Assessment of Genotoxicity in Human Cells Exposed to Modulated Electromagnetic Fields of Wireless Communication Devices.

Modulated electromagnetic fields (wEMFs), as generated by modern communication technologies, have raised concerns about adverse health effects. The International Agency for Research on Cancer (IARC) classifies them as "possibly carcinogenic to humans" (Group 2B), yet, the underlying molecular mechanisms initiating and promoting tumorigenesis remain elusive. Here, we comprehensively assess the impact of technologically relevant wEMF modulations on the genome integrity of cultured human cells, investigating cell type-specificities as well as time- and dose-dependencies. Classical and advanced methodologies of genetic toxicology and DNA repair were applied, and key experiments were performed in two separate laboratories. Overall, we found no conclusive evidence for an induction of DNA damage nor for alterations of the DNA repair capacity in cells exposed to several wEMF modulations (i.e., GSM, UMTS, WiFi, and RFID). Previously reported observations of increased DNA damage after exposure of cells to GSM-modulated signals could not be reproduced. Experimental variables, presumably underlying the discrepant observations, were investigated and are discussed. On the basis of our data, we conclude that the possible carcinogenicity of wEMF modulations cannot be explained by an effect on genome integrity through direct DNA damage. However, we cannot exclude non-genotoxic, indirect, or secondary effects of wEMF exposure that may promote tumorigenesis in other ways.

Novel ETV6-RUNX1 Mouse Model to Study the Role of ELF-MF in Childhood B-Acute Lymphoblastic Leukemia: a Pilot Study.

Exposure to extremely low-frequency magnetic fields (ELF-MFs) has been classified by the International Agency for Research on Cancer (IARC) as "possibly carcinogenic to humans," based on limited scientific evidence concerning childhood leukemia. This assessment emphasized the lack of appropriate animal models recapitulating the natural history of this disease. Childhood B-cell acute lymphoblastic leukemia (B-ALL) is the result of complex interactions between genetic susceptibility and exposure to exogenous agents. The most common chromosomal alteration is the ETV6-RUNX1 fusion gene, which confers a low risk of developing the malignancy by originating a preleukemic clone requiring secondary hits for full-blown disease to appear. To develop potential prophylactic interventions, we need to identify the environmental triggers of the second hit. Recently, we generated a B-ALL mouse model of the human ETV6-RUNX1+ preleukemic state. Here, we present the results from the ARIMMORA pilot study, obtained by exposing 34 Sca1-ETV6-RUNX1 mice (vs. 27 unexposed) to a 50 Hz magnetic field of 1.5 mT with both fundamental and harmonic content, with an on/off cycle of 10 min/5 min, for 20 h/day, from conception until 3 months of age. Mice were monitored until 2 years of age and peripheral blood was periodically analyzed by flow cytometry. One of the exposed mice developed B-ALL while none of the non-exposed did. Although the results are statistically non-significant due to the limited number of mice used in this pilot experiment, overall, the results show that the newly developed Sca1-ETV6-RUNX1 mouse can be successfully used for ELF-MF exposure studies about the etiology of childhood B-ALL. Bioelectromagnetics. 2019;40:343-353. © 2019 Bioelectromagnetics Society.

Cerium oxide and barium sulfate nanoparticle inhalation affects gene expression in alveolar epithelial cells type II.

