Effects of Radiofrequency Electromagnetic Field (RF-EMF) exposure on pregnancy and birth outcomes: A systematic review of experimental studies on non-human mammals.

BACKGROUND: The World Health Organization is coordinating an international project aimed at systematically reviewing the evidence regarding the association between radiofrequency electromagnetic field (RF-EMF) exposure and adverse health effects. Within the project, 6 topics have been prioritized by an expert group, which include reproductive health outcomes. OBJECTIVES: According to the protocol published in 2021, a systematic review and meta-analyses on the adverse effects of RF-EMF exposure during pregnancy in offspring of experimental animals were conducted. METHODS: Three electronic databases (PubMed, Scopus and EMF Portal) were last searched on September 8 or 17, 2022. Based on predefined selection criteria, the obtained references were screened by two independent reviewers. Studies were included if they met the following criteria: 1) original, sham controlled experimental study on non-human mammals exposed in utero, published in peer-reviewed journals, 2) the experimental RF-EMF exposure was within the frequency range 100 kHz-300 GHz, 3) the effects of RF-EMF exposure on fecundity (litter size, embryonic/fetal losses), on the offspring health at birth (decrease of weight or length, congenital malformations, changes of sex ratio) or on delayed effects (neurocognitive alterations, female infertility or early-onset cancer) were studied. Study characteristics and outcome data were extracted by two reviewers. Risk of bias (RoB) was assessed using the Office of Health Assessment and Translation (OHAT) guidelines. Study results were pooled in a random effects meta-analysis comparing average exposure to no-exposure and in a dose-response meta-analysis using all exposure doses, after exclusion of studies that were rated at "high concern" for RoB. Subgroup analyses were conducted for species, Specific Absorption Rate (SAR) and temperature increase. The certainty of the evidence was assessed using the Grading of Recommendations, Assessment, Development and Evaluations (GRADE) approach. RESULTS: Eighty-eight papers could be included in this review. Effects on fecundity. The meta-analysis of studies on litter size, conducted at a whole-body average SAR of 4.92 W/kg, did not show an effect of RF-EMF exposure (MD 0.05; 95% CI -0.21 to 0.30). The meta-analysis of studies on resorbed and dead fetuses, conducted at a whole-body average SAR of 20.26 W/kg, showed a significant increase of the incidence in RF-EMF exposed animals (OR 1.84; 95% CI 1.27 to 2.66). The results were similar in the dose-response analysis. Effects on the offspring health at birth. The meta-analysis of studies on fetal weight, conducted at a whole-body average SAR of 9.83 W/kg, showed a small decrease in RF-EMF exposed animals (SMD 0.31; 95% CI 0.15 to 0.48). The meta-analysis of studies on fetal length, conducted at a whole-body average SAR of 4.55 W/kg, showed a moderate decrease in length at birth (SMD 0.45; 95% CI 0.07 to 0.83). The meta-analysis of studies on the percentage of fetuses with malformations, conducted at a whole-body average SAR of 6.75 W/kg, showed a moderate increase in RF-EMF exposed animals (SMD -0.45; 95% CI -0.68 to -0.23). The meta-analysis of studies on the incidence of litters with malformed fetuses, conducted at a whole-body average SAR of 16.63 W/kg, showed a statistically significant detrimental RF-EMF effect (OR 3.22; 95% CI 1.9 to 5.46). The results were similar in the dose-response analyses. Delayed effects on the offspring health. RF-EMF exposure was not associated with detrimental effects on brain weight (SMD 0.10; 95% CI -0.09 to 0.29) and on learning and memory functions (SMD -0.54; 95% CI -1.24 to 0.17). RF-EMF exposure was associated with a large detrimental effect on motor activity functions (SMD 0.79; 95% CI 0.21 to 1.38) and a moderate detrimental effect on motor and sensory functions (SMD -0.66; 95% CI -1.18 to -0.14). RF-EMF exposure was not associated with a decrease of the size of litters conceived by F2 female offspring (SMD 0.08; 95% CI -0.39 to 0.55). Notably, meta-analyses of neurobehavioural effects were based on few studies, which suffered of lack of independent replication deriving from only few laboratories. DISCUSSION: There was high certainty in the evidence for a lack of association of RF-EMF exposure with litter size. We attributed a moderate certainty to the evidence of a small detrimental effect on fetal weight. We also attributed a moderate certainty to the evidence of a lack of delayed effects on the offspring brain weight. For most of the other endpoints assessed by the meta-analyses, detrimental RF-EMF effects were shown, however the evidence was attributed a low or very low certainty. The body of evidence had limitations that did not allow an assessment of whether RF-EMF may affect pregnancy outcomes at exposure levels below those eliciting a well-known adverse heating impact. In conclusion, in utero RF-EMF exposure does not have a detrimental effect on fecundity and likely affects offspring health at birth, based on the meta-analysis of studies in experimental mammals on litter size and fetal weight, respectively. Regarding possible delayed effects of in utero exposure, RF-EMF probably does not affect offspring brain weight and may not decrease female offspring fertility; on the other hand, RF-EMF may have a detrimental impact on neurobehavioural functions, varying in magnitude for different endpoints, but these last findings are very uncertain. Further research is needed on the effects at birth and delayed effects with sample sizes adequate for detecting a small effect. Future studies should use standardized endpoints for testing prenatal developmental toxicity and developmental neurotoxicity (OECD TG 414 and 426), improve the description of the exposure system design and exposure conditions, conduct appropriate dosimetry characterization, blind endpoint analysis and include several exposure levels to better enable the assessment of a dose-response relationship. PROTOCOL REGISTRATION AND PUBLICATION: The protocol was published in Pacchierotti et al., 2021 and registered in PROSPERO CRD42021227746 (https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=227746).

