US regulations to curb alleged cancer causes are ineffectual and compromised by scientific, constitutional and ethical violations.

The 1958 Delaney amendment to the Federal Food Drug and Cosmetics Act prohibited food additives causing cancer in animals by appropriate tests. Regulators responded by adopting chronic lifetime cancer tests in rodents, soon challenged as inappropriate, for they led to very inconsistent results depending on the subjective choice of animals, test design and conduct, and interpretive assumptions. Presently, decades of discussions and trials have come to conclude it is impossible to translate chronic animal data into verifiable prospects of cancer hazards and risks in humans. Such conclusion poses an existential crisis for official agencies in the US and abroad, which for some 65 years have used animal tests to justify massive regulations of alleged human cancer hazards, with aggregated costs of $trillions and without provable evidence of public health advantages. This article addresses suitable remedies for the US and potentially worldwide, by critically exploring the practices of regulatory agencies vis-á-vis essential criteria for validating scientific evidence. According to this analysis, regulations of alleged cancer hazards and risks have been and continue to be structured around arbitrary default assumptions at odds with basic scientific and legal tests of reliable evidence. Such practices raise a manifold ethical predicament for being incompatible with basic premises of the US Constitution, and with the ensuing public expectations of testable truth and transparency from government agencies. Potential remedies in the US include amendments to the US Administrative Procedures Act, preferably requiring agencies to justify regulations compliant with the Daubert opinion of the Daubert ruling of the US Supreme Court, which codifies the criteria defining reliable scientific evidence. International reverberations are bound to follow what remedial actions may be taken in the US, the origin of current world regulatory procedures to control alleged cancer causing agents.

Size, Surface Functionalization, and Genotoxicity of Gold Nanoparticles In Vitro.

Several studies suggested that gold nanoparticles (NPs) could be genotoxic in vitro and in vivo. However, gold NPs currently produced present a wide range of sizes and functionalization, which could affect their interactions with the environment or with biological structures and, thus, modify their toxic effects. In this study, we investigated the role of surface charge in determining the genotoxic potential of gold NPs, as measured by the induction of DNA damage (comet assay) and chromosomal damage (micronucleus assay) in human bronchial epithelial BEAS-2B cells. The cellular uptake of gold NPs was assessed by hyperspectral imaging. Two core sizes (~5 nm and ~20 nm) and three functionalizations representing negative (carboxylate), positive (ammonium), and neutral (poly(ethylene glycol) (PEG)ylated) surface charges were examined. Cationic ammonium gold NPs were clearly more cytotoxic than their anionic and neutral counterparts, but genotoxicity was not simply dependent on functionalization or size, since DNA damage was induced by 20-nm ammonium and PEGylated gold NPs, while micronucleus induction was increased by 5-nm ammonium and 20-nm PEGylated gold NPs. The 5-nm carboxylated gold NPs were not genotoxic, and evidence on the genotoxicity of the 20-nm carboxylated gold NPs was restricted to a positive result at the lowest dose in the micronucleus assay. When interpreting the results, it has to be taken into account that cytotoxicity limited the doses available for the ammonium-functionalized gold NPs and that gold NPs were earlier described to interfere with the comet assay procedure, possibly resulting in a false positive result. In conclusion, our findings show that the cellular uptake and cytotoxicity of gold NPs are clearly enhanced by positive surface charge, but neither functionalization nor size can single-handedly account for the genotoxic effects of the gold NPs.

Genotoxic and inflammatory effects of nanofibrillated cellulose in murine lungs.

Nanofibrillated cellulose (NFC) is a sustainable and renewable nanomaterial, with diverse potential applications in the paper and medical industries. As NFC consists of long fibres of high aspect ratio, we examined here whether TEMPO-(2,2,6,6-tetramethyl-piperidin-1-oxyl) oxidised NFC (length 300-1000nm, thickness 10-25nm), administrated by a single pharyngeal aspiration, could be genotoxic to mice, locally in the lungs or systemically in the bone marrow. Female C57Bl/6 mice were treated with four different doses of NFC (10, 40, 80 and 200 µg/mouse), and samples were collected 24h later. DNA damage was assessed by the comet assay in bronchoalveolar lavage (BAL) and lung cells, and chromosome damage by the bone marrow erythrocyte micronucleus assay. Inflammation was evaluated by BAL cell counts and analysis of cytokines and histopathological alterations in the lungs. A significant induction of DNA damage was observed at the two lower doses of NFC in lung cells, whereas no increase was seen in BAL cells. No effect was detected in the bone marrow micronucleus assay, either. NFC increased the recruitment of inflammatory cells to the lungs, together with a dose-dependent increase in mRNA expression of tumour necrosis factor α, interleukins 1ß and 6, and chemokine (C-X-C motif) ligand 5, although there was no effect on the levels of the respective proteins. The histological analysis showed a dose-related accumulation of NFC in the bronchi, the alveoli and some in the cytoplasm of macrophages. In addition, neutrophilic accumulation in the alveolar lung space was observed with increasing dose. Our findings showed that NFC administered by pharyngeal aspiration caused an acute inflammatory response and DNA damage in the lungs, but no systemic genotoxic effect in the bone marrow. The present experimental design did not, however, allow us to determine whether the responses were transient or could persist for a longer time.