BACKGROUND: Understanding the molecular mechanisms of nanomaterial interacting with cellular systems is important for appropriate risk assessment. The identification of early biomarkers for potential (sub-)chronic effects of nanoparticles provides a promising approach towards cost-intensive and animal consuming long-term studies. As part of a 90-day inhalation toxicity study with CeO2 NM-212 and BaSO4 NM-220 the present investigations on gene expression and immunohistochemistry should reveal details on underlying mechanisms of pulmonary effects. The role of alveolar epithelial cells type II (AEII cells) is focused since its contribution to defense against inhaled particles and potentially resulting adverse effects is assumed. Low dose levels should help to specify particle-related events, including inflammation and oxidative stress. RESULTS: Rats were exposed to clean air, 0.1, 0.3, 1.0, and 3.0 mg/m3 CeO2 NM-212 or 50.0 mg/m3 BaSO4 NM-220 and the expression of 391 genes was analyzed in AEII cells after one, 28 and 90 days exposure. A total number of 34 genes was regulated, most of them related to inflammatory mediators. Marked changes in gene expression were measured for Ccl2, Ccl7, Ccl17, Ccl22, Ccl3, Ccl4, Il-1α, Il-1ß, and Il-1rn (inflammation), Lpo and Noxo1 (oxidative stress), and Mmp12 (inflammation/lung cancer). Genes related to genotoxicity and apoptosis did not display marked regulation. Although gene expression was less affected by BaSO4 compared to CeO2 the gene pattern showed great overlap. Gene expression was further analyzed in liver and kidney tissue showing inflammatory responses in both organs and marked downregulation of oxidative stress related genes in the kidney. Increases in the amount of Ce were measured in liver but not in kidney tissue. Investigation of selected genes on protein level revealed increased Ccl2 in bronchoalveolar lavage of exposed animals and increased Lpo and Mmp12 in the alveolar epithelia. CONCLUSION: AEII cells contribute to CeO2 nanoparticle caused inflammatory and oxidative stress reactions in the respiratory tract by the release of related mediators. Effects of BaSO4 exposure are low. However, overlap between both substances were detected and support identification of potential early biomarkers for nanoparticle effects on the respiratory system. Signs for long-term effects need to be further evaluated by comparison to a respective exposure setting.

Evaluation of a human in vitro hepatocyte-NPC co-culture model for the prediction of idiosyncratic drug-induced liver injury: A pilot study.

Interactions between hepatocytes and immune cells as well as inflammatory episodes are frequently discussed to play a critical role in the alteration of the individual susceptibility to idiosyncratic drug-induced liver injury (iDILI). To evaluate this hypothesis and to face the urgent need for predictive in vitro models, we established two co-culture systems based on two human cell lines in presence or absence of pro-inflammatory factors (LPS, TNF), i.e. hepatoma HepG2 cells co-cultured with monocytic or macrophage-like THP-1 cells. HepG2 monocultures served as control scenario. Mono- or co-cultures were treated with iDILI reference substances (Troglitazone [TGZ], Trovafloxacin [TVX], Diclofenac [DcL], Ketoconazole [KC]) or their non-iDILI partner compounds (Rosiglitazone, Levofloxacin, Acetylsalicylic Acid, Fluconazole). The liver cell viability was subsequently determined via WST-Assay. An enhanced cytotoxicity (synergy) or a hormetic response compared to the drug effect in the HepG2 monoculture was considered as iDILI positive. TGZ synergized in co-cultures with monocytes without an additional pro-inflammatory stimulus, while DcL and KC showed a hormetic response. All iDILI drugs synergized with TNF in the simple HepG2 monoculture, indicating its relevance as an initiator of iDILI. KC showed a synergy when co-exposed to both, monocytes and LPS, while TVX and DcL showed a synergy under the same conditions with macrophages. All described iDILI responses were not observed with the corresponding non-iDILI partner compounds. Our first results confirm that an inflammatory environment increases the sensitivity of liver cells towards iDILI compounds and point to an involvement of pro-inflammatory factors, especially TNF, in the development of iDILI.

Effects from a 90-day inhalation toxicity study with cerium oxide and barium sulfate nanoparticles in rats.

