Cellular uptake and mutagenic potential of metal oxide nanoparticles in bacterial cells

Extensive production and consumption of nanomaterials such as ZnO and TiO₂ has increased their release and disposal into the environment. The accumulation of nanoparticles (NPs) in ecosystem is likely to pose threat to non-specific targets such as bacteria. The present study explored the effect of ZnO and TiO₂ NPs in a model bacterium, Salmonella typhimurium. The uptake of ZnO and TiO₂ bare NPs in nano range without agglomeration was observed in S. typhimurium. TEM analysis demonstrated the internalization and uniform distribution of NPs inside the cells. Flow cytometry data also demonstrates that both ZnO and TiO₂ NPs were significantly internalized in the S. typhimurium cells in a concentration dependent manner. A significant increase in uptake was observed in the S. typhimurium treated even with 8 and 80 ng mL⁻¹ of ZnO and TiO₂ NPs with S9 after 60 min, possibly the formation of micelles or protein coat facilitated entry of NPs. These NPs exhibited weak mutagenic potential in S. typhimurium strains TA98, TA1537 and Escherichia coli (WP2uvrA) of Ames test underscoring the possible carcinogenic potential similar to certain mutagenic chemicals. Our study reiterates the need for re-evaluating environmental toxicity of ZnO and TiO₂ NPs presumably considered safe in environment.     

Behavioural and biochemical responses of two marine invertebrates Scrobicularia plana and Hediste diversicolor to copper oxide nanoparticles

Engineered nano-sized Cu oxide particles are extensively used in diverse applications. Because aquatic environments are the ultimate “sink” for all contaminants, it is expected that nanoparticles (NP) will follow the same fate. In this study, two marine invertebrates Scrobicularia plana and Hediste diversicolor were chosen as ecotoxicological models. The aim was to evaluate behavioural (burrowing kinetics, feeding rate) and biochemical (biomarkers) responses of S. plana and H. diversicolor exposed in the laboratory to Cu (10 μg L⁻¹) added in natural seawater either in the form of engineered nanoparticles (NPs) of CuO or as dissolved Cu in 2% HNO₃. Exposure was characterized by considering (i) the physico-chemical fate of NP (ii) the fraction of labile Cu in experimental media and (iii) Cu bioaccumulation. Results showed high aggregation of CuO NPs in seawater and no additional bioavailable Cu concentrations. Behavioural impairments were observed in S. plana exposed to CuO NPs or soluble Cu whereas in H. diversicolor, only the exposure to soluble Cu led to a burrowing decrease. No obvious neurotoxicity effects were revealed since in both species, no changes in cholinesterasic activity occurred in response to both forms of Cu exposure. Biomarkers of oxidative-stress catalase and glutathione-S-transferase were enhanced in both species whereas superoxide dismutase was increased only in S. plana exposed to CuO NPs. Metallothionein-like protein was increased in bivalves exposed to both forms of Cu. Since, no detectable release of soluble Cu from CuO NPs occurred during the time of experiment, ecotoxicity effects seem to be related to CuO NPs themselves.     

Toxicity of lead on Ceriodaphnia dubia in the presence of nano-CeO(2) and nano-TiO(2)

Although engineered nanoparticles (NPs) could negatively impact environmental organisms, the synergistic effect of NPs and other toxic substances, which could be more significant than that of NP alone, have seldom been examined. The effect of two common NPs, nano-CeO₂ and nano-TiO₂, on the toxicity of Pb was evaluated using Ceriodaphnia dubia (C. dubia) as the model organism. Standard EPA procedures were followed in the toxicity evaluation. The toxicity of bare NPs (without Pb) was first evaluated and safe doses (levels without causing lethal effect) of NPs were used in the synergistic studies. It was found that the overall toxicity of Pb in the system containing NPs was greater than that of Pb alone, as indicted by the reduced median lethal concentration (LC₅₀) of soluble Pb. The sorption of Pb onto the NP, and the uptake of NPs in the gastrointestinal tract of C. dubia were validated. Therefore, the uptake of Pb-loaded NPs increased the exposure of C. dubia to Pb, resulting in the enhanced toxicity. Reducing the solution pH could shift Pb speciation and enhance the overall toxicity of Pb, with or without the presence of NPs.     