Genotoxic and immunotoxic effects of cellulose nanocrystals in vitro.

Nanocellulosics are among the most promising innovations for a wide-variety of applications in materials science. Although nanocellulose is presently produced only on a small scale, its possible toxic effects should be investigated at this early stage. The aim of the present study was to examine the potential genotoxicity and immunotoxicity of two celluloses in vitro - cellulose nanocrystals (CNC; mean fibril length 135 nm, mean width 7.3 nm) and a commercially available microcrystalline (non-nanoscale) cellulose (MCC; particle size ∼50 µm). Both celluloses showed 55% cytotoxicity at approximately 100 µg/ml after 4-h, 24-h, and 48-h treatment of human bronchial epithelial BEAS 2B cells, as determined by luminometric detection of ATP and cell count (dead cells identified by propidium iodide). Neither of the materials was able to induce micronuclei (MN) in binucleate or mononucleate BEAS 2B cells after a 48-h treatment (2.5-100 µg/ml). In human monocyte-derived macrophages, MCC induced a release (measured by enzyme-linked immunosorbent assay; ELISA) of the pro-inflammatory cytokines tumor necrosis factor α (TNF-α) and (after lipopolysaccharide-priming) interleukin 1ß (IL-1ß) after a 6-h exposure to a dose of 300 µg/ml, but CNC (30-300 µg/ml) did not. In conclusion, our results show that nanosized CNC is neither genotoxic nor immunotoxic under the conditions tested, whereas non-nanosized MCC is able to induce an inflammatory response. More studies are needed, especially in vivo, to further assess if CNC and other nanocelluloses induce secondary genotoxic effects mediated by inflammation.

Inhalation of rod-like carbon nanotubes causes unconventional allergic airway inflammation.

BACKGROUND: Carbon nanotubes (CNT) represent a great promise for technological and industrial development but serious concerns on their health effects have also emerged. Rod-shaped CNT are, in fact, able to induce asbestos-like pathogenicity in mice including granuloma formation in abdominal cavity and sub-pleural fibrosis. Exposure to CNT, especially in the occupational context, happens mainly by inhalation. However, little is known about the possible effects of CNT on pulmonary allergic diseases, such as asthma. METHODS: We exposed mice by inhalation to two types of multi-walled CNT, rigid rod-like and flexible tangled CNT, for four hours a day once or on four consecutive days. Early events were monitored immediately and 24 hours after the single inhalation exposure and the four day exposure mimicked an occupational work week. Mast cell deficient mice were used to evaluate the role of mast cells in the occurring inflammation. RESULTS: Here we show that even a short-term inhalation of the rod-like CNT induces novel innate immunity-mediated allergic-like airway inflammation in healthy mice. Marked eosinophilia was accompanied by mucus hypersecretion, AHR and the expression of Th2-type cytokines. Exploration of the early events by transcriptomics analysis reveals that a single 4-h exposure to rod-shaped CNT, but not to tangled CNT, causes a radical up-regulation of genes involved in innate immunity and cytokine/chemokine pathways. Mast cells were found to partially regulate the inflammation caused by rod-like CNT, but also alveaolar macrophages play an important role in the early stages. CONCLUSIONS: These observations emphasize the diverse abilities of CNT to impact the immune system, and they should be taken into account for hazard assessment.

Range-finding risk assessment of inhalation exposure to nanodiamonds in a laboratory environment.

