Abstract
Some natural clays and synthetic nanofibres present in the environment have a severe impact on human health. After several decades of research, the molecular mechanism of how asbestos induce cancers is not well understood. Different fibres, including asbestos, can penetrate the membrane and introduce DNA in both, bacterial and eukaryotic cells. Incubating Escherichia coli with sepiolite, a clayey material, and asbestos under friction forces, both fibres cause double-strand breaks in bacteria. Since antibiotics and clays are used together in animal husbandry, the mutagenic effect of these fibres might constitute a pathway to antibiotic resistance due to the friction provided by peristalsis of the gut from farm animals in addition to the previously proposed horizontal gene transfer. Moreover, we raise the possibility that the same mechanism could generate bacteria diversity in natural scenarios with a role in the evolution of species. Finally, we provide a new model on how asbestos may promote mutagenesis and cancer based on the observed mechanical genotoxicity.
Introduction
Clays such as sepiolite are jointly used with antibiotics in farming as growth promoters. This practice improves growth and animal product quality, and these additives are common in feed for broiler chickens and pigs [1,2]. Sepiolite is considered to be safe, stable and chemical inert hence being also used in tablet formulation for human medicine [3]. However, in a recent study clays used as animal feed additive can increase the risk of horizontal gene transfer (HGT) among microbes, resulting possibly in a rise of antibiotic resistance [4,5].
In this case, the transformation of bacteria by foreign DNA can be achieved when clay fibres are spread by friction or vibrations. This phenomenon is known as Yoshida effect [6] and relies on the ability of mineral nanofibres or nano-needles to adsorb DNA and to penetrate bacterial cells under sliding friction forces [7]. By its mechanical nature, the Yoshida effect can be used to transform diverse bacterial species [5,8,9]. The action of sepiolite and other clays fibres is not only capable of delivering DNA into the receptor bacteria but also able to promote the releasing of DNA by disrupting the cell envelope of the portion of the population by the abrasive action of clays [5].
Before Yoshida began his experiments with bacteria, the ability of asbestos to transform eukaryotic cells was reported at the end of the eighties [10]. In fact, fibrous clays and industrial nanofibres are considered genotoxic and carcinogenic, likely due to their ability to damage DNA [11]. They have assayed in several experimental models including bacteria and cell in cultures, but they display a poor correlation with mutagenicity or carcinogenesis found in vivo [12,13]. According to these observations, a significant concern arises from fibrous clays or industrial nanofibres which are responsible for severe human diseases such as asbestosis [14]. However, short or long periods of exposure to fibres have been failing to identify a molecular basis of DNA damage in different several genotoxicity tests [14]. Thus, nowadays the mechanisms underlying the genotoxicity and carcinogenicity of asbestos and other fibres remain obscure.
Additionally, clays may have the potential to enhance antibiotic resistance in farming activities [4]. In natural scenarios, sediments and stones (gastroliths) are frequently swallowed by animals resulting unavoidably in the exposure of their microbiota to pebbles, sand, and clays. Soils and waters are a primary source of antimicrobials, either by natural microbial production or environmental antibiotic pollution, a major selective pressure that favours resistant strains [15,16]. Even, gut microbes can produce antibiotic compounds [17].
In this study, the ability of fibrous clays such as sepiolite and asbestos to transform bacteria and to induce mutagenic DNA double-strand breaks (DSBs) when they are exposed to friction forces was experimentally shown. Additionally, a molecular mechanism of action for asbestos, which was a strong inducer of DSBs in Escherichia coli when friction is present, was proposed. Finally, the importance of this mechanism is discussed for the speciation processes of animals that use gastroliths.
