Comparative Toxicogenomics of Glyphosate and Roundup Herbicides by Mammalian Stem Cell-Based Genotoxicity Assays and Molecular Profiling in Sprague-Dawley Rats.

Whether glyphosate-based herbicides (GBHs) are more potent than glyphosate alone at activating cellular mechanisms, which drive carcinogenesis remain controversial. As GBHs are more cytotoxic than glyphosate, we reasoned they may also be more capable of activating carcinogenic pathways. We tested this hypothesis by comparing the effects of glyphosate with Roundup GBHs both in vitro and in vivo. First, glyphosate was compared with representative GBHs, namely MON 52276 (European Union), MON 76473 (United Kingdom), and MON 76207 (United States) using the mammalian stem cell-based ToxTracker system. Here, MON 52276 and MON 76473, but not glyphosate and MON 76207, activated oxidative stress and unfolded protein responses. Second, molecular profiling of liver was performed in female Sprague-Dawley rats exposed to glyphosate or MON 52276 (at 0.5, 50, and 175 mg/kg bw/day glyphosate) for 90 days. MON 52276 but not glyphosate increased hepatic steatosis and necrosis. MON 52276 and glyphosate altered the expression of genes in liver reflecting TP53 activation by DNA damage and circadian rhythm regulation. Genes most affected in liver were similarly altered in kidneys. Small RNA profiling in liver showed decreased amounts of miR-22 and miR-17 from MON 52276 ingestion. Glyphosate decreased miR-30, whereas miR-10 levels were increased. DNA methylation profiling of liver revealed 5727 and 4496 differentially methylated CpG sites between the control and glyphosate and MON 52276 exposed animals, respectively. Apurinic/apyrimidinic DNA damage formation in liver was increased with glyphosate exposure. Altogether, our results show that Roundup formulations cause more biological changes linked with carcinogenesis than glyphosate.

Use of Shotgun Metagenomics and Metabolomics to Evaluate the Impact of Glyphosate or Roundup MON 52276 on the Gut Microbiota and Serum Metabolome of Sprague-Dawley Rats.

BACKGROUND: There is intense debate on whether glyphosate can inhibit the shikimate pathway of gastrointestinal microorganisms, with potential health implications. OBJECTIVES: We tested whether glyphosate or its representative EU herbicide formulation Roundup MON 52276 affects the rat gut microbiome. METHODS: We combined cecal microbiome shotgun metagenomics with serum and cecum metabolomics to assess the effects of glyphosate [0.5, 50, 175mg/kg body weight (BW) per day] or MON 52276 at the same glyphosate-equivalent doses, in a 90-d toxicity test in rats. RESULTS: Glyphosate and MON 52276 treatment resulted in ceca accumulation of shikimic acid and 3-dehydroshikimic acid, suggesting inhibition of 5-enolpyruvylshikimate-3-phosphate synthase of the shikimate pathway in the gut microbiome. Cysteinylglycine, γ-glutamylglutamine, and valylglycine levels were elevated in the cecal microbiome following glyphosate and MON 52276 treatments. Altered cecum metabolites were not differentially expressed in serum, suggesting that the glyphosate and MON 52276 impact on gut microbial metabolism had limited consequences on physiological biochemistry. Serum metabolites differentially expressed with glyphosate treatment were associated with nicotinamide, branched-chain amino acid, methionine, cysteine, and taurine metabolism, indicative of a response to oxidative stress. MON 52276 had similar, but more pronounced, effects than glyphosate on the serum metabolome. Shotgun metagenomics of the cecum showed that treatment with glyphosate and MON 52276 resulted in higher levels of Eggerthella spp., Shinella zoogleoides, Acinetobacter johnsonii, and Akkermansia muciniphila. Shinella zoogleoides was higher only with MON 52276 exposure. In vitro culture assays with Lacticaseibacillus rhamnosus strains showed that Roundup GT plus inhibited growth at concentrations at which MON 52276 and glyphosate had no effect. DISCUSSION: Our study highlights the power of multi-omics approaches to investigate the toxic effects of pesticides. Multi-omics revealed that glyphosate and MON 52276 inhibited the shikimate pathway in the rat gut microbiome. Our findings could be used to develop biomarkers for epidemiological studies aimed at evaluating the effects of glyphosate herbicides on humans. https://doi.org/10.1289/EHP6990.