This study considers fundamental methods in occupational risk assessment of exposure to airborne engineered nanomaterials. We discuss characterization of particle emissions, exposure assessment, hazard assessment with in vitro studies, and risk range characterization using calculated inhaled doses and dose-response translated to humans from in vitro studies. Here, the methods were utilized to assess workers' risk range of inhalation exposure to nanodiamonds (NDs) during handling and sieving of ND powder. NDs were agglomerated to over 500 nm particles, and mean exposure levels of different work tasks varied from 0.24 to 4.96 µg·m(-3) (0.08 to 0.74 cm(-3)). In vitro-experiments suggested that ND exposure may cause a risk for activation of inflammatory cascade. However, risk range characterization based on in vitro dose-response was not performed because accurate assessment of delivered (settled) dose on the cells was not possible. Comparison of ND exposure with common pollutants revealed that ND exposure was below 5 µg·m(-3), which is one of the proposed exposure limits for diesel particulate matter, and the workers' calculated dose of NDs during the measurement day was 74 ng which corresponded to 0.02% of the modeled daily (24 h) dose of submicrometer urban air particles.

Genotoxicity of short single-wall and multi-wall carbon nanotubes in human bronchial epithelial and mesothelial cells in vitro.

Although some types of carbon nanotubes (CNTs) have been described to induce mesothelioma in rodents and genotoxic effects in various cell systems, there are few previous studies on the genotoxicity of CNTs in mesothelial cells. Here, we examined in vitro DNA damage induction by short multi-wall CNTs (MWCNTs; 10-30 nm × 1-2 µm) and single-wall CNTs (SWCNTs; >50% SWCNTs, ~40% other CNTs; <2 nm × 1-5 µm) in human mesothelial (MeT-5A) cells and bronchial epithelial (BEAS 2B) cells, using the single cell gel electrophoresis (comet) assay and the immunoslot blot assay for the detection of malondialdehyde (M1dG) DNA adducts. In BEAS 2B cells, we also studied the induction of micronuclei (MN) by the CNTs using the cytokinesis-block method. The cells were exposed to the CNTs (5-200 µg/cm(2), corresponding to 19-760 µg/ml) for 24 and 48h in the comet assay and for 48 and 72 h in the MN and M1dG assays. Transmission electron microscopy (TEM) showed more MWCNT fibres and SWCNT clusters in BEAS 2B than MeT-5A cells, but no significant differences were seen in intracellular dose expressed as area of SWCNT clusters between TEM sections of the cell lines. In MeT-5A cells, both CNTs caused a dose-dependent induction of DNA damage (% DNA in comet tail) in the 48-h treatment and SWCNTs additionally in the 24-h treatment, with a statistically significant increase at 40 µg/cm(2) of SWCNTs and (after 48 h) 80 µg/cm(2) of both CNTs. SWCNTs also elevated the level of M1dG DNA adducts at 1, 5, 10 and 40 µg/cm(2) after the 48-h treatment, but both CNTs decreased M1dG adduct level at several doses after the 72-h treatment. In BEAS 2B cells, SWCNTs induced a statistically significant increase in DNA damage at 80 and 120 µg/cm(2) after the 24-h treatment and in M1dG adduct level at 5 µg/cm(2) after 48 h and 10 and 40 µg/cm(2) after 72 h; MWCNTs did not affect the level of DNA damage but produced a decrease in M1dG adducts in the 72-h treatment. The CNTs did not affect the level of MN. In conclusion, MWCNTs and SWCNTs induced DNA damage in MeT-5A cells but showed a lower (SWCNTs) or no (MWCNTs) effect in BEAS 2B cells, suggesting that MeT-5A cells were more sensitive to the DNA-damaging effect of CNTs than BEAS 2B cells, despite the fact that more CNT fibres or clusters were seen in BEAS 2B than MeT-5A cells. M1dG DNA adducts were induced by SWCNTs but decreased after a 3-day exposure to MWCNTs and (in MeT-5A cells) SWCNTs, indicating that CNTs may lead to alterations in oxidative effects within the cells. Neither of the CNTs was able to produce chromosomal damage (MN).

Genotoxicity of inhaled nanosized TiO(2) in mice.