Results and Discussion
Mutant frequency in sepiolite-treated cells is higher than in non sepiolite-treated cells
Different types of clays can transform bacteria by absorbing DNA and penetrating the cell envelope. In that case, the penetration could allow the clays to interact with the intracellular DNA and promote mutations. To test whether sepiolite under friction forces (as in transformation) has an impact on bacterial mutation rate, the mutant frequency of Escherichia coli was measured by plating in the antibiotic fosfomycin and enumerating spontaneous mutants (fig 1). When the cells were merely exposed to sepiolite without any friction on agar plates surface, no significant differences in mutant frequencies were detected (Mann-Whitney U test; P=0.999). In contrast, a six-fold increase in mutant frequency was found when friction was present for two or three minutes (Mann-Whitney U test; P=0.008) and a modest increase—but not significant—when the treatment time lasted for one minute (Mann-Whitney U test; P=0.421). Interestingly, only cells in the stationary phase displayed an increase in mutant frequency (Kruskal-Wallis test; P=0.001), while no significant mutagenesis was found when bacteria came from exponential cultures (Kruskal-Wallis test; P=0.954; fig 1). Along with the mutant frequency experiments, the effect of the treatments on cell viability was checked. Sensitivity to the treatment is higher in exponential phase than in stationary phase cultures (fig 2). The observed mutant frequency differences was initially attributed to a higher sensitivity to the treatment. However, several hypotheses can explain the observed increase in mutant frequency and reduced-sensitivity in stationary phase cultures.
Heavy metals are non relevant on sepiolite mutagenesis
Many minerals containing metals such as iron, aluminium or copper are toxic for bacteria because of the generation of reactive oxygen species (ROS) via the Fenton reaction [18]. The release of metal ions inside the cell could therefore be the reason of the increase in mutagenesis. In fact, despite the addition of 2-2′ bipyridyl, a chelating agent, shortly before treatment, mutagenesis was still observed (Kruskal-Wallis test; P=0.008; fig S1). This result indicates that the mutagenic effect does not depend on the metals present in the fibres.
Sepiolite interacts with the DNA by causing double-strand breaks
A second likely explanation is the physical interaction of individual clay fibres in motion directly damaging DNA by creating DSBs. The ability of sepiolite fibres to penetrate and interact with DNA has already been stated [6,19]. Physical or mechanical stress on the DNA duplex is a relevant cause of DSBs [20]. To evaluate this possibility, E. coli DH5α strain (recA deficient) carrying the plasmid (pET-19b) was subjected to treatment with sepiolite and sliding friction. Sepiolite without friction and bacterial cells alone were used as controls. The plasmid content was extracted, and its integrity was evaluated by gel migration (fig 3A). Typically, during plasmid DNA extraction, three molecular conformations are found: the supercoiled (which migrates very fast), nicked DNA (which is also closed circular but relaxed due to single strand breaks and it has an intermediate migration rate) and linear molecules (with a lower migration speed) [21]. These latter DNA molecules were especially abundant in the friction-sepiolite treated group at the time that they are present in a low level in control groups. In fact, plasmids from the sepiolite group (under friction) presented a significantly high level of linearised molecules when compared to the control groups (One-Way ANOVA test; P=3.33×10−16). According to these results, the joint action of sepiolite and friction are responsible for induction of DSBs in the DNA. Interestingly, no increase in nicked DNA (single-strand break) was observed, indicating that if this type of lesion occurs, it happens at a non-detectable rate by this technique (fig 3B).