Identification of aspartame-induced haematopoietic and lymphoid tumours in rats after lifetime treatment.

Lymphomas and leukaemias involving the lung have in some cases been hard to distinguish from respiratory tract infection in Sprague-Dawley (SD) rats from long-term bioassays. In order to differentiate between tumours and immune cell infiltrates, updated pathological criteria and nomenclature were used and immunohistochemistry (IHC) was applied to haematopoietic and lymphoid tissue tumours (HLTs) in the original prenatal long-term Aspartame (APM) study performed by the Ramazzini Institute (RI). All 78 cases of HLTs from treated and control groups were re-examined based on light microscopic morphological characteristics and subjected to a panel of IHC markers including Ki67, CD3, PAX5, CD20, CD68, TdT, CD45, CD14 and CD33. The analysis confirmed the diagnoses of HLTs in 72 cases, identified 3 cases of preneoplastic lesions (lymphoid hyperplasia), and categorized 3 cases as inflammatory lesions. A statistically significant increase in total HLTs (p = 0.006), total lymphomas (p = 0.032) and total leukaemias (p = 0.031) in treated female rats was confirmed (high dose vs control), and a statistically significant linear trend for each HLT type was also observed. After the HLT cases re-evaluation, the results obtained are consistent with those reported in the previous RI publication and reinforce the hypothesis that APM has a leukaemogenic and lymphomatogenic effect.

Gene expression profiles for low-dose exposure to diethyl phthalate in rodents and humans: a translational study with implications for breast carcinogenesis.

Phthalates are commonly included as ingredients in personal care products such as cosmetics, shampoos and perfumes. Diethyl phthalate (DEP) has been found to be anti-androgenic and linked with adverse reproductive effects on males, but effects on females are poorly understood. We designed an integrative and translational study to experimentally examine the effects of DEP exposure at a human-equivalent dose on the mammary transcriptome in rats and to subsequently examine the DEP gene signature in breast tissues (both pre-malignant and tumor) from a population study. In Sprague-Dawley rats treated orally with DEP from birth to adulthood, we identified a signature panel of 107 genes predominantly down-regulated by DEP exposure. Univariate analysis of this 107 DEP gene signature in pre-malignant breast tissues revealed that six genes (P4HA1, MPZL3, TMC4, PLEKHA6, CA8, AREG) were inversely associated with monoethyl phthalate (MEP; the urinary metabolite of DEP) concentration (p < 0.05) among postmenopausal women; all six genes loaded on to one of seven factors identified by factor analysis. Transcription factor enrichment analysis revealed that genes in this factor were enriched for androgen receptor binding sites. These six genes were also significantly down-regulated in pre-malignant adjacent tissues compared to the corresponding tumor tissues in pair-wise analyses (p < 0.05). Results from our translational study indicate that low level exposure to diethyl phthalate results in measurable genomic changes in breast tissue with implications in breast carcinogenesis.

The Contribution of In Vivo Mammalian Studies to the Knowledge of Adverse Effects of Radiofrequency Radiation on Human Health.

The proliferation of cellular antennas and other radiofrequency radiation (RFR) generating devices of the last decades has led to more and more concerns about the potential health effects from RFR exposure. Since the 2011 classification as a possible carcinogen by the International Agency for Research on Cancer (IARC), more experimental studies have been published that support a causal association between RFR exposure and health hazards. As regard cancer risk, two long-term experimental studies have been recently published by the US National Toxicology Program (NTP) and the Italian Ramazzini Institute (RI). Despite important experimental differences, both studies found statistically significant increases in the development of the same type of very rare glial malignant tumors. In addition to carcinogenicity, reproductive organs might be particularly exposed, as well as sensitive to RFR. In this work, we reviewed the currently available evidence from in vivo studies on carcinogenicity and reproductive toxicity studies in order to summarize the contribution of experimental research to the prevention of the adverse effects of RFR on human health.

