Publications by authors named "Fiorella Belpoggi"

54 Publications

Multi-omics phenotyping of the gut-liver axis reveals metabolic perturbations from a low-dose pesticide mixture in rats.

Commun Biol 2021 Apr 14;4(1):471. Epub 2021 Apr 14.

Gene Expression and Therapy Group, King's College London, Faculty of Life Sciences & Medicine, Department of Medical and Molecular Genetics, Guy's Hospital, London, UK.

Health effects of pesticides are not always accurately detected using the current battery of regulatory toxicity tests. We compared standard histopathology and serum biochemistry measures and multi-omics analyses in a subchronic toxicity test of a mixture of six pesticides frequently detected in foodstuffs (azoxystrobin, boscalid, chlorpyrifos, glyphosate, imidacloprid and thiabendazole) in Sprague-Dawley rats. Analysis of water and feed consumption, body weight, histopathology and serum biochemistry showed little effect. Contrastingly, serum and caecum metabolomics revealed that nicotinamide and tryptophan metabolism were affected, which suggested activation of an oxidative stress response. This was not reflected by gut microbial community composition changes evaluated by shotgun metagenomics. Transcriptomics of the liver showed that 257 genes had their expression changed. Gene functions affected included the regulation of response to steroid hormones and the activation of stress response pathways. Genome-wide DNA methylation analysis of the same liver samples showed that 4,255 CpG sites were differentially methylated. Overall, we demonstrated that in-depth molecular profiling in laboratory animals exposed to low concentrations of pesticides allows the detection of metabolic perturbations that would remain undetected by standard regulatory biochemical measures and which could thus improve the predictability of health risks from exposure to chemical pollutants.
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http://dx.doi.org/10.1038/s42003-021-01990-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8046807PMC
April 2021

Maternal urinary levels of glyphosate during pregnancy and anogenital distance in newborns in a US multicenter pregnancy cohort.

Environ Pollut 2021 Mar 22;280:117002. Epub 2021 Mar 22.

Department of Environmental Medicine and Public Heath, Icahn School of Medicine at Mount Sinai, New York, NY, USA. Electronic address:

Human exposure to glyphosate has become ubiquitous because of its increasing agricultural use. Recent studies suggest endocrine disrupting effects of glyphosate. Specifically, in our work in rodents, low-dose early-life exposure to Roundup® (glyphosate-based herbicide) lengthened anogenital distance (AGD) in male and female offspring. AGD is a marker of the prenatal hormone milieu in rodents and humans. The relationship between glyphosate exposure and AGD has not been studied in humans. We conducted a pilot study in 94 mother-infant pairs (45 female and 49 male) from The Infant Development and the Environment Study (TIDES). For each infant, two AGD measurements were collected after birth; the anopenile (AGD-AP) and anoscrotal (AGD-AS) distances for males, and anoclitoral (AGD-AC) and anofourchette distances (AGD-AF) for females. We measured levels of glyphosate and its degradation product aminomethylphosphonic acid (AMPA) in 2nd trimester maternal urine samples using ultra-high-performance liquid chromatography-tandem mass spectrometry. We assessed the relationship between exposure and AGD using sex-stratified multivariable linear regression models. Glyphosate and AMPA were detected in 95% and 93% of the samples (median 0.22 ng/mL and 0.14 ng/mL, respectively). Their concentrations were moderately correlated (r = 0.55, p = 5.7 × 10). In female infants, high maternal urinary glyphosate (above the median) was associated with longer AGD-AC (β = 1.48, 95%CI (-0.01, 3.0), p = 0.05), but this was not significant after covariate adjustment. Increased AMPA was associated with longer AGD-AF (β = 1.96, 95%CI (0.44, 3.5), p = 0.01) after adjusting for infant size and age at AGD exam. No associations were detected in male offspring. These preliminary findings partially reproduce our previous results in rodents and suggest that glyphosate is a sex-specific endocrine disruptor with androgenic effects in humans. Given the increasing glyphosate exposures in the US population, larger studies should evaluate potential developmental effects on endocrine and reproductive systems.
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http://dx.doi.org/10.1016/j.envpol.2021.117002DOI Listing
March 2021

Response to "Cancerogenic effects of radiofrequency radiation: A statistical reappraisal".