Epigenetic-based antioxidant effect of an ethanolic extract of Corylus avellana L. on THLE-2 human primary hepatocytes.

The ethanolic extract of Corylus avellana L hazelnut, prepared in our laboratories, has been previously characterized by liquid chromatography coupled to high resolution mass spectrometry. We here aimed at testing the antioxidant effect of such extract in H2O2-challenged THLE-2 human primary hepatocytes and verified whether it might be based on microRNA-34b/c expression changes. We here demonstrate that miR-34b/miR-34c undergo significant stimulation (≥2-fold change, p < 0.05) in THLE-2 when treated for 72h with not-toxic hazelnut concentrations (0.04-0.4 mg/ml), when compared with 0.06% ethanol control. When administered with H2O2 (1000-2000 µM, 24h), THLE-2 are significantly protected from oxidative stress if pre-treated with hazelnut, the H2O2-driven cytotoxicity and reactive oxygen species generation being recovered by hazelnut extract, through miR-34b/c stimulation. Although preliminary, our findings pave the way for further preclinical studies aimed at validating the possible health-related application of hazelnut matrix, and/or its metabolites, as powerful epigenetic-based drugs, food supplements or nutraceuticals.

Effects of sub-chronic oral exposure to pyrogenic synthetic amorphous silica (NM-203) in male and female Sprague-Dawley rats: focus on reproductive systems.

Synthetic amorphous silica (SAS) consists of agglomerates and aggregates of primary particles in the nanorange (<100 nm) and it is the E551 authorized food additive. The potential risks for human health associated to dietary exposure to SAS are not completely assessed; in particular, data on male and female reproductive systems are lacking. A 90-day oral toxicity study with pyrogenic SAS nanomaterial NM-203 was carried out on the basis of the OECD test guideline 408 in the frame of the NANoREG project. Adult Sprague-Dawley rats of both sexes were orally treated for 90 days with 0, 2, 5, 10, 20 and 50 mg SAS/kg bw per day. Dose levels were selected to be as close as possible to the expected human exposure to food additive E551. The present paper provides specific information on potential effects on male and female reproductive systems, through the evaluation of serum biomarkers, sperm count, histopathological analysis of testis, epididymis, ovary and uterus and real-time PCR on uterus; potential genotoxic alterations were evaluated by comet assay on testis, sperm and ovary. NM-203 did not induce histophatological and genotoxic effects in male reproductive system. In female rats, ovary is not target of NM-203 and only tissue-specific effects on uterus were recorded up to 10 mg/kg bw per day. To our best knowledge, this is the first study providing data on male and female reproductive systems after long-term, repeated oral exposure at dose levels close to dietary human exposure, which identifies a limited concern only for female reproductive health.

Comet assay: a versatile but complex tool in genotoxicity testing.

