Exploring occupational toxicant exposures in patients with metabolic dysfunction-associated steatotic liver disease: A prospective pilot study.

BACKGROUND: Metabolic dysfunction-associated steatotic liver disease (MASLD) has been traditionally associated with insulin resistance and obesity. Recently, pollutants have been shown to contribute to the development of MASLD. Given the global burden of MASLD, understanding whether pollutants are merely associated with steatosis or contribute to its progression to advanced chronic liver disease (ACLD) and hepatocellular carcinoma (HCC) is critical. Workers exposed to occupational toxicants represent an ideal population for assessing the potentially hazardous consequences of professional exposure. Confirming a link between occupational exposure and ACLD/HCC may not only provide further elements in understanding MASLD, but also contribute to preventive strategies for exposed workers. OBJECTIVE: This study aimed to assess the prevalence of self-reported occupational exposure to toxicants in patients with MASLD. METHODS: This hospital-based prospective pilot study included 201 patients with MASLD. Data on workplace toxicant exposure were collected systematically using a structured questionnaire. Subsequently, patients with ACLD and/or HCC (n = 55) were compared to controls (n = 146). Logistic regression analysis and propensity score models were used to investigate the associations between self-reported occupational exposure and ACLD and/or HCC. RESULTS: Patients with ACLD/HCC reported exposure to metals, halogenated refrigerants, pain/resins, and fuel emissions more often than the controls. After controlling for confounders, durations of 21-30 years and >30 years of occupational exposure to toxicants showed odds ratios (ORs) of 2.31 (95 % confidence interval [CI]: 1.09-4.88, p = 0.029) and 4.47 (95 % CI: 2.57-7.78, p<0.001), respectively. CONCLUSIONS: In this pilot study, patients with MASLD complications were more likely to report workplace toxicant exposure. Our results warrant future multicentre confirmatory studies, as implementing prevention policies may reduce the risk of life-threatening diseases among exposed populations.

Evaluation of Toxicant-Associated Fatty Liver Disease and Liver Neoplastic Progress in Sprague-Dawley Rats Treated with Low Doses of Aflatoxin B1 Alone or in Combination with Extremely Low Frequency Electromagnetic Fields.

The term toxicant-associated fatty liver disease (TAFLD) has been proposed to describe fatty liver diseases connected to toxicants other than alcohol. Aflatoxins are mycotoxins commonly found as contaminants in foods and feeds, which are known liver toxicants and potential candidates as potential causes of TAFLD. Aflatoxin B1 (AFB1) was administered at low doses to Sprague-Dawley (SD) rats, alone or in combination with S-50 Hz an extremely low frequency electromagnetic field (ELFEMF), to study the evolution of TAFLD, preneoplastic and neoplastic lesions of the liver and the potential enhancing effect of lifespan exposure to ELFEMF. Steatosis, inflammation and foci of different types were significantly increased in both aflatoxin-treated males and females, which is consistent with a pattern of TAFLD. A significant increase in adenomas, cystic dilation of biliary ducts, hepatocellular hyperplasia and hypertrophy and oval cell hyperplasia were also observed in treated females only. The administration of low doses of AFB1 caused TAFLD in SD rats, inducing liver lesions encompassing fatty infiltration, foci of different types and adenomas. Furthermore, the pattern of change observed in preneoplastic liver lesions often included liver steatosis and steatohepatitis (TASH). ELFEMF did not result in any enhancing or toxic effect in the liver of SD rats.

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

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 × 10-9). In female infants, high maternal urinary glyphosate (above the median) was associated with longer AGD-AC (ß = 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 (ß = 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.

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

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.

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.

Historical evidence of glyphosate exposure from a US agricultural cohort.

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.

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

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.

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.

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.

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.

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

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.

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

[This corrects the article DOI: 10.1289/EHP419.].

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.

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

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.

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

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.

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

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.

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.

Paired Serum and Urine Concentrations of Biomarkers of Diethyl Phthalate, Methyl Paraben, and Triclosan in Rats.

BACKGROUND: Exposure to environmental chemicals, including phthalates and phenols such as parabens and triclosan, is ubiquitous within the U.S. general population. OBJECTIVE: This proof-of-concept rodent study examined the relationship between oral doses of three widely used personal care product ingredients [diethyl phthalate (DEP), methyl paraben (MPB), and triclosan] and urine and serum concentrations of their respective biomarkers. METHODS: Using female Sprague-Dawley rats, we carried out two rounds of experiments with oral gavage doses selected in accordance with no observed adverse effect levels (NOAELs) derived from previous studies: 1,735 (DEP), 1,050 (MPB), 50 (triclosan) mg/kg/day. Administered doses ranged from 0.005 to 173 mg/kg/day, 10-100,000 times below the NOAEL for each chemical. Controls for the MPB and triclosan experiments were animals treated with olive oil (vehicle) only; controls for the DEP serum experiments were animals treated with the lowest doses of MPB and triclosan. Doses were administered for 5 days with five rats in each treatment group. Urine and blood serum, collected on the last day of exposure, were analyzed for biomarkers. Relationships between oral dose and biomarker concentrations were assessed using linear regression. RESULTS: Biomarkers were detected in all control urine samples at parts-per-billion levels, suggesting a low endemic environmental exposure to the three chemicals that could not be controlled even with all of the precautionary measures undertaken. Among the exposed animals, urinary concentrations of all three biomarkers were orders of magnitude higher than those in serum. A consistently positive linear relationship between oral dose and urinary concentration was observed (R2 > 0.80); this relationship was inconsistent in serum. CONCLUSIONS: Our study highlights the importance of carefully considering the oral dose used in animal experiments and provides useful information in selecting doses for future studies. CITATION: Teitelbaum SL, Li Q, Lambertini L, Belpoggi F, Manservisi F, Falcioni L, Bua L, Silva MJ, Ye X, Calafat AM, Chen J. 2016. Paired serum and urine concentrations of biomarkers of diethyl phthalate, methyl paraben, and triclosan in rats. Environ Health Perspect 124:39-45; http://dx.doi.org/10.1289/ehp.1409586.