Environ Res 2021 Mar 26;197:111067. Epub 2021 Mar 26.

Ramazzini Institute, Bologna, Italy. Electronic address:

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http://dx.doi.org/10.1016/j.envres.2021.111067DOI Listing
March 2021

Low-dose exposure of glyphosate-based herbicides disrupt the urine metabolome and its interaction with gut microbiota.

Sci Rep 2021 Feb 5;11(1):3265. Epub 2021 Feb 5.

Department of Environmental Medicine and Public Health, Icahn School of Medicine at Mount Sinai, 1425 Madison Avenue, New York, NY, USA.

Glyphosate-based herbicides (GBHs) can disrupt the host microbiota and influence human health. In this study, we explored the potential effects of GBHs on urinary metabolites and their interactions with gut microbiome using a rodent model. Glyphosate and Roundup (equal molar for glyphosate) were administered at the USA glyphosate ADI guideline (1.75 mg/kg bw/day) to the dams and their pups. The urine metabolites were profiled using non-targeted liquid chromatography-high resolution mass spectrometry (LC-HRMS). Our results found that overall urine metabolite profiles significantly differed between dams and pups and between female and male pups. Specifically, we identified a significant increase of homocysteine, a known risk factor of cardiovascular disease in both Roundup and glyphosate exposed pups, but in males only. Correlation network analysis between gut microbiome and urine metabolome pointed to Prevotella to be negatively correlated with the level of homocysteine. Our study provides initial evidence that exposures to commonly used GBH, at a currently acceptable human exposure dose, is capable of modifying urine metabolites in both rat adults and pups. The link between Prevotella-homocysteine suggests the potential role of GBHs in modifying the susceptibility of homocysteine, which is a metabolite that has been dysregulated in related diseases like cardiovascular disease or inflammation, through commensal microbiome.
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http://dx.doi.org/10.1038/s41598-021-82552-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7864973PMC
February 2021

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.

Environ Health Perspect 2021 Jan 27;129(1):17005. Epub 2021 Jan 27.

Gene Expression and Therapy Group, Department of Medical and Molecular Genetics, King's College London, Faculty of Life Sciences & Medicine, Guy's Hospital, London, UK.

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, ] 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, , 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 spp., , , and . was higher only with MON 52276 exposure. culture assays with 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.
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http://dx.doi.org/10.1289/EHP6990DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7839352PMC
January 2021

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

Acta Histochem 2020 Jul 20;122(5):151548. Epub 2020 May 20.

Cesare Maltoni Cancer Research Center, Ramazzini Institute, via Saliceto 3, 40010 Bentivoglio, Bologna, Italy. Electronic address:

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.
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http://dx.doi.org/10.1016/j.acthis.2020.151548DOI Listing
July 2020

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

Sci Rep 2020 04 27;10(1):7067. Epub 2020 Apr 27.

Department of Environmental Medicine and Public Health, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

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.
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http://dx.doi.org/10.1038/s41598-020-63904-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7184607PMC
April 2020

Protocol for a systematic review and meta-analysis of human exposure to pesticide residues in honey and other bees' products.

Environ Res 2020 07 3;186:109470. Epub 2020 Apr 3.

Cesare Maltoni Cancer Research Center, Ramazzini Institute, Bologna, Italy.

Background: The presence of pesticides in honey and related products is an increasing concern for consumers and producers, although there is lack of data on the current burden of exposure of the general human population through these products. We present a protocol for a systematic review and meta-analysis of contamination to insecticides, herbicides and fungicides of products from honeybees, and an estimation of how much the consumption of these products contributes to the ADI (Acceptable Daily Intake) of selected substances.

Objectives: We aim to systematically review and meta-analyse studies on the contamination to plant protection products in honey, royal jelly, beeswax and propolis, applying the Navigation Guide and WHO-ILO systematic review methodology as an organizing framework.

Data Sources: We will search electronic academic databases for potentially relevant records from PubMed, TOXNET and EMBASE. We will include quantitative studies analysing the contamination from insecticides, herbicides and fungicides in honey, propolis, royal jelly and beeswax. In particular, we will evaluate the presence of the following substances and classes of pesticides: Glyphosate, Chlorpyrifos, pyrethroid and neonicotinoid pesticides, fungicides and acaricides.