The view of mutagenic DSBs by mechanical shearing is very consistent with the absence of mutagenic effect in exponentially growing bacteria. If the organism is diploid (even if the diploidy is only transient, as in replicating bacteria or replicating haploid yeast), then homology-directed repair can be used [20]. Because E. coli lacks a pathway to join non-homologous ends, homologous recombination is the only mechanism to salvage broken chromosomes [22]. But how can E. coli repair DSBs in stationary phase by homologous recombination? Stationary-phase cultures contain cells with several chromosome copies [23]. In exponentially growing E. coli DSB repair is non-mutagenic [24,25]. However, break repair becomes mutagenic during the stationary phase and requires the Sigma S factor (RpoS), the SOS response, and the error-prone DNA polymerase PolIV. The change from one situation to the other has been described as a switch from high-fidelity repair in the exponential phase to error-prone DNA double-strand breaks during the stationary phase [24,25]. Because DSBs are lethal unless repaired, and repair action requires RecA protein [24,25], the experiment of sepiolite mutagenesis was repeated with E. coli DH5α that is impaired in the SOS response triggering to confirm this notion. In such analysis, sepiolite mutagenesis was completely abolished by recA gene inactivation in stationary phase (Kruskal-Wallis test; P=0.011; fig 4). Thus, the lower level of mutant frequency in the recA deficient strain could be explained by the death of cells that suffered DSBs and were unable to repair them. Mutations introduced by DSB repair are considered a mechanism of diversity via mutagenic repair in bacteria [26,27].
Potentially, the mutagenicity of clay treatment is also enhanced in stationary phase cells due to DNA being more tightly compacted than in the exponential phase [28]. Indeed, in Escherichia coli, DNA goes to a co-crystallization state with the stress-induced protein Dps offering protection to several types of stress, ordinarily chemical damage [29]. However, while crystallization is often associated with less flexibility or added fragility to direct physical contact, less compacted DNA of proliferating E. coli is elastic and soft [30], which may limit the number of DSBs. It is then possible that mineral fibres under friction can break DNA strands more easily in the stationary than in the exponential phase.
Sepiolite fibres can penetrate bacteria when friction forces are present
To reunite more evidence that penetration and interaction of fibres with DNA cause DSBs inside the cell, a direct observation of sepiolite-treated bacteria by scanning electron microscopy (SEM) was performed. Fibres look compatible in dimensions able to penetrate bacteria without completely destroying the envelope. Additionally, bacteria were directly penetrated by fibres while those that were exposed to mineral without friction were not (fig 5). This observation is in concordance with previous studies, whereas sepiolite and other nano-sized acicular materials can penetrate bacterial cells under friction forces on a hydrogel [6]. In fact, the partial destruction of the cell wall and the presence of mutants after adding 2-2′ bipyridyl point to the mechanical action as causing agent of the damage. The notion of mechanical breaks is in good agreement with the results in cell-free systems. In these experiments, breakage of plasmid DNA was not directly associated with the amount of iron released by asbestos fibres when they are incubated together [14].
Sepiolite fibre length matters to cause significant DNA damage in the cell
Sepiolite also contains very short fibres (fig S2). In the case of asbestos, there is a certainty that long fibres are much more dangerous by their carcinogenic potential. We designed an experiment to test the influence sepiolite fibre length for mutagenesis in bacteria. The exposure of stationary phase bacteria to a suspension of short fibres (less than 1 μm) did not cause any significant DNA damage when compared with the control and in contrast with the long-fibre original mineral suspension (Kruskal-Wallis test; P=0.005; fig 6).
Asbestos fibres increase the mutant frequency in the same way as sepiolite do
Bacterial genotoxicity experiments are considered a key step in the assessment of mutagenic properties of chemicals, drugs or materials in general [31]. Because asbestos fibres resemble sepiolite ones, an experiment to test if asbestos fibres provoke an increase in mutagenesis was designed using crocidolite asbestos (fig S2). In our assay, the addition of asbestos to bacteria in the plates without friction did not increase the mutant frequency. In contrast, the application of friction when the fibres were present increased the mutant frequency even more than sepiolite alone (Kruskal-Wallis test; P=0.002; fig 7), probably by the same mode of action. Yoshida et al. have suggested that asbestos and other clays can be potentially mutagenic based on integrity analysis of genomic DNA from treated bacteria [32]. A clear antecedent of the ability of fibrous nanoclays to penetrate bacteria was the transformation of monkey cells in culture by exogenous plasmid DNA using chrysotile (a type of asbestos) [10]. Although procedures are not described in details, we think that this transformation requires penetration of the cell membrane.