The Ramazzini Institute 13-week pilot study glyphosate-based herbicides administered at human-equivalent dose to Sprague Dawley rats: effects on development and endocrine system.

BACKGROUND: Glyphosate-based herbicides (GBHs) are broad-spectrum herbicides that act on the shikimate pathway in bacteria, fungi, and plants. The possible effects of GBHs on human health are the subject of an intense public debate for both its potential carcinogenic and non-carcinogenic effects, including potential effects on the endocrine system The present pilot study examine whether exposure to GBHs at the dose of glyphosate considered to be "safe" (the US Acceptable Daily Intake - ADI - of 1.75 mg/kg bw/day), starting from in utero life, affect the development and endocrine system across different life stages in Sprague Dawley (SD) rats. METHODS: Glyphosate alone and Roundup Bioflow, a commercial brand of GBHs, were administered in drinking water at 1.75 mg/kg bw/day to F0 dams starting from the gestational day (GD) 6 (in utero) up to postnatal day (PND) 120. After weaning, offspring were randomly distributed in two cohorts: 8 M + 8F/group animals belonging to the 6-week cohort were sacrificed after puberty at PND 73 ± 2; 10 M + 10F/group animals belonging to the 13-week cohort were sacrificed at adulthood at PND 125 ± 2. Effects of glyphosate or Roundup exposure were assessed on developmental landmarks and sexual characteristics of pups. RESULTS: In pups, anogenital distance (AGD) at PND 4 was statistically significantly increased both in Roundup-treated males and females and in glyphosate-treated males. Age at first estrous (FE) was significantly delayed in the Roundup-exposed group and serum testosterone concentration significantly increased in Roundup-treated female offspring from the 13-week cohort compared to control animals. A statistically significant increase in plasma TSH concentration was observed in glyphosate-treated males compared with control animals as well as a statistically significant decrease in DHT and increase in BDNF in Roundup-treated males. Hormonal status imbalances were more pronounced in Roundup-treated rats after prolonged exposure. CONCLUSIONS: The present pilot study demonstrate that GBHs exposure, from prenatal period to adulthood, induced endocrine effects and altered reproductive developmental parameters in male and female SD rats. In particular, it was associated with androgen-like effects, including a statistically significant increase of AGDs in both males and females, delay of FE and increased testosterone in female.

Histology and Transcriptome Profiles of the Mammary Gland across Critical Windows of Development in Sprague Dawley Rats.

Breast development occurs through well-defined stages representing 'windows of susceptibility' to adverse environmental exposures that potentially modify breast cancer risk. Systematic characterization of morphology and transcriptome during normal breast development lays the foundation of our understanding of cancer etiology. We examined mammary glands in female Sprague Dawley rats across six developmental stages - pre-pubertal, peri-pubertal, pubertal, lactation, adult parous and adult nulliparous. We investigated histology by Hematoxylin and Eosin and Mallory's Trichrome stain, proliferative and apoptotic rate by immunohistochemistry and whole-transcriptome by microarrays. We identified differentially expressed genes between adjacent developmental stages by linear models, underlying pathways by gene ontology analysis and gene networks and hubs active across developmental stages by coexpression network analysis. Mammary gland development was associated with large-scale changes in the transcriptome; particularly from pre-pubertal to peri-pubertal period and the lactation period were characterized by distinct patterns of gene expression with unique biological functions such as immune processes during pre-pubertal development and cholesterol biosynthesis during lactation. These changes were reflective of the shift in mammary gland histology, from a rudimentary organ during early stages to a secretory organ during lactation followed by regression with age. Hub genes within mammary gene networks included metabolic genes such as Pparg during the pre-pubertal stage and tight junction-related genes claudins and occludins in lactating mammary glands. Transcriptome profile paired with histology enhanced our understanding of mammary development, which is fundamental in understanding the etiologic mechanism of breast cancer, especially pertaining to windows of susceptibility to environmental exposures that may alter breast cancer risk.

The Ramazzini Institute 13-week pilot study on glyphosate and Roundup administered at human-equivalent dose to Sprague Dawley rats: effects on the microbiome.