In vitro studies have suggested that nanosized titanium dioxide (TiO(2)) is genotoxic. The significance of these findings with respect to in vivo effects is unclear, as few in vivo studies on TiO(2) genotoxicity exist. Recently, nanosized TiO(2) administered in drinking water was reported to increase, e.g., micronuclei (MN) in peripheral blood polychromatic erythrocytes (PCEs) and DNA damage in leukocytes. Induction of micronuclei in mouse PCEs was earlier also described for pigment-grade TiO(2) administered intraperitoneally. The apparent systemic genotoxic effects have been suggested to reflect secondary genotoxicity of TiO(2) due to inflammation. However, a recent study suggested that induction of DNA damage in mouse bronchoalveolar lavage (BAL) cells after intratracheal instillation of nanosized or fine TiO(2) is independent of inflammation. We examined here, if inhalation of freshly generated nanosized TiO(2) (74% anatase, 26% brookite; 5 days, 4 h/day) at 0.8, 7.2, and (the highest concentration allowing stable aerosol production) 28.5 mg/m(3) could induce genotoxic effects in C57BL/6J mice locally in the lungs or systematically in peripheral PCEs. DNA damage was assessed by the comet assay in lung epithelial alveolar type II and Clara cells sampled immediately following the exposure. MN were analyzed by acridine orange staining in blood PCEs collected 48 h after the last exposure. A dose-dependent deposition of Ti in lung tissue was seen. Although the highest exposure level produced a clear increase in neutrophils in BAL fluid, indicating an inflammatory effect, no significant effect on the level of DNA damage in lung epithelial cells or micronuclei in PCEs was observed, suggesting no genotoxic effects by the 5-day inhalation exposure to nanosized TiO(2) anatase. Our inhalation exposure resulted in much lower systemic TiO(2) doses than the previous oral and intraperitoneal treatments, and lung epithelial cells probably received considerably less TiO(2) than BAL cells in the earlier intratracheal study.

Glutamate increases toxicity of inorganic lead in GT1-7 neurons: partial protection induced by flunarizine.

Recent studies point to an interaction between the glutamatergic neurotransmitter system and inorganic lead (Pb) neurotoxicity. Pb (1-100 microM) evoked cytotoxicity over the period of 72 h in mouse hypothalamic GT1-7 neurons. Glutamate (0.1 or 1 mM) on its own did not have any effect on cell viability. However, 1 mM glutamate clearly increased Pb-induced cell death at 48 and 72 h. Although flunarizine (0.1-10 microM), an antagonist of L- and T-type voltage-sensitive calcium channels (VSCCs), partially protected from the cytotoxicity induced by co-exposure to Pb (10 or 100 micro M) and glutamate (1 mM), it had no protective effect on cytotoxicity induced by Pb alone. The flunarizine-induced protection was dependent on time and observed only at 48 h. Neither verapamil, an antagonist of L-type VSCCs, nor DIDS, an inhibitor of anion exchange, at non-toxic concentrations (0.1-10 microM) had any effect on cytotoxicity induced by Pb alone or together with glutamate at any studied time point. Co-exposure to Pb and glutamate also resulted in more prominent production of reactive oxygen species (ROS) than either of the compounds alone. Interestingly, we observed an increase in intracellular glutathione (GSH) levels in cells exposed to micromolar concentrations of Pb. Glutamate decreased the levels of intracellular GSH and also partially reduced the Pb-induced increase in GSH levels. These results suggest that the interaction of glutamate and Pb results in increased neuronal cell death via mechanisms that involve an increase in ROS production, a decrease in intracellular GSH defense against oxidative stress and probably T-type VSCCs.

Pb2+-induced toxicity is associated with p53-independent apoptosis and enhanced by glutamate in GT1-7 neurons.

Recent studies indicate that the glutamatergic neurotransmitter system is involved in neurotoxicity caused by inorganic lead (Pb2+). We studied the role of apoptosis in the effects induced by Pb2+ (0.01-100 microM) and glutamate (0.1 and 1 mM) in mouse hypothalamic GT1-7 neurons. Although glutamate alone had no effect on cell viability, it enhanced neuronal cell death induced by Pb2+ (1-100 microM) within 72 h. Glutamate alone neither induced caspase-3-like protease activity nor promoted internucleosomal DNA fragmentation, both biochemical hallmarks of apoptosis. However, concurrent exposure to Pb2+ (10 or 100 microM) and glutamate (1 mM) resulted in more prominent cleavage of the fluorogenic caspase-3 substrate (Ac-DEVD-AMC) than caused by the same Pb2+ concentrations alone at 24-72 h. The highest caspase-3-like protease activities were measured at 48 h. Internucleosomal DNA fragmentation caused by Pb2+ (10 or 100 microM) alone or together with glutamate (1 mM) was evident at 96 h, less clear at 72 h and absent at 48 h. Immunoblotting did not reveal any changes in p53 protein levels in cells exposed to Pb2+, glutamate or their combination at any studied time point (3-72 h). Our results suggest that Pb2+-induced neurotoxicity may partially be mediated through p53-independent apoptosis and enhanced by glutamate.