Further discussion
The poor correlation between DNA damage in vivo and in vitro described in previous studies [12] may be explained by the limited or lack of penetration of asbestos in experimental designs. Thus, the introduction of some friction or shaking can in determining if penetration of cells by asbestos and other fibres underly a molecular mechanism of carcinogenesis. The mechanism(s) underlying asbestos toxicity associated with the pathogenesis of mesothelioma has been a challenge to unravel for more than six decades [33]. According to our results and the current knowledge about asbestos-induced carcinomas, we speculate about a model that explains a potential path leading to carcinomas. Briefly, we think that people exposed to asbestos fibres during prolonged periods accumulate them in the respiratory tract. It is frequent to find asbestos fibres into the pleural cavity, and maybe they increase the friction coefficient in the pleural space, a parameter with a very small value in in physiological conditions [34]. The coelomic movement (a cyclical mechanical movement between the parietal pleura—covering membrane of the inner surface of the thoracic cavity—and the visceral pleura—covering membrane of the lung surface—) provokes the movement of asbestos, trespassing occasionally the mesothelial cell membranes or floating mesothelial cells, physically interacting and disrrupting the DNA or spindle. This physical interaction, with adequate intensity, could induce DSBs, which generate chromosome aberrations or fragmentations in eukaryotic cells as we found here for bacteria. After years of exposure, DSBs or spindle disruption can cause chromosome damages or losses or aneuploidy that increase the probability of malignancy. The proposed model for eukaryote cells would need in vitro validation with epithelial cells but this is beyond the scope of the current study and left for future research. Moreover, this model does not exclude other toxic and genotoxic mechanisms of asbestosis such as reactive species arising from metal action or inflammatory response.
One of the most important limitations of our study is the lack of an animal model to test if our finding of mutagenicity in bacteria by clays occurs in vivo. In theory, clays present in livestock feed could promote antibiotic resistance and virulence in pathogenic bacteria by not only their transformation ability but also via mutations. However, testing conditions are hindered by the fact that experiments would require at least S1 security level, and this is difficult to achieve with livestock animals [4]. Transformation of plasmid DNA requires penetration and sepiolite and other clays have shown this capacity in a wide range of concentrations although it diminishes at high concentration due to the killing of bacteria [9,35–37]. In a previous study, the values of pressure in the gut of many animal species were discussed, meeting the criteria very well [4]. The presence of a hydrogel does not seem to be a problem since both mucin layer of the gut or mucoid secretion in the respiratory tract can play that role, particularly if fibres have the capacity to change viscosity locally or gradients of viscosity exist across these body compartments.
An implication of our study is the consideration of other factors (such the friction forces) in assessing of genotoxicity and carcinogenesis by certain fibrous materials. Until now, many studies associate clay-induced damage mostly with ROS [14]. DNA damage can be produced by oxido-reduction processes generated by metal containing-fibres. Asbestos fibres are carcinogenic for both, humans and experimental animals, because asbestos produce DNA breaks leading to the formation of micronucleus (a type of chromosomal aberration) [38]. This kind of damage seems to be caused more by mechanical action rather than ROS generation, which can worsen the situation but not necessarily has to be determinant. In other words, we think that ROS is more a symptom than a cause. Another example of a potentially dangerous material are the carbon nanotubes (CNTs), a novel industrial material with many applications. The genetic alterations provoked by these nanotubes in rat malignant mesothelioma were similar to those induced by asbestos [39]. Interestingly, CNTs lack heavy metals in their composition. The nanoscale size and needle-like rigid structure of CNTs appear to be associated with their pathogenicity in mammalian cells [38]. Coincidentally, CNTs can be used to transform bacteria with plasmids [40] in a similar fashion that asbestos [10,41] and sepiolite do [6,42]. It would not be surprising that all these fibrous nanomaterials share their ability to mechanically induce DSBs.