The comet assay is a versatile method for measuring DNA strand breaks in individual cells. It can also be applied to cells isolated from treated animals. In this review, we highlight advantages and limitations of this in vivo comet assay in a regulatory context. Modified versions of the standard protocol detect oxidized DNA bases and may be used to reveal sites of DNA base loss, DNA interstrand crosslinks, and the extent of DNA damage induced indirectly by reactive oxygen species elicited by chemical-induced oxidative stress. The assay is, however, at best semi-quantitative, and we discuss possible approaches to improving DNA damage quantitation and highlight the necessity of optimizing protocol standardization to enhance the comparability of results between laboratories. As a genotoxicity test in vivo, the in vivo comet assay has the advantage over the better established micronucleus erythrocyte test that it can be applied to any organ, including those that are specific targets of chemical carcinogens or those that are the first sites of contact of ingested or inhaled mutagens. We illustrate this by examples of its use in risk assessment for the food contaminants ochratoxin and furan. We suggest that improved quantitation is required to reveal the full potential of the comet assay and enhance its role in the battery of in vivo approaches to characterize the mechanisms of toxicity and carcinogenicity of chemicals and to aid the determination of safe human exposure limits.

Chemically induced aneuploidy in germ cells. Part II of the report of the 2017 IWGT workgroup on assessing the risk of aneugens for carcinogenesis and hereditary diseases.

As part of the 7th International Workshops on Genotoxicity Testing held in Tokyo, Japan in November 2017, a workgroup of experts reviewed and assessed the risk of aneugens for human health. The present manuscript is one of three manuscripts from the workgroup and reports on the unanimous consensus reached on the evidence for aneugens affecting germ cells, their mechanisms of action and role in hereditary diseases. There are 24 chemicals with strong or sufficient evidence for germ cell aneugenicity providing robust support for the ability of chemicals to induce germ cell aneuploidy. Interference with microtubule dynamics or inhibition of topoisomerase II function are clear characteristics of germ cell aneugens. Although there are mechanisms of chromosome segregation that are unique to germ cells, there is currently no evidence for germ cell-specific aneugens. However, the available data are heavily skewed toward chemicals that are aneugenic in somatic cells. Development of high-throughput screening assays in suitable animal models for exploring additional targets for aneuploidy induction, such as meiosis-specific proteins, and to prioritize chemicals for the potential to be germ cell aneugens is encouraged. Evidence in animal models support that: oocytes are more sensitive than spermatocytes and somatic cells to aneugens; exposure to aneugens leads to aneuploid conceptuses; and, the frequencies of aneuploidy are similar in germ cells and zygotes. Although aneuploidy in germ cells is a significant cause of infertility and pregnancy loss in humans, there is currently limited evidence that aneugens induce hereditary diseases in human populations because the great majority of aneuploid conceptuses die in utero. Overall, the present work underscores the importance of protecting the human population from exposure to chemicals that can induce aneuploidy in germ cells that, in contrast to carcinogenicity, is directly linked to an adverse outcome.

Targets and mechanisms of chemically induced aneuploidy. Part 1 of the report of the 2017 IWGT workgroup on assessing the risk of aneugens for carcinogenesis and hereditary diseases.

An aneuploidy workgroup was established as part of the 7th International Workshops on Genotoxicity Testing. The workgroup conducted a review of the scientific literature on the biological mechanisms of aneuploidy in mammalian cells and methods used to detect chemical aneugens. In addition, the current regulatory framework was discussed, with the objective to arrive at consensus statements on the ramifications of exposure to chemical aneugens for human health risk assessment. As part of these efforts, the workgroup explored the use of adverse outcome pathways (AOPs) to document mechanisms of chemically induced aneuploidy in mammalian somatic cells. The group worked on two molecular initiating events (MIEs), tubulin binding and binding to the catalytic domain of aurora kinase B, which result in several adverse outcomes, including aneuploidy. The workgroup agreed that the AOP framework provides a useful approach to link evidence for MIEs with aneuploidy on a cellular level. The evidence linking chemically induced aneuploidy with carcinogenicity and hereditary disease was also reviewed and is presented in two companion papers. In addition, the group came to the consensus that the current regulatory test batteries, while not ideal, are sufficient for the identification of aneugens and human risk assessment. While it is obvious that there are many different MIEs that could lead to the induction of aneuploidy, the most commonly observed mechanisms involving chemical aneugens are related to tubulin binding and, to a lesser extent, inhibition of mitotic kinases. The comprehensive review presented here should help with the identification and risk management of aneugenic agents.