Study Appraisal And Synthesis Methods: At least two authors will independently screen titles and abstracts at a first stage of review, and full texts at a second stage, of potentially eligible records against the eligibility criteria; data extraction of included studies will then be performed by at least two authors, in blind. At least two authors will assess risk of bias and the quality of evidence, using the most suited tools currently available. The data on prevalence of contaminated samples and concentration of pesticides in the products will be combined using meta-analysis: when more than three studies reporting the necessary measures to fit the models are available, meta-analysis will be performed separately by product and by exposure; otherwise, weighted descriptive analysis will be performed. We will report the results using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines (PRISMA).
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http://dx.doi.org/10.1016/j.envres.2020.109470DOI Listing
July 2020

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

Int J Environ Res Public Health 2019 09 12;16(18). Epub 2019 Sep 12.

Cesare Maltoni Cancer Research Center, Ramazzini Institute, Castello di Bentivoglio, via Saliceto 3, Bentivoglio, 40010 Bologna, Italy.

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.
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http://dx.doi.org/10.3390/ijerph16183379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6765993PMC
September 2019

Effects of short and long-term alcohol-based fixation on Sprague-Dawley rat tissue morphology, protein and nucleic acid preservation.

Acta Histochem 2019 Aug 3;121(6):750-760. Epub 2019 Jul 3.

Cesare Maltoni Cancer Research Center (CMCRC), Ramazzini Institute (RI), Bentivoglio, Bologna, Italy. Electronic address:

Safety concerns on the toxic and carcinogenic effects of formalin exposure have drawn increasing attention to the search for alternative low risk fixatives for processing tissue specimens in laboratories worldwide. Alcohol-based fixatives are considered some of the most promising alternatives. We evaluated the performance of alcohol-fixed paraffin-embedded (AFPE) samples from Sprague-Dawley (SD) rats analyzing tissue morphology, protein and nucleic acid preservation after short and extremely long fixation times (up to 7 years), using formalin-fixed paraffin-embedded (FFPE) samples as a comparator fixative. Following short and long-term alcohol fixation, tissue morphology and cellular details in tissues, evaluated by scoring stained sections (Hematoxylin-Eosin and Mallory's trichrome), were optimally preserved if compared to formalin fixation. Immunoreactivity of proteins (Ki67, CD3, PAX5, CD68), evaluated by immunohistochemistry, showed satisfactory results when the fixation period did not exceed 1 year. Finally, we confirm the superiority of alcohol fixation compared to formalin, in terms of quantity of nucleic acid extracted from paraffin blocks, even after an extremely long time of alcohol fixation. Our results confirm that alcohol fixation is a suitable and safe alternative to formalin for pathological evaluations. There is a need for standardization of formalin-free methods and harmonization of diagnosis in pathology department worldwide.
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http://dx.doi.org/10.1016/j.acthis.2019.05.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6939362PMC
August 2019

Historical evidence of glyphosate exposure from a US agricultural cohort.

Environ Health 2019 05 7;18(1):42. Epub 2019 May 7.

Department of Environmental and Occupational Health, Milken Institute School of Public Health, The George Washington University, 950 New Hampshire Ave, Washington, DC, 20052, USA.

In response to the recent review by Gillezeau et al., The evidence of human exposure to glyphosate: A review, Environmental Health 1/19/19, here we report additional glyphosate biomonitoring data from a repository of urine samples collected from United States farmers in 1997-98. To determine if glyphosate exposure could be identified historically, we examined urine samples from a biorepository of specimens collected from US dairy farmers between 1997 and 98. We compared samples from farmers who self-reported glyphosate application in the 8 h prior to sample collection to samples from farm applicators who did not report using glyphosate. Of 18 applicator samples tested, 39% showed detectable levels of glyphosate (mean concentration 4.04 μg/kg; range:1.3-12) compared to 0% detections among 17 non glyphosate applicator samples (p-value < 0.01). One of the applicator samples that tested positive for glyphosate also tested positive for AMPA. Concentrations of glyphosate were consistent with levels reported in the prior occupational biomonitoring studies reviewed by Gillezeau et al.Accurately detecting both glyphosate and AMPA in this small sample of Wisconsin farmers demonstrates a) glyphosate exposures among farmers were occurring 20 years ago, which was prior to the widespread planting of genetically engineered glyphosate tolerant crops first approved in 1996; and b) liquid chromatography tandem mass spectrometry (LC-MS/MS) can be used for sensitive characterization in cryopreserved urine samples. These data offer an important historical benchmark to which urinary levels from current and future biomonitoring studies can be compared.
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http://dx.doi.org/10.1186/s12940-019-0474-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6503538PMC
May 2019