Recently, a possible link between talcum powder and ovarian cancer risk associated with asbestos contamination in talc is under discussion. Although the risk is small, some studies suggested a low or moderate but significant chance of cancer, while other rejected/discarded this correlation [43–45]. It is necessary to advance the understanding of molecular base of DNA damage by asbestos and other industrial fibres. If the proposed model of mechanical/physical DNA breaks is validated in future studies, some genotoxicity assays intended to unveil mutagenic properties of materials (e.g. the test of Ames) should be modified accordingly to include a standardised procedure of friction or promoting some sort of shaking during incubation steps. Similarly, several in vitro test, with both bacteria and eukaryotic cells, were modified by researchers and regulatory agencies where introduced the metabolic activation by fraction S9 of liver homogenate [46].
Other implications of the induction of DSBs by nanofibres in bacteria could be related with the microbiota of animals that use gastroliths. It has been suggested that gut microbes play a crucial role in keeping species apart or enhance the speciation [47]. It is tempting to speculate that animals that use gastroliths or sediment ingestion expose their microbiota to the abrasive action of stone derivative fibres. Therefore, the shaping of their own microbes is expected to contribute to their own speciation trajectories. Among animals that use or used gastroliths in their evolutionary trajectories, we find several branches of fishes, amphibians, reptiles (including dinosaurs) and birds. Gastroliths also regularly occur in several groups of invertebrates [48]. Wings (2007) recommends making a distinction between lithophagy and geophagy. Lithophagy (stones larger than 0.063 mm in diameter) is defined as the deliberate consumption of stones that turn into gastroliths after their ingestion. Geophagy is the consumption of soil and it is known for reptiles, birds, and mammals. These soils, rich in clays, salts or fat, serve mainly as a food supplement for the supply of specific minerals or for medical purposes [48]. Both concepts can contribute to getting together all the components that this mechanism needs to operate: gut microbiota, gut mucin mucoid layer (hydrogel) and friction forces provided by the peristaltic pressure of digestive tract in animals, especially the gizzard and the stomach. An interesting question is why sepiolite from limestone gastroliths does not damage the animal gut. A convincing explanation is that the mucoid layer in the gut protects it from the action of these sharp fibres at the time that serve as a protective layer for gut epithelium. In mammals, this mucoid layer is around 200 μm thick and is under continuous renovation [49]. Sepiolite is a natural clay mineral characterised by a nanofibre structure with average dimensions less than or equal to 0.2 micrometers in diameter, and from 2 to 5 micrometers in length, although longer fibres can be present.
Concluding remarks
Overall, one of the most significant contributions of this article is the proposition for the first time of a bacterial model to test genotoxicity of nanofibres and uncover a new mechanism of action for asbestos that correlates better with in vivo observations. Asbestosis is a global health and environmental problem, which molecular basis has been a challenge for several decades [33]. Although asbestos fibres are widely distributed in the anatomy of patients [33,50], the most common cancers caused by asbestos originate in lungs (mostly mesothelioma). If the most explored mechanism of action is based on reactive radicals (chemical damage), why is not there significant differences in the frequencies of other types of carcinoma such as leukaemia, lymphoma, liver or kidney cancer among exposed populations? In the last place, and not less important, is the tighter contact of slippery membranes (a monolayer of flattened epithelial-like cells) of the mesothelium. The pleural space is in continuous movement and constitute preferential target of asbestos-induced carcinogenesis. Of particular interest are free-floating mesothelial cells of the cavity, that even proliferate under damaging conditions [51]. The free-floating cells are the ideal candidates to be penetrated by asbestos in the pleural space. They may be more sensitive to suffer direct (physical) or indirect (chemical) DNA damage and become into a mesothelioma. Finally, sepiolite transformation technique gained some popularity in the last years because there is no need to prepare competence cells [9,19,42,52]. In that case, diverse bacteria can be transformed [4] in both stationary and exponentially growing phases. However, to prevent undesired mutations in both, plasmid and genomic DNA, it is highly recommendable to use exponential phase bacteria, where mutagenesis is not significant, at least in E. coli.