Role of aneuploidy in the carcinogenic process: Part 3 of the report of the 2017 IWGT workgroup on assessing the risk of aneugens for carcinogenesis and hereditary diseases.

Aneuploidy is regarded as a hallmark of cancer, however, its role is complex with both pro- and anti-carcinogenic effects evident. In this IWGT review, we consider the role of aneuploidy in cancer biology; cancer risk associated with constitutive aneuploidy; rodent carcinogenesis with known chemical aneugens; and chemotherapy-related malignant neoplasms. Aneuploidy is seen at various stages in carcinogenesis. However, the relationship between induced aneuploidy occurring after exposure and clonal aneuploidy present in tumours is not clear. Recent evidence indicates that the induction of chromosomal instability (CIN), may be more important than aneuploidy per se, in the carcinogenic process. Down Syndrome, trisomy 21, is associated with altered hematopoiesis in utero which, in combination with subsequent mutations, results in an increased risk for acute megakaryoblastic and lymphoblastic leukemias. In contrast, there is reduced cancer risk for most solid tumours in Down Syndrome. Mouse models with high levels of aneuploidy are also associated with increased cancer risk for particular tumours with long latencies, but paradoxically other types of tumour often show decreased incidence. The aneugens reviewed that induce cancer in humans and animals all possess other carcinogenic properties, such as mutagenicity, clastogenicity, cytotoxicity, organ toxicities, hormonal and epigenetic changes which likely account for, or interact with aneuploidy, to cause carcinogenesis. Although the role that aneuploidy plays in carcinogenesis has not been fully established, in many cases, it may not play a primary causative role. Tubulin-disrupting aneugens that do not possess other properties linked to carcinogenesis, were not carcinogenic in rodents. Similarly, in humans, for the tubulin-disrupting aneugens colchicine and albendazole, there is no reported association with increased cancer risk. There is a need for further mechanistic studies on agents that induce aneuploidy, particularly by mechanisms other than tubulin disruption and to determine the role of aneuploidy in pre-neoplastic events and in early and late stage neoplasia.

Risks of aneuploidy induction from chemical exposure: Twenty years of collaborative research in Europe from basic science to regulatory implications.

Although Theodor Boveri linked abnormal chromosome numbers and disease more than a century ago, an in-depth understanding of the impact of mitotic and meiotic chromosome segregation errors on cell proliferation and diseases is still lacking. This review reflects on the efforts and results of a large European research network that, from the 1980's until 2004, focused on protection against aneuploidy-inducing factors and tackled the following problems: 1) the origin and consequences of chromosome imbalance in somatic and germ cells; 2) aneuploidy as a result of environmental factors; 3) dose-effect relationships; 4) the need for validated assays to identify aneugenic factors and classify them according to their modes of action; 5) the need for reliable, quantitative data suitable for regulating exposure and preventing aneuploidy induction; 6) the need for mechanistic insight into the consequences of aneuploidy for human health. This activity brought together a consortium of experts from basic science and applied genetic toxicology to prepare the basis for defining guidelines and to encourage regulatory activities for the prevention of induced aneuploidy. Major strengths of the EU research programmes on aneuploidy were having a valuable scientific approach based on well-selected compounds and accurate methods that allow the determination of precise dose-effect relationships, reproducibility and inter-laboratory comparisons. The work was conducted by experienced scientists stimulated by a fascination with the complex scientific issues surrounding aneuploidy; a key strength was asking the right questions at the right time. The strength of the data permitted evaluation at the regulatory level. Finally, the entire enterprise benefited from a solid partnership under the lead of an inspired and stimulating coordinator. The research programme elucidated the major modes of action of aneugens, developed scientifically sound assays to assess aneugens in different tissues, and achieved the international validation of relevant assays with the goal of protecting human populations from aneugenic chemicals. The role of aneuploidy in tumorigenesis will require additional research, and the study of effects of exposure to multiple agents should become a priority. It is hoped that these reflections will stimulate the implementation of aneuploidy testing in national and OECD guidelines.

Impact of modelled PM2.5, NO2 and O3 annual air concentrations on some causes of mortality in Tuscany municipalities.

BACKGROUND: In 2014, the European Environment Agency estimated 59 630 premature deaths in Italy attributable to long-term exposure to PM2.5, 17 290 to NO2 and 2900 to O3. The aim of this study was to test an approach for assessing health impact of the above pollutants analyzing possible associations between annual municipal concentrations, estimated by the national dispersion model developed by ENEA, and mortality rates for trachea, bronchus and lung (TBL) cancer, total respiratory diseases (RD) and chronic obstructive pulmonary diseases (COPD). Tuscany was selected as test case. METHODS: For the 287 municipalities, 2009-13 standardized mortality rates (SMRates) for each cause of death were calculated by the ENEA epidemiological database. The SMRates of municipalities, aggregated on the basis of the 2003 or 2010 estimated pollutant concentration tertiles, were also computed. RESULTS: TBL cancer SMRate in municipalities with 2003 PM2.5 levels >15.2 µg/m3 was significantly higher than the SMRates of the two lowest tertiles and COPD SMRates in the two highest O3 tertiles were significantly higher than that of the lower tertile. No association between PM2.5 or NO2 concentrations and RD and COPD was detected. Approximately 625 TBL cancer deaths attributable to PM2.5 levels above 10 µg/m3 in 2003 were estimated in the region. Smoking habits and deprivation index were homogeneously distributed among municipalities. CONCLUSION: This methodological approach allowed detecting associations between mortality and specific air pollutants even at levels below the Italian normative limits and could be employed to evaluate the potential health impact of air pollution in areas where direct measures of concentration are unavailable.

Clinical and genomic safety of treatment with Ginkgo biloba L. leaf extract (IDN 5933/Ginkgoselect®Plus) in elderly: a randomised placebo-controlled clinical trial [GiBiEx].

BACKGROUND: Numerous health benefits have been attributed to the Ginkgo biloba leaf extract (GBLE), one of the most extensively used phytopharmaceutical drugs worldwide. Recently, concerns of the safety of the extract have been raised after a report from US National Toxicology Program (NTP) claimed high doses of GBLE increased liver and thyroid cancer incidence in mice and rats. A safety study has been designed to assess, in a population of elderly residents in nursing homes, clinical and genomic risks associated to GBLE treatment. METHODS: GiBiEx is a multicentre randomized clinical trial, placebo controlled, double blinded, which compared subjects randomized to twice-daily doses of either 120-mg of IDN 5933 (also known as Ginkgoselect®Plus) or to placebo for a 6-months period. IDN 5933 is extracted from dried leaves and contains 24.3% flavone glycosides and 6.1% of terpene lactones (2.9% bilobalide, 1.38% ginkgolide A, 0.66% ginkgolide B, 1.12% ginkgolide C) as determined by HPLC. The study was completed by 47 subjects, 20 in the placebo group and 27 in the treatment group. Clinical (adverse clinical effect and liver injury) and genomic (micronucleus frequency, comet assay, c-myc, p53, and ctnnb1 expression profile in lymphocytes) endpoints were assessed at the start and at the end of the study. RESULTS: No adverse clinical effects or increase of liver injury markers were reported in the treatment group. The frequency of micronuclei [Mean Ratio (MR) = 1.01, 95% Confidence Intervals (95% CI) 0.86-1.18), and DNA breaks (comet assay) (MR = 0.91; 95% CI 0.58-1.43), did not differ in the two study groups. No significant difference was found in the expression profile of the three genes investigated. CONCLUSIONS: None of the markers investigated revealed a higher risk in the treatment group, supporting the safety of IDN 5933 at doses prescribed and for duration of six months. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03004508 , December 20, 2016. Trial retrospectively registered.

Air quality modeling and inhalation health risk assessment for a new generation coal-fired power plant in Central Italy.

An assessment of potential carcinogenic and toxic health outcomes related to atmospheric emissions from the new-generation coal fired power plant of Torrevaldaliga Nord, in Central Italy, has been conducted. A chemical-transport model was applied on the reference year 2010 in the area of the plant, in order to calculate airborne concentrations of a set of 17 emitted pollutants of health concern. Inhalation cancer risks and hazard quotients, for each pollutant and for each target organ impacted via the inhalation pathway, were calculated and mapped on the study domain for the overall ambient concentrations and for the sole contribution of the plant to airborne concentrations, allowing to assess the relative contribution of the power plant to the risk from all sources. Cancer risks, cumulated on all pollutants, resulted around 5 × 10-5 for the concentrations from all sources and below 3 × 10-7 for the plant contribution, mainly targeting the respiratory system. On each part of the study domain, the plant contributed for less than 6% to the overall cancer risk. Hazard quotients from all sources, cumulated on all pollutants, reached values of 2.5 for the respiratory and 1.5 for the cardiovascular systems. Hazard quotients of non-carcinogenic risks from the plant, cumulated on all pollutants, resulted below 0.03 for the respiratory system and 0.02 for the cardiovascular system. On each part of the study domain, the plant contributed for less than 5% to the respiratory and cardiovascular risks. Both cancer risks and hazard quotients related to the plant are far below international thresholds for human health protection, while the values from all sources require consideration. The proposed method provides an instrument for prospective health risk assessment of large industrial sources, with some limitations presented and discussed.

Cytotoxicity, genotoxicity and gene expression changes elicited by exposure of human hepatic cells to Ginkgo biloba leaf extract.

The use of Ginkgo biloba leaf extract as nutraceutical is becoming increasingly common. As a consequence, the definition of a reliable toxicological profile is a priority for its safe utilization. Recently, contrasting data have been reported on the carcinogenic potential of Ginkgo biloba extract in rodent liver. We measured viability, Reactive Oxygen Species (ROS), apoptosis, colony-forming efficiency, genotoxicity by comet assay, and gene expression changes associated with hepato-carcinogenicity in human cells of hepatic origin (HepG2 and THLE-2) treated with different concentrations (0.0005-1.2 mg/mL) of Ginkgoselect®Plus. Our analyses highlighted a decrease of cell viability, not due to apoptosis, after treatment with high doses of the extract, which was likely due to ROS generation by a chemical reaction between extract polyphenols and some components of the culture medium. Comet assay did not detect genotoxic effect at any extract concentration. Finally, the array analysis detected a slight decrease in the expression of only one gene (IGFBP3) in Ginkgo-treated THLE-2 cells as opposed to changes in 28 genes in Aflatoxin B1 treated-cells. In conclusion, our results did not detect any significant genotoxic or biologically relevant cytotoxic effects and gross changes in gene expression using the Ginkgo extract in the hepatic cells tested.

Study of TiO2 P25 Nanoparticles Genotoxicity on Lung, Blood, and Liver Cells in Lung Overload and Non-Overload Conditions After Repeated Respiratory Exposure in Rats.

Inhaled titanium dioxide (TiO2) nanoparticles (NPs) can have negative health effects, and have been shown to cause respiratory tract cancer in rats. Inflammation has been linked to oxidative stress, and both have been described as possible mechanisms for genotoxicity of NPs, but rarely examined side-by-side in animal studies. In the present study, a wide range of complementary endpoints have been performed to study TiO2 P25 NP-induced genotoxicity in lung overload and non-overload conditions. Additionally, lung burden, inflammation, cytotoxicity and oxidative stress have also been evaluated in order to link genotoxicity with these responses. To assess quick and delayed responses after recovery, endpoints were evaluated at two time points: 2 h and 35 days after three repeated instillations. This study confirmed the previously described lung overload threshold at approximately 200-300 cm2 of lung burden for total particle surface area lung deposition or 4.2 µl/kg for volume-based cumulative lung exposure dose, above which lung clearance is impaired and inflammation is induced. Our results went on to show that these overload doses induced delayed genotoxicity in lung, associated with persistent inflammation only at the highest dose. The lowest tested doses had no toxicity or genotoxicity effects in the lung. In blood, no lymphocyte DNA damage, erythrocytes chromosomal damage or gene mutation could be detected. Our data also demonstrated that only overload doses induced liver DNA lesions irrespective of the recovery time. Tested doses of TiO2 P25 NPs did not induce glutathione changes in lung, blood or liver at both recovery times.

No genotoxicity in rat blood cells upon 3- or 6-month inhalation exposure to CeO2 or BaSO4 nanomaterials.

In the course of a 2-year combined chronic toxicity-carcinogenicity study performed according to Organisation for Economic Co-operation and Development (OECD) Test Guideline 453, systemic (blood cell) genotoxicity of two OECD representative nanomaterials, CeO2 NM-212 and BaSO4 upon 3- or 6-month inhalation exposure to rats was assessed. DNA effects were analysed in leukocytes using the alkaline Comet assay, gene mutations and chromosome aberrations were measured in erythrocytes using the flow cytometric Pig-a gene mutation assay and the micronucleus test (applying both microscopic and flow cytometric evaluation), respectively. Since nano-sized CeO2 elicited lung effects at concentrations of 5mg/m3 (burdens of 0.5mg/lung) in the preceding range-finding study, whereas nano-sized BaSO4 did not induce any effect, female rats were exposed to aerosol concentrations of 0.1 up to 3mg/m3 CeO2 or 50mg/m3 BaSO4 nanomaterials (6h/day; 5 days/week; whole-body exposure). The blood of animals treated with clean air served as negative control, whereas blood samples from rats treated orally with three doses of 20mg/kg body weight ethylnitrosourea at 24h intervals were used as positive controls. As expected, ethylnitrosourea elicited significant genotoxicity in the alkaline Comet and Pig-a gene mutation assays and in the micronucleus test. By contrast, 3- and 6-month CeO2 or BaSO4 nanomaterial inhalation exposure did not elicit significant findings in any of the genotoxicity tests. The results demonstrate that subchronic inhalation exposure to different low doses of CeO2 or to a high dose of BaSO4 nanomaterials does not induce genotoxicity on the rat hematopoietic system at the DNA, gene or chromosome levels.

Genotoxicity Induced by Foetal and Infant Exposure to Magnetic Fields and Modulation of Ionising Radiation Effects.

BACKGROUND: Few studies have investigated the toxicity and genotoxicity of extremely low frequency magnetic fields (ELF-MF) during prenatal and neonatal development. These phases of life are characterized by cell proliferation and differentiation, which might make them sensitive to environmental stressors. Although in vitro evidences suggest that ELF-MF may modify the effects of ionizing radiation, no research has been conducted so far in vivo on the genotoxic effects of ELF-MF combined with X-rays. AIM AND METHODS: Aim of this study was to investigate in somatic and germ cells the effects of chronic ELF-MF exposure from mid gestation until weaning, and any possible modulation produced by ELF-MF exposure on ionizing radiation-induced damage. Mice were exposed to 50 Hz, 65 µT magnetic field, 24 hours/day, for a total of 30 days, starting from 12 days post-conception. Another group was irradiated with 1 Gy X-rays immediately before ELF-MF exposure, other groups were only X-irradiated or sham-exposed. Micronucleus test on blood erythrocytes was performed at multiple times from 1 to 140 days after birth. Additionally, 42 days after birth, genotoxic and cytotoxic effects on male germ cells were assessed by comet assay and flow cytometric analysis. RESULTS: ELF-MF exposure had no teratogenic effect and did not affect survival, growth and development. The micronucleus test indicated that ELF-MF induced a slight genotoxic damage only after the maximum exposure time and that this effect faded away in the months following the end of exposure. ELF-MF had no effects on ionizing radiation (IR)-induced genotoxicity in erythrocytes. Differently, ELF-MF appeared to modulate the response of male germ cells to X-rays with an impact on proliferation/differentiation processes. These results point to the importance of tissue specificity and development on the impact of ELF-MF on the early stages of life and indicate the need of further research on the molecular mechanisms underlying ELF-MF biological effects.

Transgenerational inheritance of enhanced susceptibility to radiation-induced medulloblastoma in newborn Ptch1⁺/⁻ mice after paternal irradiation.

The hypothesis of transgenerational induction of increased cancer susceptibility after paternal radiation exposure has long been controversial because of inconsistent results and the lack of a mechanistic interpretation. Here, exploiting Ptch1 heterozygous knockout mice, susceptible to spontaneous and radiation-induced medulloblastoma, we show that exposure of paternal germ cells to 1 Gy X-rays, at the spermatogonial stage, increased by a considerable 1.4-fold the offspring susceptibility to medulloblastoma induced by neonatal irradiation. This effect gained further biological significance thanks to a number of supporting data on the immunohistochemical characterization of the target tissue and preneoplastic lesions (PNLs). These results altogether pointed to increased proliferation of cerebellar granule cell precursors and PNLs cells, which favoured the development of frank tumours. The LOH analysis of tumor DNA showed Ptch1 biallelic loss in all tumor samples, suggesting that mechanisms other than interstitial deletions, typical of radiation-induced medulloblastoma, did not account for the observed increased cancer risk. This data was supported by comet analysis showing no differences in DNA damage induction and repair in cerebellar cells as a function of paternal irradiation. Finally, we provide biological plausibility to our results offering evidence of a possible epigenetic mechanism of inheritance based on radiation-induced changes of the microRNA profile of paternal sperm.

Environmental Impact on DNA Methylation in the Germline: State of the Art and Gaps of Knowledge.

The epigenome consists of chemical changes in DNA and chromatin that without modifying the DNA sequence modulate gene expression and cellular phenotype. The epigenome is highly plastic and reacts to changing external conditions with modifications that can be inherited to daughter cells and across generations. Whereas this innate plasticity allows for adaptation to a changing environment, it also implies the potential of epigenetic derailment leading to so-called epimutations. DNA methylation is the most studied epigenetic mark. DNA methylation changes have been associated with cancer, infertility, cardiovascular, respiratory, metabolic, immunologic, and neurodegenerative pathologies. Experiments in rodents demonstrate that exposure to a variety of chemical stressors, occurring during the prenatal or the adult life, may induce DNA methylation changes in germ cells, which may be transmitted across generations with phenotypic consequences. An increasing number of human biomonitoring studies show environmentally related DNA methylation changes mainly in blood leukocytes, whereas very few data have been so far collected on possible epigenetic changes induced in the germline, even by the analysis of easily accessible sperm. In this paper, we review the state of the art on factors impinging on DNA methylation in the germline, highlight gaps of knowledge, and propose priorities for future studies.

Genomic instability: Crossing pathways at the origin of structural and numerical chromosome changes.

Genomic instability leads to a wide spectrum of genetic changes, including single nucleotide mutations, structural chromosome alterations, and numerical chromosome changes. The accepted view on how these events are generated predicts that separate cellular mechanisms and genetic events explain the occurrence of these types of genetic variation. Recently, new findings have shed light on the complexity of the mechanisms leading to structural and numerical chromosome aberrations, their intertwining pathways, and their dynamic evolution, in somatic as well as in germ cells. In this review, we present a critical analysis of these recent discoveries in this area, with the aim to contribute to a deeper knowledge of the molecular networks leading to adverse outcomes in humans following exposure to environmental factors. The review illustrates how several technological advances, including DNA sequencing methods, bioinformatics, and live-cell imaging approaches, have contributed to produce a renewed concept of the mechanisms causing genomic instability. Special attention is also given to the specific pathways causing genomic instability in mammalian germ cells. Remarkably, the same scenario emerged from some pioneering studies published in the 1980s to 1990s, when the evolution of polyploidy, the chromosomal effects of spindle poisons, the fate of micronuclei, were intuitively proposed to share mechanisms and pathways. Thus, an old working hypothesis has eventually found proper validation.

Biodistribution and acute toxicity of a nanofluid containing manganese iron oxide nanoparticles produced by a mechanochemical process.

Superparamagnetic iron oxide nanoparticles are candidate contrast agents for magnetic resonance imaging and targeted drug delivery. Biodistribution and toxicity assessment are critical for the development of nanoparticle-based drugs, because of nanoparticle-enhanced biological reactivity. Here, we investigated the uptake, in vivo biodistribution, and in vitro and in vivo potential toxicity of manganese ferrite (MnFe2O4) nanoparticles, synthesized by an original high-yield, low-cost mechanochemical process. Cultures of murine Balb/3T3 fibroblasts were exposed for 24, 48, or 72 hours to increasing ferrofluid concentrations. Nanoparticle cellular uptake was assessed by flow-cytometry scatter-light measurements and microscopy imaging after Prussian blue staining; cytotoxicity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony-forming assays. After a single intravenous injection, in vivo nanoparticle biodistribution and clearance were evaluated in mice by Mn spectrophotometric determination and Prussian blue staining in the liver, kidneys, spleen, and brain at different posttreatment times up to 21 days. The same organs were analyzed for any possible histopathological change. The in vitro study demonstrated dose-dependent nanoparticle uptake and statistically significant cytotoxic effects from a concentration of 50 µg/mL for the MTT assay and 20 µg/mL for the colony-forming assay. Significant increases in Mn concentrations were detected in all analyzed organs, peaking at 6 hours after injection and then gradually declining. Clearance appeared complete at 7 days in the kidneys, spleen, and brain, whereas in the liver Mn levels remained statistically higher than in vehicle-treated mice up to 3 weeks postinjection. No evidence of irreversible histopathological damage to any of the tested organs was observed. A comparison of the lowest in vitro toxic concentration with the intravenously injected dose and the administered dose of other ferrofluid drugs currently in clinical practice suggests that there might be sufficient safety margins for further development of our formulation.