BACKGROUND: Nanomaterials like cerium oxide and barium sulfate are frequently processed in industrial and consumer products and exposure of humans and other organisms is likely. Generally less information is given on health effects and toxicity, especially regarding long-term exposure to low nanoparticle doses. Since inhalation is still the major route of uptake the present study focused on pulmonary effects of CeO2NM-212 (0.1, 0.3, 1.0, 3.0 mg/m3) and BaSO4NM-220 nanoparticles (50.0 mg/m3) in a 90-day exposure setup. To define particle-related effects and potential mechanisms of action, observations in histopathology, bronchoalveolar lavage and immunohistochemistry were linked to pulmonary deposition and clearance rates. This further allows evaluation of potential overload related effects. RESULTS: Lung burden values increased with increasing nanoparticle dose levels and ongoing exposure. At higher doses, cerium clearance was impaired, suggesting lung overload. Barium elimination was extremely rapid and without any signs of overload. Bronchoalveolar lavage fluid analysis and histopathology revealed lung tissue inflammation with increasing severity and post-exposure persistency for CeO2. Also, marker levels for genotoxicity and cell proliferation were significantly increased. BaSO4 showed less inflammation or persistency of effects and particularly affected the nasal cavity. CONCLUSION: CeO2 nanoparticles penetrate the alveolar space and affect the respiratory tract after inhalation mainly in terms of inflammation. Effects at low dose levels and post-exposure persistency suggest potential long-term effects and a notable relevance for human health. The generated data might be useful to improve nanoparticle risk assessment and threshold value generation. Mechanistic investigations at conditions of non-overload and absent inflammation should be further investigated in future studies.

Maintenance of high quality rat precision cut liver slices during culture to study hepatotoxic responses: Acetaminophen as a model compound.

Precision cut liver slices (PCLiS) represent a promising tool in reflecting hepatotoxic responses. However, the culture of PCLiS varies considerably between laboratories, which can affect the performance of the liver slices and thus the experimental outcome. In this study, we describe an easily accessible culture method, which ensures optimal slice viability and functionality, in order to set the basis for reproducible and comparable PCLiS studies. The quality of the incubated rat PCLiS was assessed during a 24h culture period using ten readouts, which covered viability (lactate dehydrogenase-, aspartate transaminase- and glutamate dehydrogenase-leakage, ATP content) and functionality parameters (urea, albumin production) as well as histomorphology and other descriptive characteristics (protein content, wet weight, slice thickness). The present culture method resulted in high quality liver slices for 24h. Finally, PCLiS were exposed to increasing concentrations of acetaminophen to assess the suitability of the model for the detection of hepatotoxic responses. Six out of ten readouts revealed a toxic effect and showed an excellent mutual correlation. ATP, albumin and histomorphology measurements were identified as the most sensitive readouts. In conclusion, our results indicate that rat PCLiS are a valuable liver model for hepatotoxicity studies, particularly if they are cultured under optimal standardized conditions.

Comparative dosimetry for children and rodents exposed to extremely low-frequency magnetic fields.

We describe a method to correlate E-fields induced by exposure to extremely low frequency magnetic fields in laboratory mice and rats during in vivo experiments to those induced in children. Four different approaches of mapping relative dose rates between humans and rodents are herein proposed and analyzed. Based on these mapping methods and volume averaging guidelines published by the International Commission on Non-Ionizing Radiation Protection (ICNRP) in 2010, maximum and median induced field values for whole body and for tissues of children and rodents were evaluated and compared. Median induced electric fields in children younger than 10 years old are in the range 5.9-8.5 V/m per T (±0.4 dB). Maximum induced electric fields, generally in the skin, are between 48 V/m and 228 V/m per T (±4 dB). To achieve induced electric fields of comparable magnitude in rodents, external magnetic field must be increased by a factor of 4.0 (±2.6 dB) for rats and 7.4 (±1.8 dB) for mice. Meanwhile, to achieve comparable magnetic field dose in rodents, ratio is close to one. These induced field dose rates for children and rodents can be used to quantifiably compare experimental data from in vivo studies with data on exposure of children from epidemiological studies, such as for leukemia. Bioelectromagnetics. 37:310-322, 2016. © 2016 Wiley Periodicals, Inc.