Methods
Bacteria and growth conditions
The E. coli MG1655 wild-type strain and its derivative mutants were cultured in Lysogenic Broth (LB). All experiments were performed at 37°C, with shaking in liquid culture. All solid cultures were grown in LB agar 1.5% for standard procedures and 2% for the sepiolite treatment. All cultures were supplemented with antibiotics when appropriate.
Mutant frequency estimation of sepiolite treated cells
Approximately 2 × 109 bacterial cells per ml of E. coli MG1655 and its derivative mutants from overnight or mid-exponential growing cultures were centrifuged and resuspended in 100 μl of sterilised transformation mixture, consisting of sepiolite (Kremer Pigmente, Germany) suspended in aqueous solution at a final concentration of 0.1 mg/ml. Resuspended cells were spread on plates containing fresh Müller-Hinton-Agar (Sigma-Aldrich, Germany) medium solidified with 2% agar, and Petri dishes were pre-dried in a biological safety flow cabinet for 20 minutes before use. Friction force was provided by streaking bacterial cultures plus sepiolite with sterile glass stir sticks gently pressed onto the medium surface for one, two and three minutes, applying as much pressure as possible without breaking the agar gel. Petri dishes were incubated at 37°C for 2 hours to allow DNA repair if any damage occurred. The plates were gently washed four times with 5 ml of 0.9% sodium chloride solution using a 5 ml pipette. The bacterial suspensions were transferred to 10 ml tubes to recover the cells by centrifugation at 3000 g for 10 minutes. The resulting pellets were resuspended in a final volume of 1 ml of fresh LB an incubate during 1 hour at 37°C to allow the cells to recover. Appropriate dilutions were plated onto LB plates to estimate bacterial viability and in LB plus fosfomycin (50 μg/ml) to estimate the number of resistant mutants. Plates were incubated overnight at 37°C. Each experiment consisted of 5 replicates and was repeated at twice. Mutant frequencies were calculated by using the FALCOR web-tool [53].
Influence of 2-2′ bipyridyl on sepiolite mutagenesis
The effect of 2-2′ bipyridyl, a metal chelating agent [54], on sepiolite mutagenesis was determined by measuring its influence on the mutant frequency for a selected concentration of sepiolite, where mutagenesis was observed. The experiment consisted of adding a titrating concentration of 2-2′ bipyridyl (200 μM) to chelate metals five minutes before the treatment. Cultures treated with sepiolite and friction without the addition of 2-2′ bipyridyl and bacteria alone without sepiolite were used as a control. The mutant frequencies for these groups were determined as described elsewhere in this section.
Assessing double-strand breaks with a plasmid system
To evaluate if sepiolite under friction treatment induces double-strand breaks in plasmid DNA, the strain Escherichia coli DH5α (fhuA2 lac(del)U169 phoA glnV44 Φ80’ lacZ(del)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17) carrying the plasmid pET-19b (Novagen, Germany) was treated with sepiolite and sliding friction forces during one minute. Several samples were recovered from the plates and pooled to compensate viability losses due to friction. The recovery was done by washing the surface with 5 ml 0.9 % NaCl saline solution four times as described for mutagenesis experiments. The recovered pellets were washed with 1 ml of TE buffer and the OD600 adjusted to 1 for each type of sample. Plasmid DNA samples were extracted using a Qiagen mini plasmid extraction kit (Qiagen, Germany). Added sepiolite with or without friction and no sepiolite groups were used as a control group. Each experiment consisted of five replicates. The same amount of plasmid DNA per replicate was applied per well to an agarose gel that was stained with SYBR® Gold Nucleic Acid Gel Stain kit (Molecular Probes, USA). A NdeI (Promega, USA) digested aliquot of pET-19b was used as control of linear migration rate. The proportion of linear molecules of the plasmid were compared among groups using a densitometry analysis by ImageJ [55].