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.

Environ Health 2019 03 12;18(1):15. Epub 2019 Mar 12.

Cesare Maltoni Cancer Research Center (CMCRC), Ramazzini Institute (RI), Via Saliceto, 3, 40010, Bentivoglio, Bologna, Italy.

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.
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http://dx.doi.org/10.1186/s12940-019-0453-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6413565PMC
March 2019

Letter to the Editor (February 14, 2018) concerning the paper "Histological findings and lung dust analysis as the basis for occupational disease compensation in asbestos-related lung cancer in Germany".

Int J Occup Med Environ Health 2018 12 13;31(6):837-839. Epub 2018 Nov 13.

Justus Liebig University, Giessen, Germany (University Hospital of Giessen and Marburg, Institute and Outpatients' Clinic for Occupational and Social Medicine (IPAS)).

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http://dx.doi.org/10.13075/ijomeh.1896.01332DOI Listing
December 2018

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

J Mammary Gland Biol Neoplasia 2018 09 28;23(3):149-163. Epub 2018 Jun 28.

Department of Environmental Medicine and Public Health, Icahn School of Medicine at Mount Sinai, Box 1057, 1 Gustave Levy Place, New York, NY, 10029, USA.

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.
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http://dx.doi.org/10.1007/s10911-018-9401-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6103804PMC
September 2018

The need for independent research on the health effects of glyphosate-based herbicides.

Environ Health 2018 05 29;17(1):51. Epub 2018 May 29.

Cesare Maltoni Cancer Research Center Ramazzini Institute, Via Saliceto, 3, 40010, Bentivoglio, Bologna, Italy.

Background: Glyphosate, formulated as Roundup, is the world's most widely used herbicide. Glyphosate is used extensively on genetically modified (GM) food crops designed to tolerate the herbicide, and global use is increasing rapidly. Two recent reviews of glyphosate's health hazards report conflicting results. An independent review by the International Agency for Research on Cancer (IARC) found that glyphosate is a "probable human carcinogen". A review by the European Food Safety Agency (EFSA) found no evidence of carcinogenic hazard. These differing findings have produced regulatory uncertainty.

Regulatory Actions: Reflecting this regulatory uncertainty, the European Commission on November 27 2017, extended authorization for glyphosate for another 5 years, while the European Parliament opposed this decision and issued a call that pesticide approvals be based on peer-reviewed studies by independent scientists rather than on the current system that relies on proprietary industry studies.

Ramazzini Institute Response: The Ramazzini Institute has initiated a pilot study of glyphosate's health hazards that will be followed by an integrated experimental research project. This evaluation will be independent of industry support and entirely sponsored by worldwide crowdfunding. The aim of the Ramazzini Institute project is to explore comprehensively the effects of exposures to glyphosate-based herbicides at current real-world levels on several toxicological endpoints, including carcinogenicity, long-term toxicity, neurotoxicity, endocrine disrupting effects, prenatal developmental toxicity, the microbiome and multi-generational effects.
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http://dx.doi.org/10.1186/s12940-018-0392-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5972398PMC
May 2018

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

Environ Health 2018 05 29;17(1):50. Epub 2018 May 29.

Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, 1428 Madison, New York, NY, 10029, USA.

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.
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http://dx.doi.org/10.1186/s12940-018-0394-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5972442PMC
May 2018

The Ramazzini Institute 13-week study on glyphosate-based herbicides at human-equivalent dose in Sprague Dawley rats: study design and first in-life endpoints evaluation.