Extremely low-frequency magnetic fields and risk of childhood leukemia: A risk assessment by the ARIMMORA consortium.

Exposure to extremely low-frequency magnetic fields (ELF-MF) was evaluated in an International Agency for Research on Cancer (IARC) Monographs as "possibly carcinogenic to humans" in 2001, based on increased childhood leukemia risk observed in epidemiological studies. We conducted a hazard assessment using available scientific evidence published before March 2015, with inclusion of new research findings from the Advanced Research on Interaction Mechanisms of electroMagnetic exposures with Organisms for Risk Assessment (ARIMMORA) project. The IARC Monograph evaluation scheme was applied to hazard identification. In ARIMMORA for the first time, a transgenic mouse model was used to mimic the most common childhood leukemia: new pathogenic mechanisms were indicated, but more data are needed to draw definitive conclusions. Although experiments in different animal strains showed exposure-related decreases of CD8+ T-cells, a role in carcinogenesis must be further established. No direct damage of DNA by exposure was observed. Overall in the literature, there is limited evidence of carcinogenicity in humans and inadequate evidence of carcinogenicity in experimental animals, with only weak supporting evidence from mechanistic studies. New exposure data from ARIMMORA confirmed that if the association is nevertheless causal, up to 2% of childhood leukemias in Europe, as previously estimated, may be attributable to ELF-MF. In summary, ARIMMORA concludes that the relationship between ELF-MF and childhood leukemia remains consistent with possible carcinogenicity in humans. While this scientific uncertainty is dissatisfactory for science and public health, new mechanistic insight from ARIMMORA experiments points to future research that could provide a step-change in future assessments. Bioelectromagnetics. 37:183-189, 2016. © 2016 Wiley Periodicals, Inc.

Desktop exposure system and dosimetry for small scale in vivo radiofrequency exposure experiments.

This paper describes a new approach to the risk assessment of exposure from wireless network devices, including an exposure setup and dosimetric assessment for in vivo studies. A novel desktop reverberation chamber has been developed for well-controlled exposure of mice for up to 24 h per day to address the biological impact of human exposure scenarios by wireless networks. The carrier frequency of 2.45 GHz corresponds to one of the major bands used in data communication networks and is modulated by various modulation schemes, including Global System for Mobile Communications (GSM), Universal Mobile Telecommunications System (UMTS), Radio Frequency Identification (RFID), and wireless local area network, etc. The system has been designed to enable exposures of whole-body averaged specific absorption rate (SAR) of up to 15 W/kg for six mice of an average weight of 25 g or of up to 320 V/m incident time-averaged fields under loaded conditions without distortion of the signal. The dosimetry for whole-body SAR and organ-averaged SAR of the exposed mice, with analysis of uncertainty and variation analysis, is assessed. The experimental dosimetry based on temperature measurement agrees well with the numerical dosimetry, with a very good SAR uniformity of 0.4 dB in the chamber. Furthermore, a thermal analysis and measurements were performed to provide better understanding of the temperature load and distribution in the mice during exposure.

A pilot study on the refinement of acute inhalation toxicity studies: the isolated perfused rat lung as a screening tool for surface-active substances.

New surface-active agents in waterproofing sprays are frequently tested for acute inhalation toxicity in vivo according to OECD Test Guideline 403. In order to refine and reduce the number of acute inhalation tests performed, we propose a screening test that uses isolated lungs. The test consists of the exposure of isolated, ventilated and perfused rat lungs, to aerosolised formulations of waterproofing agents (mass median aerodynamic diameter = 1µm), and on-line monitoring of respiratory parameters and gross pathology analysis. A pilot evaluation of the isolated perfused rat lung model for use in a screening test was carried out by blind testing 12 surface-active substances. The results obtained compared well with data available from in vivo acute inhalation studies. Substances that triggered harmful effects, such as impaired lung compliance and atelectasis of the isolated perfused lung, were also found to cause changes in respiratory parameters, some of which would be severe enough to lead to death in in vivo tests with rats. The changes in respiratory parameters suggest that the mode-of-action is associated with impairment of the surfactant layer. Therefore, pre-testing in the isolated perfused rat lung allows the identification of surface-active substances with the potential for causing acute inhalation toxicity.