RecA deficient strain construction
The recA null mutant was constructed following a previously described methodology [56] with the primers 5′-CAGAACATATTGACTATCCGGTATTACCCG-GCAT GACAGGAGT AAAAAT GGT-GT AGGCT GGAGCT GCTTC-3′ and 5-ATGCGACCCTTGTGTATCAAACAAGACGATTAAAAATCTTCGTTAGTTTCATGGGAAT-TAGCCATGGTCC-3′ (forward and reverse respectively) using the pKD3 plasmid as template. The mutant was checked by PCR amplification using the primers c1 5′-TTATACGCAAGGCGACAAGG-3′ and c2 5′-GATCTTCCGTCACAGGTAGG-3′ in combination with specific primers for upstream and downstream regions of recA gene: 5′-ATTGCAGACCTTGTGGCAAC-3′ and 5′-CGATCCAACAGGCGAGCATAT-3′ respectively. Additionally, the increased susceptibility to UV light and mitomycin C was tested phenotypically in comparison to the parental strain. The antibiotic resistance gene was eliminated using the pCP20 plasmid as described previously [56].
SEM of E. coli treated with sepiolite
Approximately 2×109 CFU of stationary phase E. coli MG1655 were treated with sepiolite and friction force was applied for one minute as described for the mutagenesis experiment. Circular agar blocks were taken from agar plates with a sterile cork borer (1 cm of diameter). Then, a thin surface layer was cut off, placed on a circular glass cover slip (1.5 cm of diameter) and incubated for 45 minutes at room temperature in a laminar flow cabinet to allow air drying of the samples. The cover glasses with dehydrated agar sections were mounted on aluminium stubs using double-sided adhesive tape and coated with gold in a sputter coater (SCD-040; Balzers, Union, Liechtenstein). The specimens were examined with a FEI Quanta 200 SEM (FEI Co., Hillsboro, OR) operating at an accelerating voltage of 15 kV under high vacuum mode at different magnifications. At least 5 sections from independent plates were observed to check physical penetration by the mineral. Some samples of sepiolite or asbestos (crocidotiles) alone were processed and observed in the same way.
Long fibre-depleted sepiolite mutagenesis experiment
To assess the role of long fibre of sepiolite in mutagenesis, a sepiolite preparation depleted of fibres longer than 1 μm was obtained. A 100 ml sepiolite suspension (1 mg/ml) in distilled water was passed though Pall® Acrodisc® glass fibre syringe filters (Sigma, USA) several times. The resulting suspension was desiccated by evaporation at 70°C overnight. A non-filtered solution was used as a control. From the obtained powder, two suspensions were prepared to a final proportion of 0.1 mg/ml. These two solutions were used for a mutagenesis experiment plating in fosfomycin as indicated previously, using a friction time of two minutes.
Mutant frequency estimation of asbestos treated cells
The procedure was carried out identically that the one described for sepiolite in this section. The time was set to two minutes and the same concentration that was used, 0.1 mg/ml. We used the crocidolite asbestos analytical standard (SPI Supplies, USA). The asbestos fibres were resuspended in distilled water, autoclaved and sonicated in bath during 10 minutes before use to render a homogeneous suspension.
Statistical analysis
To compare experimental groups, Kruskal-Wallis test or One-way ANOVA test were performed. In case of significance, Bonferroni-corrected one-tailed Mann-Whitney U test or Tukey HSD Test were used respectively. P values less than or equal to 0.05, after correction if needed, were considered statistically significant. All tests were performed with the statistic software R v. 3.4.2 [57].
Acknowledgements
ARR was supported by The Collaborative Research Centre (CRC) via project 973 “Priming and Memory of Organismic Responses to Stress”, project C5. J.B. was supported by Plan Nacional de I+D+i and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD15/0012)-co-financed by European Development Regional Fund “A way to achieve Europe” ERDF and Fondo de Investigación Sanitaria Grant PI13/00063. We are grateful to Alejandro Couce, Olga Makarova and Jens Rolff for critical comments about the manuscript.