Environ Health 2018 05 29;17(1):52. Epub 2018 May 29.

Cesare Maltoni Cancer Research Center (CMCRC), Ramazzini Institute (RI), Via Saliceto, 3, 40010 Bentivoglio, Bologna, Italy.

Background: Glyphosate-based herbicides (GBHs) are the most widely used pesticides worldwide, and glyphosate is the active ingredient of such herbicides, including the formulation known as Roundup. The massive and increasing use of GBHs results in not only the global burden of occupational exposures, but also increased exposure to the general population. The current pilot study represents the first phase of a long-term investigation of GBHs that we are conducting over the next 5 years. In this paper, we present the study design, the first evaluation of in vivo parameters and the determination of glyphosate and its major metabolite aminomethylphosphonic acid (AMPA) in urine.

Methods: We exposed Sprague-Dawley (SD) rats orally via drinking water to a dose of glyphosate equivalent to the United States Acceptable Daily Intake (US ADI) of 1.75 mg/kg bw/day, defined as the chronic Reference Dose (cRfD) determined by the US EPA, starting from prenatal life, i.e. gestational day (GD) 6 of their mothers. One cohort was continuously dosed until sexual maturity (6-week cohort) and another cohort was continuously dosed until adulthood (13-week cohort). Here we present data on general toxicity and urinary concentrations of glyphosate and its major metabolite AMPA.

Results: Survival, body weight, food and water consumption of the animals were not affected by the treatment with either glyphosate or Roundup. The concentration of both glyphosate and AMPA detected in the urine of SD rats treated with glyphosate were comparable to that observed in animals treated with Roundup, with an increase in relation to the duration of treatment. The majority of glyphosate was excreted unchanged. Urinary levels of the parent compound, glyphosate, were around 100-fold higher than the level of its metabolite, AMPA.

Conclusions: Glyphosate concentrations in urine showed that most part of the administered dose was excreted as unchanged parent compound upon glyphosate and Roundup exposure, with an increasing pattern of glyphosate excreted in urine in relation to the duration of treatment. The adjuvants and the other substances present in Roundup did not seem to exert a major effect on the absorption and excretion of glyphosate. Our results demonstrate that urinary glyphosate is a more relevant marker of exposure than AMPA in the rodent model.
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http://dx.doi.org/10.1186/s12940-018-0393-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5972408PMC
May 2018

Diagnosis, monitoring and prevention of exposure-related non-communicable diseases in the living and working environment: DiMoPEx-project is designed to determine the impacts of environmental exposure on human health.

J Occup Med Toxicol 2018 5;13. Epub 2018 Feb 5.

20Center for Human Genetics, University of Leuven, Leuven, Belgium.

The WHO has ranked environmental hazardous exposures in the living and working environment among the top risk factors for chronic disease mortality. Worldwide, about 40 million people die each year from noncommunicable diseases (NCDs) including cancer, diabetes, and chronic cardiovascular, neurological and lung diseases. The exposure to ambient pollution in the living and working environment is exacerbated by individual susceptibilities and lifestyle-driven factors to produce complex and complicated NCD etiologies. Research addressing the links between environmental exposure and disease prevalence is key for prevention of the pandemic increase in NCD morbidity and mortality. However, the long latency, the chronic course of some diseases and the necessity to address cumulative exposures over very long periods does mean that it is often difficult to identify causal environmental exposures. EU-funded COST Action DiMoPEx is developing new concepts for a better understanding of health-environment (including gene-environment) interactions in the etiology of NCDs. The overarching idea is to teach and train scientists and physicians to learn how to include efficient and valid exposure assessments in their research and in their clinical practice in current and future cooperative projects. DiMoPEx partners have identified some of the emerging research needs, which include the lack of evidence-based exposure data and the need for human-equivalent animal models mirroring human lifespan and low-dose cumulative exposures. Utilizing an interdisciplinary approach incorporating seven working groups, DiMoPEx will focus on aspects of air pollution with particulate matter including dust and fibers and on exposure to low doses of solvents and sensitizing agents. Biomarkers of early exposure and their associated effects as indicators of disease-derived information will be tested and standardized within individual projects. Risks arising from some NCDs, like pneumoconioses, cancers and allergies, are predictable and preventable. Consequently, preventative action could lead to decreasing disease morbidity and mortality for many of the NCDs that are of major public concern. DiMoPEx plans to catalyze and stimulate interaction of scientists with policy-makers in attacking these exposure-related diseases.
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http://dx.doi.org/10.1186/s12995-018-0186-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5800006PMC
February 2018

A transcriptomic analysis of turmeric: Curcumin represses the expression of cholesterol biosynthetic genes and synergizes with simvastatin.