Indication of cocarcinogenic potential of chronic UMTS-modulated radiofrequency exposure in an ethylnitrosourea mouse model.

PURPOSE: To evaluate putative effects on tumour susceptibility in mice exposed to a UMTS (universal mobile telecommunications system) test signal for up to 24 months, commencing with embryo-fetal exposure. MATERIAL AND METHODS: Animals were exposed to UMTS fields with intensities of 0, 4.8, and 48 W/m(2), the low-dose group (4.8 W/m(2)) was subjected to additional prenatal ethylnitrosourea treatment (40 mg ENU/kg body weight). RESULTS: The high-level UMTS exposure (48 W/m(2)), the sham exposure, and the cage control groups showed comparable tumour incidences in the protocol organs. In contrast, the ENU-treated group UMTS-exposed at 4.8 W/m(2) displayed an enhanced lung tumour rate and an increased incidence of lung carcinomas as compared to the controls treated with ENU only. Furthermore, tumour multiplicity of the lung carcinomas was increased and the number of metastasising lung tumours was doubled in the ENU/UMTS group as compared to the ENU control group. CONCLUSION: This pilot study indicates a cocarcinogenic effect of lifelong UMTS exposure (4.8 W/m(2)) in female B6C3F1 descendants subjected to pretreatment with ethylnitrosourea.

Absence of genotoxic potential of 902 MHz (GSM) and 1747 MHz (DCS) wireless communication signals: In vivo two-year bioassay in B6C3F1 mice.

PURPOSE: The aim of the present investigation was to determine the incidence of micronuclei in peripheral blood erythrocytes of B6C3F1 mice that had been chronically exposed to radiofrequencies (RF) used for mobile communication. MATERIALS AND METHODS: 'Ferris wheels' were used to expose tube-restrained male and female mice to simulated environmental RF signals of the Global System for Mobile Communications (GSM, 902 MHz) or Digital Cellular System (DCS, 1747 MHz). RF signals were applied to the mice for 2 hours/day on 5 days/week for two years, at maximal whole-body-averaged specific absorption rates of 0.4, 1.3, and 4.0 W/kg body weight. Concurrent sham-exposed mice, cage controls, and positive controls injected with mitomycin C were included in this investigation. At necropsy, peripheral blood smears were prepared, and coded slides were stained using May-Grunwald-Giemsa or acridine orange. The incidence of micronuclei was recorded for each mouse in 2000 polychromatic and 2000 normochromatic erythrocytes. RESULTS: There were no significant differences in the frequency of micronuclei between RF-exposed, sham-exposed, and cage control mice, irrespective of the staining/counting method used. Micronuclei were, however, significantly increased in polychromatic erythrocytes of the positive control mice. CONCLUSIONS: In conclusion, the data did not indicate RF-induced genotoxicity in mice after two years of exposure.

Carcinogenicity study of GSM and DCS wireless communication signals in B6C3F1 mice.

The purpose of this study using a total of 1170 B6C3F1 mice was to detect and evaluate possible carcinogenic effects in mice exposed to radio-frequency-radiation (RFR) from Global System for Mobile Communication (GSM) and Digital Personal Communications System (DCS) handsets as emitted by handsets operating in the center of the communication band, that is, at 902 MHz (GSM) and 1747 MHz (DCS). Restrained mice were exposed for 2 h per day, 5 days per week over a period of 2 years to three different whole-body averaged specific absorption rate (SAR) levels of 0.4, 1.3, 4.0 mW/g bw (SAR), or were sham exposed. Regarding the organ-related tumor incidence, pairwise Fisher's test did not show any significant increase in the incidence of any particular tumor type in the RF exposed groups as compared to the sham exposed group. Interestingly, while the incidences of hepatocellular carcinomas were similar in EMF and sham exposed groups, in both studies the incidences of liver adenomas in males decreased with increasing dose levels; the incidences in the high dose groups were statistically significantly different from those in the sham exposed groups. Comparison to published tumor rates in untreated mice revealed that the observed tumor rates were within the range of historical control data. In conclusion, the present study produced no evidence that the exposure of male and female B6C3F1 mice to wireless GSM and DCS radio frequency signals at a whole body absorption rate of up to 4.0 W/kg resulted in any adverse health effect or had any cumulative influence on the incidence or severity of neoplastic and non-neoplastic background lesions, and thus the study did not provide any evidence of RF possessing a carcinogenic potential.