Impact of nano-CuO stress on rice (Oryza sativa L.) seedlings

Indiscriminate release of metal oxide nanoparticles (NPs) into the environment due to anthropogenic activities has become a serious threat to the ecological system including plants. The present study assesses the toxicity of nano-CuO on rice (Oryza sativa cv. Swarna) seedlings. Three different levels of stress (0.5 mM, 1.0 mM and 1.5 mM suspensions of copper II oxide, <50 nm particle size) were imposed and seedling growth performance was studied along control at 7 and 14 d of experiment. Modulation of ascorbate–glutathione cycle, membrane damage, in vivo ROS detection, foliar H₂O₂ and proline accumulation under nano-CuO stress were investigated in detail to get an overview of nano-stress response of rice. Seed germination percentage was significantly reduced under stress. Higher uptake of Evans blue by nano-CuO stressed roots over control indicates loss of root cells viability. Presence of dark blue and deep brown spots on leaves evident after histochemical staining with NBT and DAB respectively indicate severe oxidative burst under nano-copper stress. APX activity was found to be significantly increased in 1.0 and 1.5 mM CuO treatments. Nevertheless, elevated APX activity might be insufficient to scavenge all H₂O₂ produced in excess under nano-CuO stress. That may be the reason why stressed leaves accumulated significantly higher H₂O₂ instead of having enhanced APX activity. In addition, increased GR activity coupled with isolated increase in GSH/GSSG ratio does not seem to prevent cells from oxidative damages, as evident from higher MDA level in leaves of nano-CuO stressed seedlings over control. Enhanced proline accumulation also does not give much protection against nano-CuO stress. Decline in carotenoids level might be another determining factor of meager performance of rice seedlings in combating nano-CuO stress induced oxidative damages.     

Nano-TiO2 enhances the toxicity of copper in natural water to Daphnia magna

The acute toxicity of engineered nanoparticles (NPs) in aquatic environments at high concentrations has been well-established. This study demonstrates that, at a concentration generally considered to be safe in the environment, nano-TiO₂ remarkably enhanced the toxicity of copper to Daphnia magna by increasing the copper bioaccumulation. Specifically, at 2 mg L⁻¹ nano-TiO₂, the (LC₅₀) of Cu²⁺ concentration observed to kill half the population, decreased from 111 μg L⁻¹ to 42 μg L⁻¹. Correspondingly, the level of metallothionein decreased from 135 μg g⁻¹ wet weight to 99 μg g⁻¹ wet weight at a Cu²⁺ level of 100 μg L⁻¹. The copper was found to be adsorbed onto the nano-TiO₂, and ingested and accumulated in the animals, thereby causing toxic injury. The nano-TiO₂ may compete for free copper ions with sulfhydryl groups, causing the inhibition of the detoxification by metallothioneins.     

Responses of a soil bacterium, Pseudomonas chlororaphis O6 to commercial metal oxide nanoparticles compared with responses to metal ions

The toxicity of commercially-available CuO and ZnO nanoparticles (NPs) to pathogenic bacteria was compared for a beneficial rhizosphere isolate, Pseudomonas chlororaphis O6. The NPs aggregated, released ions to different extents under the conditions used for bacterial exposure, and associated with bacterial cell surface. Bacterial surface charge was neutralized by NPs, dependent on pH. The CuO NPs were more toxic than the ZnO NPs. The negative surface charge on colloids of extracellular polymeric substances (EPS) was reduced by Cu ions but not by CuO NPs; the EPS protected cells from CuO NPs-toxicity. CuO NPs-toxicity was eliminated by a Cu ion chelator, suggesting that ion release was involved. Neither NPs released alkaline phosphatase from the cells’ periplasm, indicating minimal outer membrane damage. Accumulation of intracellular reactive oxygen species was correlated with CuO NPs lethality. Environmental deposition of NPs could create niches for ion release, with impacts on susceptible soil microbes.     

Toxicity of nanoparticulate and bulk ZnO, Al2O3 and TiO2 to the nematode Caenorhabditis elegans

Limited information is available on the environmental behavior and associated potential risk of manufactured oxide nanoparticles (NPs). In this research, toxicity of nanoparticulate and bulk ZnO, Al₂O₃ and TiO₂ were examined to the nematode Caenorhabditis elegans with Escherichia coli as a food source. Parallel experiments with dissolved metal ions from NPs were also conducted. The 24-h median lethal concentration (LC₅₀) and sublethal endpoints were assessed. Both NPs and their bulk counterparts were toxic, inhibiting growth and especially the reproductive capability of the nematode. The 24-h LC₅₀ for ZnO NPs (2.3 mg L⁻¹) and bulk ZnO was not significantly different, but significantly different between Al₂O₃ NPs (82 mg L⁻¹) and bulk Al₂O₃ (153 mg L⁻¹), and between TiO₂ NPs (80 mg L⁻¹) and bulk TiO₂ (136 mg L⁻¹). Oxide solubility influenced the toxicity of ZnO and Al₂O₃ NPs, but nanoparticle-dependent toxicity was indeed observed for the investigated NPs.     