Pharmacol Res 2018 06 3;132:176-187. Epub 2018 Feb 3.

Iconix Biosciences, Mountain View, CA 94043, United States.

The spice turmeric (Curcuma longa L.) has a long history of use as an anti-inflammatory agent. The active component curcumin induces a variety of diverse biological effects and forms a series of degradation and metabolic products in vivo. Our hypothesis is that the field of toxicogenomics provides tools that can be used to characterize the mode of action and toxicity of turmeric components and to predict turmeric-drug interactions. Male Sprague-Dawley rats were treated for 4 days with turmeric root containing about 3% curcumin (comparable to what people consume in the fresh or dried root) or a fraction of turmeric enriched for curcumin (∼74%) and liver tissue collected for gene expression analysis. Two doses of each agent were added to the diet, corresponding to 540 and 2700 mg/kg body weight/day of turmeric. The transcriptomic effects of turmeric on rat liver tissue were examined using 3 programs, ToxFx Analysis Suite, in the context of a large drug database, Ingenuity Pathway and NextBio analyses. ToxFx analysis indicates that turmeric containing about 3% or 74% curcumin represses the expression of cholesterol biosynthetic genes. The dose of 400 mg/kg b.w./day curcumin induced the Drug Signature associated with hepatic inflammatory infiltrate. Ingenuity analysis confirmed that all 4 turmeric treatments had a significant effect on cholesterol biosynthesis, specifically the Cholesterol biosynthesis superpathway and Cholesterol biosynthesis 1 and 2. Among the top 10 up or downregulated genes, all 4 treatments downregulated PDK4; while 3 treatments downregulated ANGPTL4 or FASN. These findings suggest curcumin may enhance the anticancer effects of certain classes of statins, which we confirmed with biological assays. Given this enhancement, lower levels of statins may be required, and even be desirable. Our findings also warn of possible safety issues, such as potential inflammatory liver effects, for patients who ingest a combination of certain classes of statins and curcumin. Transcriptomic analysis suggests that turmeric is worthwhile to study to prevent and treat cancer and lipid disorders. Our approach lays new groundwork for studies of the mode of action and safety of herbal medicines and can also be used to develop a methodology to standardize herbal medicines.
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http://dx.doi.org/10.1016/j.phrs.2018.01.023DOI Listing
June 2018

New insights into the toxicity of mineral fibres: A combined in situ synchrotron μ-XRD and HR-TEM study of chrysotile, crocidolite, and erionite fibres found in the tissues of Sprague-Dawley rats.

Toxicol Lett 2017 May 12;274:20-30. Epub 2017 Apr 12.

Laboratory for Material Chemistry, National Institute of Chemistry, Hajdrihova 19, Ljubljana, Slovenia.

Along the line of the recent research topic aimed at understanding the in vivo activity of mineral fibres and their mechanisms of toxicity, this work describes the morpho-chemical characteristics of the mineral fibres found in the tissues of Sprague-Dawley rats subjected to intraperitoneal/intrapleural injection of UICC chrysotile, UICC crocidolite and erionite-Na from Nevada (USA). The fibres are studied with in situ synchrotron powder diffraction and high resolution transmission electron microscopy to improve our understanding of the mechanisms of toxicity of these mineral fibres. In contact with the tissues of the rats, chrysotile fibres are prone to dissolve, with leaching of Mg and production of a silica rich relict. On the other hand, crocidolite and erionite-Na fibres are stable even for very long contact times within the tissues of the rats, showing just a thin dissolution amorphous halo. These findings support the model of a lower biopersistence of chrysotile with respect to crocidolite and erionite-Na but the formation of a silica-rich fibrous residue after the pseudo-amorphization of chrysotile may justify a higher cytotoxic potential and intense inflammatory activity of chrysotile in the short term in contact with the lung tissues.
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http://dx.doi.org/10.1016/j.toxlet.2017.04.004DOI Listing
May 2017

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

Environ Res 2017 Jan 29;152:233-243. Epub 2016 Oct 29.