Effects of 1-week and 6-week exposure to GSM/DCS radiofrequency radiation on micronucleus formation in B6C3F1 mice.

The aim of this study was to examine the possible induction of micronuclei in erythrocytes of the peripheral blood and bone marrow and in keratinocytes and spleen lymphocytes of mice exposed to radiofrequency (RF) radiation for 2 h per day over periods of 1 and 6 weeks, respectively. The applied signal simulated the exposure from GSM900 and DCS1800 handsets, including the low-frequency amplitude-modulation components as they occur during speaking (GSM Basic), listening (DTX) and moving within the environment (handovers, power control). The carrier frequency was set to the center of the system's uplink band, i.e., 902 MHz for GSM and 1747 MHz for DCS. Uniform whole-body exposure was achieved by restraining the mice in tubes at fixed positions in the exposure setup. Mice were exposed to slot-averaged whole-body SARs of 33.2, 11.0, 3.7 and 0 mW/g during the 1-week study and 24.9, 8.3, 2.8 and 0 mW/g during the 6-week study. Exposure levels for the 1- and 6-week studies were determined in a pretest to confirm that no thermal effect was present that could influence the genotoxic end points. During both experiments and for both frequencies, no clinical abnormalities were detected in the animals. Cells of the bone marrow from the femur (1-week study), erythrocytes of the peripheral blood (6-week study), keratinocytes from the tail root, and lymphocytes from the spleen (both studies) were isolated on slides and stained for micronucleus analysis. Two thousand cells per animal were scored in erythrocyte and keratinocyte samples. In spleen lymphocytes, 1000 binucleated lymphocytes were scored for each animal. The RF-field exposure had no influence on the formation of red blood cells. After 1 week of exposure, the ratio of polychromatic to normochromatic erythrocytes was unchanged in the treated groups compared to the sham-exposed groups. Furthermore, the RF-field exposure of mice did not induce an increase in the number of micronuclei in erythrocytes of the bone marrow or peripheral blood, in keratinocytes, or in spleen lymphocytes compared to the sham-treated control.

Animal carcinogenicity studies on radiofrequency fields related to mobile phones and base stations.

Since a report in 1997 on an increased lymphoma incidence in mice chronically exposed to a mobile phone radiofrequency signal, none of the subsequent long-term studies in rodents have confirmed these results. On the other hand, several of the follow-up co- and carcinogenicity studies are still underway or are presently being initiated. Most of the published long-term studies used 1 exposure level only and suffer from a poor dosimetry which does not consider the animal's growth. Additional points of criticism are a limited, in some cases, questionable histopathology and inadequate group sizes. Overall, if dealing with new chemicals or drugs, these studies would not be acceptable for registration with the responsible authorities. The major critical points are taken into consideration within the European co- and carcinogenicity projects (CEMFEC and PERFORM-A), which are in their final stages and in the US long-term studies in mice and rats which are about to be initiated. Nevertheless, the WHO evaluation for health risk assessment of long-term telephone use and base station exposure will start in late 2005.

Maternal effects and cancer risk in the progeny of mice exposed to X-rays before conception.