Growth, photosynthesis and antioxidant responses of two microalgal species, Chlorella vulgaris and Selenastrum capricornutum, to nonylphenol stress

The effect of nonylphenol (NP) on growth, photochemistry and biochemistry of two green microalgae, Chlorella vulgaris and Selenanstrum capricornutum, and their ability to degrade NP were compared. The 96 h EC₅₀ of C. vulgaris and S. capricornutum were greater than 4.0 and 1.0 mg L⁻¹ NP, respectively, suggesting that the former species was more tolerant to NP. Both microalgae acclimated to NP stress through down-regulating their photosynthetic activities, including antenna size (chlorophyll a content), maximal photochemistry (Fv/Fm) and the light absorbed by PSII (ABS/CS₀), but the dissipation of energy from reaction centres (DI₀/RC) increased with the increase of NP concentrations. In C. vulgaris, the changes of these parameters were more significant than in S. capricornutum and recovered completely after a 96 h exposure. The antioxidant responses, such as GSH content, CAT and POD activities in C. vulgaris increased with the increase of NP concentrations after a 24 h exposure, but these changes disappeared with exposure time and recovered to the control levels after 96 h. In S. capricornutum, although GSH content, CAT and POD activities also increased when exposed to low- to moderate-NP concentrations, these values were significantly reduced at a high concentration (4 mg L⁻¹) even after a 96 h exposure, indicating its antioxidant responses were significantly delayed. It is clear that the more NP-tolerant species, C. vulgaris, acclimated better with a faster recovery of its photosynthetic activity from the NP-induced damage, and exhibited more efficient and rapid responses to NP-induced oxidative stress. C. vulgaris also had a higher NP degradation ability than S. capricornutum.     

Influence of liberated silver from silver nanoparticles on nitrification inhibition of Nitrosomonas europaea

The ecotoxicity of silver nanoparticles (Ag-NPs) to wastewater biota, including ammonia oxidizing bacteria (AOB), is gaining increasing interest as the number of products containing Ag-NPs continues to rise exponentially and they are expected to accumulate in wastewater treatment plants. This research demonstrated that the addition order of Ag-NP and the media constituents had a profound influence on the stability of the Ag-NP suspension and the corresponding repeatability of results and sensitivity of Nitrosomonas europaea. N. europaea, a model AOB, was found to be extremely sensitive to ionic silver (Ag⁺) and two sizes of Ag-NPs (20 and 80 nm). Ag⁺ exposures resulted in the highest level of toxicity with smaller Ag-NPs (20 nm) being more toxic than larger Ag-NPs (80 nm). The increased sensitivity of N. europaea to smaller Ag-NPs was caused by their higher rates of dissolved silver (dAg) release, via dissolution, due to a greater surface area to volume ratio. dAg was shown to be responsible for the vast majority of the observed Ag-NP toxicity, as determined by abiotic Ag-NP dissolution tests. For the sizes of Ag-NP studied (20 and 80 nm), there appears to be a negligible nanoparticle-specific toxicity. This was further supported by similarities in inhibition mechanisms between Ag⁺ and Ag-NP, with both causing decreases in AMO activity and destabilization of the outer-membrane of N. europaea. Finally, equal concentrations of total silver were found to be tightly associated to both Ag⁺ and Ag-NP-exposed cells despite Ag-NP concentrations being five times greater, by mass, than Ag⁺ concentrations.     

Impact of silver nanoparticles on natural marine biofilm bacteria

There has been a recent increase in the use of silver nanoparticles (Ag NPs) in a wide range of consumer products due to their highly effective antimicrobial properties. However, Ag NPs give cause for concern since their wide use makes them likely to be released into aquatic ecosystems and potentially affect natural bacterial communities. In this study marine biofilms were grown in situ in a coastal site (Singapore Harbour) and exposed in the laboratory for a further 24 h to 0-2000 μg L⁻¹ of well characterised Ag NPs. Increasing concentrations of Ag NPs caused a significant decrease in biofilm volume and biomass, and Ag uptake by biofilms per unit of volume was also dependent on concentration. Terminal fragment length polymorphisms and subsequent cluster and phylogenetic analysis showed the presence of major bacterial groups in biofilms irrespective of treatment with Ag NPs. This implies that even at the highest concentrations studied these taxonomic groups were not displaced. Nevertheless, biofilm succession was impeded on Ag NP treated biofilms, affecting the relative abundance of major bacterial groups in the biofilm community, with potential longer term effects on biofilm development and function.     

Biosafety assessment of titanium dioxide nanoparticles in acutely exposed nematode Caenorhabditis elegans with mutations of genes required for oxidative stress or stress response

We used Caenorhabditis elegans to investigate whether acute exposure to TiO₂-NPs at the concentration of 20 μg L⁻¹ reflecting predicted environmental relevant concentration and 25 mg L⁻¹ reflecting concentration in food can cause toxicity on nematodes with mutations of susceptible genes. Among examined mutants associated with oxidative stress and stress response, we found that genes of sod-2, sod-3, mtl-2, and hsp-16.48 might be susceptible for TiO₂-NPs toxicity. Mutations of these genes altered functions of both possible primary and secondary targeted organs in nematodes exposed to 25 mg L⁻¹ of TiO₂-NPs for 24-h. Mutations of these genes caused similar expression patterns of genes required for oxidative stress in TiO₂-NPs exposed mutant nematodes, implying their similar mechanisms to form the susceptible property. Nevertheless, acute exposure to 20 μg L⁻¹ of TiO₂-NPs for 24-h and 25 mg L⁻¹ of TiO₂-NPs for 0.48-h or 5.71-h did not influence functions of both possible primary and secondary targeted organs in sod-2, sod-3, mtl-2, and hsp-16.48 mutants. Therefore, our results suggest the relatively safe property of acute exposure to TiO₂-NPs with certain durations at predicted environmental relevant concentrations or concentrations comparable to those in food in nematodes with mutations of some susceptible genes.     

Converging hazard assessment of gold nanoparticles to aquatic organisms

The gold nanoparticles (Au-NPs) are being increasingly used because of their huge diversity of applications, and consequently, elevated levels in the environment are expected. However, due to their physico-chemical properties and functionalization a high variety of Au-NPs can be found, and complete toxicological information for each type of Au-NPs still lacks, and even, the toxicological information for the same species is sometimes contradictory. Therefore, hazard assessment should be done case by case. Hence, the objective of this study was to obtain ecotoxicological information of the same Au-NPs in aquatic organisms and to find a rationale for Au-NPs toxicity. For such a purpose, bare and hyaluronic acid capped Au-NPs (12.5 nm) along with Au-NPs bulk material were tested on freshwater algae, Daphnia and zebrafish. Results showed that while gold nanoparticles were found to be harmless to the tested organisms, the soluble gold showed to be toxic to algae and Daphnia, with an LC₅₀ between 1 and 2 mg L⁻¹. Comparing our results with those gathered in the literature, it appears that a common hazard assessment of Au-NPs on the studied organisms can be elucidated.     

Effects of suspended common-scale and nanoscale particles on the survival,growth and reproduction of Daphnia magna

Suspended particles are a natural component of aquatic ecosystems. This study provides a report on the survival, growth and reproduction of common-scale and nanoscale particles of Daphnia magna Straus exposed to five types of particles (i.e. KN (kaolinite), MN (montmorillonite), MNn (nanoscale MN), NP (natural particles), and NPn (nanoscale NP)). The results of the study show that the suspended particles elicited a dose-dependent toxicity in KN, MN and MNn, with the following toxicity pattern: MN > KN > MNn. On the contrary, NP and NPn did not show any harmful effects on the animals. Instead, NP and NPn, especially NPn, contributed to the survivorship of the animals. The animals were able to survive throughout the 21-day period of bioassays, and when the particle concentration reached 600 mg L⁻¹, they produced many neonates without any addition of food. This experimental results also indicated that the nanoscale particles were less toxic than the common-scale ones, both for MN and NP. Moreover, the results of the 21-day period of bioassays indicated that when the organisms were exposed to particles of different size scales, they showed different selection patterns for allocating resources. This may be due to the generation of different assimilation and digestion patterns.     

Polyaniline nanofibers: acute toxicity and teratogenic effect on Rhinella arenarum embryos

The fate and effect of nanomaterials in the environment is of paramount importance towards the technological application of the materials. This work shows the ecotoxicological potential of polyaniline (PANI) nanofibers in the larvae Rhinella arenarum by means of AMPHITOX test. Acute toxicity of PANI nanofibers towards embryos of the common South American toad R. arenarum (Anura: bufonidae) was evaluated in the premetamorphosis (stage 25) larvae. The exposure of R. arenarum larvae to at dose of 150, 250 and 400 mg L⁻¹ resulted in 100% viability within 96 h exposure. The embryos at 2-4 blastomers stage (early life stage teratogenic test) revealed that embryos were not killed and no teratogenic effects were observed when embryos were incubated with PANI nanofibers (150 and 250 mg L⁻¹), while only a growth retardation of embryos was induced at levels of 250 mg PANI nanofibers L⁻¹. On the other hand, at 400 mg L⁻¹ concentration, a reduction in the body length of larvae and tail malformation was observed. This results suggest that a concentration-dependent toxicity is operative, typified by phenotypes that had abnormal body axes. The presence of PANI nanofibers in gut contents and its excretion by larval stages of R. arenarum was confirmed by UV-visible spectroscopy.     

Effects of the organophosphate insecticide azinphos-methyl on the reproduction and cholinesterase activity of Biomphalaria glabrata

Azinphos-methyl is an organophosphate insecticide used for pest control on a number of food crops in many parts of the world. The snail Biomphalaria glabrata is a freshwater gastropod widely distributed in South America, