissues collected was routinely performed on each animal. The results demonstrate that in our experimental conditions NaAsO2 induces sparse benign and malignant tumors among treated rats. The types of tumors observed are infrequent in the strain of Sprague‐Dawley rats of the colony used in our laboratory, namely, lung adenomas and carcinomas, kidney adenomas/papillomas and carcinomas, and bladder carcinomas. Notably, an elevated incidence of these types of oncological lesions is also observed among people living in geographical areas where As is present at higher concentrations in drinking water.

Pharmacological mechanisms of black cohosh in Sprague-Dawley rats.

BACKGROUND Studies indicate that extracts and purified components from black cohosh inhibit the growth of human breast cancer cells, but the molecular targets and signaling pathways have not yet been defined. PURPOSE This study examines the pharmacological mechanisms and toxicological effects in the short term of the herb black cohosh on female Sprague-Dawley rats. MATERIALS AND METHODS To assess effects on gene activity and lipid content, we treated female Sprague-Dawley rats with an extract of black cohosh enriched in triterpene glycosides (27%) at 35.7 or 0mg/kg. Four animals for each group were sacrificed at 1, 6 and 24h after treatment; liver tissue and serum samples were obtained for gene expression and lipid analysis. RESULTS Microarray analysis of rat liver tissue indicated that black cohosh markedly downregulated mitochondrial oxidative phosphorylation genes. Phospholipid biosynthesis and remodeling, PI3-Kinase and sphingosine signaling were upregulated, driven largely by an upregulation of several isoforms of phospholipase C. Hierarchical clustering indicated that black cohosh clustered with antiproliferative compounds, specifically tubulin binding vinca alkaloids and DNA alkylators. In support of this, black cohosh repressed the expression of cyclin D1 and ID3, and inhibited the proliferation of HepG2, p53 positive, liver cancer cells. Black cohosh reduced the level of free fatty acids at 6 and 24h and triglycerides at 6h in the serum, but increased the free fatty acid and triglyceride content of the treated livers at 24h. CONCLUSION Our results suggest that black cohosh warrants further study for breast cancer prevention and therapy.

A transcriptomic analysis of black cohosh: Actein alters cholesterol biosynthesis pathways and synergizes with simvastatin

Previous studies indicate that the herb black cohosh (Actaea racemosa L.) and the triterpene glycoside actein inhibit the growth of human breast cancer cells and activate stress-associated responses. This study assessed the transcriptomic effects of black cohosh and actein on rat liver tissue, using Ingenuity and ToxFX analyses. Sprague-Dawley rats were treated with an extract of black cohosh enriched in triterpene glycosides (27%) for 24 h or actein for 6 and 24 h, at 35.7 mg/kg, and liver tissue collected for gene expression analysis. Ingenuity analysis indicates the top canonical pathways are, for black cohosh, RAR Activation, and, for actein, Superpathway of Cholesterol Biosynthesis, at 24 h. Actein alters the expression of cholesterol biosynthetic genes, but does not inhibit HMG-CoA reductase activity. Black cohosh and actein inhibited the growth of human breast and colon cancer cells and synergized with the statin simvastatin. Combinations of black cohosh with certain classes of statins could enhance their activity, as well as toxic, such as inflammatory liver, side effects. Transcriptomic analysis indicates black cohosh and actein warrant further study to prevent and treat cancer and lipid disorders. This study lays the basis for an approach to characterize the mode of action and toxicity of herbal medicines.