Department of Environmental Medicine and Public Health, Icahn School of Medicine at Mount Sinai, Box 1057, 1 Gustave Levy Place, New York, NY 10029, USA; Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, Box 1057, 1 Gustave Levy Place, New York, NY 10029, USA; Department of Medicine, Hematology and Medical Oncology, Icahn School of Medicine at Mount Sinai, Box 1057, 1 Gustave Levy Place, New York, NY 10029, USA; Department of Pediatrics, Icahn School of Medicine at Mount Sinai, Box 1057, 1 Gustave Levy Place, New York, NY 10029, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.envres.2016.10.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5135583PMC
January 2017

Aneuploidy: a common and early evidence-based biomarker for carcinogens and reproductive toxicants.

Environ Health 2016 10 12;15(1):97. Epub 2016 Oct 12.

Department of Environmental and Occupational Health, Milken Institute School of Public Health, The George Washington University, 950 New Hampshire Ave. NW, 4th Floor, Washington, DC, 20052, USA.

Aneuploidy, defined as structural and numerical aberrations of chromosomes, continues to draw attention as an informative effect biomarker for carcinogens and male reproductive toxicants. It has been well documented that aneuploidy is a hallmark of cancer. Aneuploidies in oocytes and spermatozoa contribute to infertility, pregnancy loss and a number of congenital abnormalities, and sperm aneuploidy is associated with testicular cancer. It is striking that several carcinogens induce aneuploidy in somatic cells, and also adversely affect the chromosome compliment of germ cells. In this paper we review 1) the contributions of aneuploidy to cancer, infertility, and developmental abnormalities; 2) techniques for assessing aneuploidy in precancerous and malignant lesions and in sperm; and 3) the utility of aneuploidy as a biomarker for integrated chemical assessments of carcinogenicity, and reproductive and developmental toxicity.
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http://dx.doi.org/10.1186/s12940-016-0180-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5059969PMC
October 2016

Changes in the Metabolome in Response to Low-Dose Exposure to Environmental Chemicals Used in Personal Care Products during Different Windows of Susceptibility.

PLoS One 2016 28;11(7):e0159919. Epub 2016 Jul 28.

Department of Preventive Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, United States of America.

The consequences of ubiquitous exposure to environmental chemicals remain poorly defined. Non-targeted metabolomic profiling is an emerging method to identify biomarkers of the physiological response to such exposures. We investigated the effect of three commonly used ingredients in personal care products, diethyl phthalate (DEP), methylparaben (MPB) and triclosan (TCS), on the blood metabolome of female Sprague-Dawley rats. Animals were treated with low levels of these chemicals comparable to human exposures during prepubertal and pubertal windows as well as chronically from birth to adulthood. Non-targeted metabolomic profiling revealed that most of the variation in the metabolites was associated with developmental stage. The low-dose exposure to DEP, MPB and TCS had a relatively small, but detectable impact on the metabolome. Multiple metabolites that were affected by chemical exposure belonged to the same biochemical pathways including phenol sulfonation and metabolism of pyruvate, lyso-plasmalogens, unsaturated fatty acids and serotonin. Changes in phenol sulfonation and pyruvate metabolism were most pronounced in rats exposed to DEP during the prepubertal period. Our metabolomics analysis demonstrates that human level exposure to personal care product ingredients has detectable effects on the rat metabolome. We highlight specific pathways such as sulfonation that warrant further study.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0159919PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4965097PMC
August 2017

An Integrated Experimental Design for the Assessment of Multiple Toxicological End Points in Rat Bioassays.

Environ Health Perspect 2017 03 22;125(3):289-295. Epub 2016 Jul 22.

Cesare Maltoni Cancer Research Center, Ramazzini Institute, Bentivoglio, Bologna, Italy.

Background: For nearly five decades long-term studies in rodents have been the accepted benchmark for assessing chronic long-term toxic effects, particularly carcinogenicity, of chemicals. The European Food Safety Authority (EFSA) and the World Health Organization (WHO) have pointed out that the current set of internationally utilized test methods capture only some of the potential adverse effects associated with exposures to these agents over the lifetime.

Objectives: In this paper, we propose the adaption of the carcinogenicity bioassay to integrate additional protocols for comprehensive long-term toxicity assessment that includes developmental exposures and long-term outcomes, capable of generating information on a broad spectrum of different end points.

Discussion: An integrated study design based on a stepwise process is described that includes the priority end points of the Economic Co-operation and Development and the National Toxicology Program guidelines on carcinogenicity and chronic toxicity and developmental and reproductive toxicity. Integrating a comprehensive set of relevant toxicological end points in a single protocol represents an opportunity to optimize animal use in accordance with the 3Rs (replacement, reduction and refinement). This strategy has the potential to provide sufficient data on multiple windows of susceptibility of specific interest for risk assessments and public health decision-making by including prenatal, lactational, neonatal exposures and evaluating outcomes over the lifespan.

Conclusion: This integrated study design is efficient in that the same generational cohort of rats used for evaluating long-term outcomes can be monitored in satellite parallel experiments to measure biomarkers and other parameters related to system-specific responses including metabolic alterations and endocrine disturbances. Citation: Manservisi F, Babot Marquillas C, Buscaroli A, Huff J, Lauriola M, Mandrioli D, Manservigi M, Panzacchi S, Silbergeld EK, Belpoggi F. 2017. An integrated experimental design for the assessment of multiple toxicological end points in rat bioassays. Environ Health Perspect 125:289-295; http://dx.doi.org/10.1289/EHP419.
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http://dx.doi.org/10.1289/EHP419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5332192PMC
March 2017

Effect of postnatal low-dose exposure to environmental chemicals on the gut microbiome in a rodent model.

Microbiome 2016 06 14;4(1):26. Epub 2016 Jun 14.

Department of Preventive Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

Background: This proof-of-principle study examines whether postnatal, low-dose exposure to environmental chemicals modifies the composition of gut microbiome. Three chemicals that are widely used in personal care products-diethyl phthalate (DEP), methylparaben (MPB), triclosan (TCS)-and their mixture (MIX) were administered at doses comparable to human exposure to Sprague-Dawley rats from birth through adulthood. Fecal samples were collected at two time points: postnatal day (PND) 62 (adolescence) and PND 181 (adulthood). The gut microbiome was profiled by 16S ribosomal RNA gene sequencing, taxonomically assigned and assessed for diversity.

Results: Metagenomic profiling revealed that the low-dose chemical exposure resulted in significant changes in the overall bacterial composition, but in adolescent rats only. Specifically, the individual taxon relative abundance for Bacteroidetes (Prevotella) was increased while the relative abundance of Firmicutes (Bacilli) was reduced in all treated rats compared to controls. Increased abundance was observed for Elusimicrobia in DEP and MPB groups, Betaproteobacteria in MPB and MIX groups, and Deltaproteobacteria in TCS group. Surprisingly, these differences diminished by adulthood (PND 181) despite continuous exposure, suggesting that exposure to the environmental chemicals produced a more profound effect on the gut microbiome in adolescents. We also observed a small but consistent reduction in the bodyweight of exposed rats in adolescence, especially with DEP and MPB treatment (p < 0.05), which is consistent with our findings of a reduced Firmicutes/Bacteroidetes ratio at PND 62 in exposed rats.

Conclusions: This study provides initial evidence that postnatal exposure to commonly used environmental chemicals at doses comparable to human exposure is capable of modifying the gut microbiota in adolescent rats; whether these changes lead to downstream health effects requires further investigation.
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http://dx.doi.org/10.1186/s40168-016-0173-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4906585PMC
June 2016

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

Am J Ind Med 2016 07 24;59(7):509-21. Epub 2016 May 24.

Cesare Maltoni Cancer Research Center, Ramazzini Institute, Bologna, Italy.

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.
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http://dx.doi.org/10.1002/ajim.22598DOI Listing
July 2016