BACKGROUND: Glyphosate-based herbicides (GBHs) are broad-spectrum herbicides that act on the shikimate pathway in bacteria, fungi, and plants. The possible effects of GBHs on human health are the subject of an intense public debate for both its potential carcinogenic and non-carcinogenic effects, including its effects on microbiome. The present pilot study examines whether exposure to GBHs at doses of glyphosate considered to be "safe" (the US Acceptable Daily Intake - ADI - of 1.75 mg/kg bw/day), starting from in utero, may modify the composition of gut microbiome in Sprague Dawley (SD) rats. METHODS: Glyphosate alone and Roundup, a commercial brand of GBHs, were administered in drinking water at doses comparable to the US glyphosate ADI (1.75 mg/kg bw/day) to F0 dams starting from the gestational day (GD) 6 up to postnatal day (PND) 125. Animal feces were collected at multiple time points from both F0 dams and F1 pups. The gut microbiota of 433 fecal samples were profiled at V3-V4 region of 16S ribosomal RNA gene and further taxonomically assigned and assessed for diversity analysis. We tested the effect of exposure on overall microbiome diversity using PERMANOVA and on individual taxa by LEfSe analysis. RESULTS: Microbiome profiling revealed that low-dose exposure to Roundup and glyphosate resulted in significant and distinctive changes in overall bacterial composition in F1 pups only. Specifically, at PND31, corresponding to pre-pubertal age in humans, relative abundance for Bacteriodetes (Prevotella) was increased while the Firmicutes (Lactobacillus) was reduced in both Roundup and glyphosate exposed F1 pups compared to controls. CONCLUSIONS: This study provides initial evidence that exposures to commonly used GBHs, at doses considered safe, are capable of modifying the gut microbiota in early development, particularly before the onset of puberty. These findings warrant future studies on potential health effects of GBHs in early development such as childhood.

Changes in mammary histology and transcriptome profiles by low-dose exposure to environmental phenols at critical windows of development.

Exposure to environmental chemicals has been linked to altered mammary development and cancer risk at high doses using animal models. Effects at low doses comparable to human exposure remain poorly understood, especially during critical developmental windows. We investigated the effects of two environmental phenols commonly used in personal care products - methyl paraben (MPB) and triclosan (TCS) - on the histology and transcriptome of normal mammary glands at low doses mimicking human exposure during critical windows of development. Sprague-Dawley rats were exposed during perinatal, prepubertal and pubertal windows, as well as from birth to lactation. Low-dose exposure to MPB and TCS induced measurable changes in both mammary histology (by Masson's Trichrome Stain) and transcriptome (by microarrays) in a window-specific fashion. Puberty represented a window of heightened sensitivity to MPB, with increased glandular tissue and changes of expression in 295 genes with significant enrichment in functions such as DNA replication and cell cycle regulation. Long-term exposure to TCS from birth to lactation was associated with increased adipose and reduced glandular and secretory tissue, with expression alterations in 993 genes enriched in pathways such as cholesterol synthesis and adipogenesis. Finally, enrichment analyses revealed that genes modified by MPB and TCS were over-represented in human breast cancer gene signatures, suggesting possible links with breast carcinogenesis. These findings highlight the issues of critical windows of susceptibility that may confer heightened sensitivity to environmental insults and implicate the potential health effects of these ubiquitous environmental chemicals in breast cancer.

Synergism between sinusoidal-50 Hz magnetic field and formaldehyde in triggering carcinogenic effects in male Sprague-Dawley rats.

BACKGROUND: Experimental rodent bioassays performed up to now have failed to provide conclusive confirmation of the carcinogenicity of extremely low frequency magnetic fields (ELFMF). OBJECTIVES: To evaluate the potential synergistic carcinogenic effects of concurrent exposure to ELFMF and formaldehyde in four groups of male and female Sprague-Dawley rats. METHODS: One group was exposed from prenatal life until natural death to S-50 Hz MF and to formaldehyde in drinking water from 6 weeks of age for 104 weeks, two groups were treated only with formaldehyde or only with MF and one group served as untreated control. RESULTS: Compared to untreated controls, exposure to MF and formaldehyde causes in males a statistically significant increased incidence of malignant tumors (P ≤ 0.01), thyroid C-cell carcinomas (P ≤ 0.01), and hemolymphoreticular neoplasias (P ≤ 0.05). No statistically significant differences were observed among female groups. CONCLUSIONS: Life-span exposure to MF and formaldehyde induces statistically significant carcinogenic effects in male rats. Am. J. Ind. Med. 59:509-521, 2016. © 2016 Wiley Periodicals, Inc.

Life-span exposure to sinusoidal-50 Hz magnetic field and acute low-dose γ radiation induce carcinogenic effects in Sprague-Dawley rats.

Background In 2002 the International Agency for Research on Cancer classified extremely low frequency magnetic fields (ELFMF) as a possible carcinogen on the basis of epidemiological evidence. Experimental bioassays on rats and mice performed up to now on ELFMF alone or in association with known carcinogens have failed to provide conclusive confirmation. Objectives To study the carcinogenic effects of combined exposure to sinusoidal-50 Hz (S-50 Hz) magnetic fields and acute γ radiation in Sprague-Dawley rats. Methods We studied groups of male and female Sprague-Dawley rats exposed from prenatal life until natural death to 20 or 1000 µT S-50 Hz MF and also to 0.1 Gy γ radiation delivered as a single acute exposure at 6 weeks of age. Results The results of the study showed significant carcinogenic effects for the mammary gland in males and females and a significant increased incidence of malignant schwannomas of the heart as well as increased incidence of lymphomas/leukemias in males. Conclusions These results call for a re-evaluation of the safety of non-ionizing radiation.

Life-span carcinogenicity studies on Sprague-Dawley rats exposed to γ-radiation: design of the project and report on the tumor occurrence after post-natal radiation exposure (6 weeks of age) delivered in a single acute exposure.

BACKGROUND: Experimental long-term carcinogenicity bioassays conducted on rats and mice proved that ionizing radiation can induce a variety of tumor types. However few studies have been conducted on rats. METHODS: This report deals with the effects of γ-radiation in groups of 416-1,051 6-weeks old Sprague-Dawley rats exposed to 0, 0.1, 1, or 3 Gy of γ-radiation delivered in a single acute exposure. The experiment lasted for the animals' lifespan and all were necropsied and underwent full histopathological evaluation. RESULTS: The results confirm the dose-related carcinogenic effects of γ-radiation for several organs and tissues. Moreover they indicate that exposure to 0.1 Gy induces a statistically significant increased incidence in Zymbal gland carcinomas and pancreas islet cell carcinomas in females. CONCLUSIONS: Our data show that exposure to γ-radiation induces carcinogenic effects at all tested doses.

The carcinogenic effects of aspartame: The urgent need for regulatory re-evaluation.

Aspartame (APM) is an artificial sweetener used since the 1980s, now present in >6,000 products, including over 500 pharmaceuticals. Since its discovery in 1965, and its first approval by the US Food and Drugs Administration (FDA) in 1981, the safety of APM, and in particular its carcinogenicity potential, has been controversial. The present commentary reviews the adequacy of the design and conduct of carcinogenicity bioassays on rodents submitted by G.D. Searle, in the 1970s, to the FDA for market approval. We also review how experimental and epidemiological data on the carcinogenic risks of APM, that became available in 2005 motivated the European Commission (EC) to call the European Food and Safety Authority (EFSA) for urgent re-examination of the available scientific documentation (including the Searle studies). The EC has further requested that, if the results of the evaluation should suggest carcinogenicity, major changes must be made to the current APM specific regulations. Taken together, the studies performed by G.D. Searle in the 1970s and other chronic bioassays do not provide adequate scientific support for APM safety. In contrast, recent results of life-span carcinogenicity bioassays on rats and mice published in peer-reviewed journals, and a prospective epidemiological study, provide consistent evidence of APM's carcinogenic potential. On the basis of the evidence of the potential carcinogenic effects of APM herein reported, a re-evaluation of the current position of international regulatory agencies must be considered an urgent matter of public health.

Aspartame administered in feed, beginning prenatally through life span, induces cancers of the liver and lung in male Swiss mice.

BACKGROUND: Aspartame (APM) is a well-known intense artificial sweetener used in more than 6,000 products. Among the major users of aspartame are children and women of childbearing age. In previous lifespan experiments conducted on Sprague-Dawley rats we have shown that APM is a carcinogenic agent in multiple sites and that its effects are increased when exposure starts from prenatal life. OBJECTIVE: The aim of this study is to evaluate the potential of APM to induce carcinogenic effects in mice. METHODS: Six groups of 62-122 male and female Swiss mice were treated with APM in feed at doses of 32,000, 16,000, 8,000, 2,000, or 0 ppm from prenatal life (12 days of gestation) until death. At death each animal underwent complete necropsy and all tissues and organs of all animals in the experiment were microscopically examined. RESULTS: APM in our experimental conditions induces in males a significant dose-related increased incidence of hepatocellular carcinomas (P < 0.01), and a significant increase at the dose levels of 32,000 ppm (P < 0.01) and 16,000 ppm (P < 0.05). Moreover, the results show a significant dose-related increased incidence of alveolar/bronchiolar carcinomas in males (P < 0.05), and a significant increase at 32,000 ppm (P < 0.05). CONCLUSIONS: The results of the present study confirm that APM is a carcinogenic agent in multiple sites in rodents, and that this effect is induced in two species, rats (males and females) and mice (males). No carcinogenic effects were observed in female mice. Am. J. Ind. Med. 53:1197-1206, 2010. © 2010 Wiley-Liss, Inc.

Effects of different electromagnetic fields on circadian rhythms of some haematochemical parameters in rats.

OBJECTIVE: To investigate the effects of different electromagnetic fields on some haematochemical parameters of circadian rhythms in Sprague-Dawley rats. METHODS: The study was carried out in 18 male and 18 female rats in good health conditions exposed to 50 Hz magnetic sinusoid fields at the intensity of 1000 microT, 100 microT, and 0 microT (control group) respectively, and in 18 male and 18 female rats in good health conditions exposed to 1.8 GHz electromagnetic fields at the intensity of 50 V/m, 25 V/m and 0 V/m (control group), respectively. Following haematochemical parameters for glucose, triglycerides, and total cholesterol were measured. RESULTS: Different effects of electromagnetic fields on circadian rhythms of both male and female rats were observed. Different changes occurred in some haematochemical parameters for glucose, triglycerides, and total cholesterol (P < 0.05). CONCLUSION: Exposure to different electromagnetic fields is responsible for the variations of some haematochemical parameters in rats.

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 bioassays on Coca-Cola administered to Sprague-Dawley rats.

Coca-Cola was invented in May 1886 in Atlanta, Georgia by a pharmacist who, by accident or design, mixed carbonated water with the syrup of sugar, phosphoric acid, caffeine, and other natural flavors to create what is known as "the world's favorite soft drink." Coca-Cola is currently sold in more than 200 countries and in early 2000, the company sold its 10 billionth unit case of Coca-Cola branded products. Given the worldwide consumption of Coca-Cola, a project of experimental bioassays to study its long-term effects when administered as substitute for drinking water on male and female Sprague-Dawley rats was planned and executed. The objective of the project was to study whether and how long-term consumption of Coca-Cola affects the basic tumorigram of test animals. The bioassays were performed on rats beginning at different ages, namely: (a) on males and females exposed since embryonic life or from 7 weeks of age; and (b) on males and females exposed from 30, 39, or 55 weeks of age. Overall, the project included 1999 rats. During the biophase, data were collected on fluid and feed consumption, body weight, and survival. Animals were kept under observation until spontaneous death and underwent complete necropsy. The results indicate: (a) an increase in body weight in all treated animals; (b) a statistically significant increase of the incidence in females, both breeders and offspring, bearing malignant mammary tumors; (c) a statistically significant increase in the incidence of exocrine ademonas of the pancreas in both male and female breeders and offspring; and (d) an increased incidence, albeit not statistically significant, of pancreatic islet cell carcinomas in females, a malignant tumor which occurs very rarely in our historical controls. On the basis of the results of this study, excessive consumption of regular soft-drinks should be generally discouraged, in particular for children and adolescents.