To investigate in an animal model whether preconceptual X-ray exposure leads to an altered tumor rate and spectrum in the offspring, a transgeneration carcinogenesis study was carried out. Female mice received X-ray irradiation (2 x 2 Gray) 2 weeks prior to mating with untreated males. After weaning, half of the descendants were exposed for 6 months to the immunomodulating and tumor-promoting compound cyclosporine A (CsA) by diet, the others remained untreated. The animals were maintained for their entire lifespan, terminal sacrifices were carried out after 28 months. Complete autopsy was performed, and three protocol organs (lung, liver and spleen) were examined histologically, together with any suspicious lesions in other organs. Fertility and the lifetime of the maternal mice were reduced by the X-ray irradiation, and their incidence of lung and liver tumors was increased as compared to non-irradiated mice. The descendants of all groups revealed comparable body weights and mortality rates. The incidence of hematopoietic/lymphoreticular tissue tumors increased in the female hybrids by 6 months of CsA-treatment. A higher incidence of lung and liver tumors in the sham-treated male progeny of irradiated mothers was detected, pointing to a possible germ cell-transmitted alteration initiated by the preconceptual maternal X-ray exposure.

Effects of di-n-butylamine on the respiratory system of Wistar (WU) rats after subchronic inhalation.

Aim of the study was to investigate the potential toxic effects of di-n-butylamine (DBA), a known skin and eye irritating compound, on the respiratory tract after inhalation exposure for up to 91 days in male and female rats [Crl:(WI)WU BR]. To check whether and to what degree the no-observed-(adverse)-effect level (NO(A)EL) decreases with increasing study duration, serial sacrifices were performed after 3 and 28 days, respectively. Based on two dose range-finding studies, the concentrations for this study were determined with 0 (clean air), 50, 150, and 450 mg/m(3). Animals were exposed for 3 days (6 h/day) 28, and 91 days (5 days/wk, 6 h/day), respectively, and immediately sacrificed thereafter. The results show clear irritating effects only in the upper part of the respiratory tract, that is, the nasal cavities. While after 3 and 28 days effects were found only in the high-dose group, slight adaptive effects, expressed as mucous (goblet) cell hyperplasia, could be diagnosed in the medium- and low-dose groups after 91 days of exposure. Pathological changes were most prominent after 3 days of exposure. In the lung, only marginal effects could be observed (increased relative lung weight only in females of the high concentration after 28 days, slight, not statistically significant histopathological effects in the high concentration after 3 days, no effects on parameters of bronchoalveolar lavage fluid), while no effects were found in the remaining groups.

Pulmonary inflammation in rats after intratracheal instillation of quartz, amorphous SiO2, carbon black, and coal dust and the influence of poly-2-vinylpyridine-N-oxide (PVNO).

Effects of poly-2-vinylpyridine-N-oxide (PVNO) were investigated in numerous in vivo and in vitro studies published in the nineteen sixties and seventies. These studies showed that PVNO inhibited development of fibrosis from quartz dust and improved lung clearance of quartz after inhalation exposure. Ameliorating effects of PVNO were observed also for pulmonary damage from colloidal SiO2 and organic substances, and the fibrogenic inflammation caused by carrageenan. Although it is not proven that silicosis is a precondition for quartz-induced lung tumours, we investigated the hypothesis that PVNO could reduce the lung tumour risk from quartz in rats. A carcinogenicity study was therefore started in rats with the main focus on the quantitative relationships among pulmonary inflammation, fibrosis and neoplasia caused by intratracheal instillation of 3 mg quartz DQ 12 with or without additional subcutaneous PVNO treatment. Other study groups were treated with multiple dust instillations, i.e. 30 instillations of 0.5 mg amorphous SiO2 at intervals of 2 weeks, 10 instillations of 0.5 mg of ultrafine carbon black or 1 mg coal at weekly intervals. The analyses of the bronchoalveolar lavage fluid (BALF) 9 months after start of the life-time study showed that the aim of producing similar levels of increased enzyme concentrations in the four groups treated with quartz/PVNO, amorphous SiO2, carbon black and coal was achieved. A 2.5- to 7.7-fold increase for lactate dehydrogenase (LDH), total protein, alkaline phosphatase and gamma-glutamyl transferase (gamma-GT) was found in these groups as compared to the control. In contrast, quartz treatment without PVNO increased the LDH level up to 24-fold and of total protein to 13-fold. However, the cell counts in the BALF were not so much different in all five groups, i.e. quartz without PVNO (leukocytes: 480.000, PMN: 190.000), quartz with PVNO (leukocytes: 300.000, PMN: 100.000), amorphous SiO2 (leukocytes: 570.000, PMN: 315.000), carbon black (leukocytes: 390.000, PMN: 150.000) and coal (leukocytes: 200.000, PMN: 65.000). Histopathological investigations after four weeks and three months revealed that the used PVNO sample was active in the quartz and amorphous SiO2 groups and markedly reduced the incidences or severity of several pulmonary changes such as macrophage accumulation, inflammatory cell infiltration, interstitial fibrosis, bronchiolo-alveolar hyperplasia, alveolar lipoproteinosis and amorphous SiO2 -induced granulomatous alveolitis/interstitial fibrotic granulomas. Also in the lung-associated lymph nodes (LALN), PVNO treatment significantly reduced the incidence and severity of inflammation in both quartz and amorphous SiO2 groups as evidenced by the presence of well-circumscribed aggregates of intact particle-laden macrophages without signs of degeneration and accompanying granulocytic infiltration and fibrosis. Immunological investigations at the 9 months timepoint on the in vitro production of reactive nitrogen (RNI) or oxygen (ROI) intermediates and tumour necrosis factor (TNF-alpha) from BALF-derived cells indicated a diminished responsiveness to LPS in all particle treatment groups. A diminished production of ROI was also found in the quartz, carbon black, and coal dust groups, respectively, as compared to the values seen in the quartz/PVNO- and amorphous SiO2 treated groups. Treatment with quartz plus PVNO restored the capability of the cells to respond to LPS as compared to the treatment with quartz alone. TNF-alpha production was diminished in the groups treated with quartz, carbon black, and coal dust alone whereas in the quartz/PVNO- and amorphous SiO2-treated groups an elevated TNF-alpha production was seen. These results led to the conclusion that only amorphous SiO2 did not affect the "normal" ability of the cells to respond to LPS and that PVNO protected the cells from a toxic effect of the quartz particles.

Lack of promotion of estrogen-dependent mammary gland tumors in vivo by an isopropanolic Cimicifuga racemosa extract.

Cimicifuga racemosa (CR) is widely used in the treatment of menopausal symptoms. Mechanistic studies suggest that unlike hormone-replacement therapy, CR does not stimulate estrogen-receptor positive breast cancer cells. To evaluate CR safety, we performed an in vivo investigation of a clinically tested isopropanolic CR extract. Mammary tumors were induced in Sprague Dawley rats (n = 75) by the application of 7,12-dimethylbenz[a]anthracene. Five to nine weeks later, the animals were ovariectomized, allowed to recover, and administered daily doses of CR extract (0.714, 7.14, or 71.4 mg/kg body weight per day) or control substances (estrogen/positive control: 450 microg/kg/day mestranol; or CR vehicle/negative control). The animals were sacrificed 6 weeks later, and tumor number, size, plasma hormone levels, and the weight of estrogen-sensitive organs were analyzed. In contrast to mestranol treatment, CR treatment did not stimulate cancerous growth. There were no significant differences in tumor number or size between the CR groups and the vehicle control. Likewise, prolactin, follicle-stimulating hormone, and luteinizing hormone levels and organ weights and endometrial proliferation were unaffected. The lack of mammary tumor-stimulating effects of this extract is of great significance in establishing the safety of CR extracts for treatment of menopausal symptoms in women with a history of breast cancer in which hormone-replacement therapy is contraindicated.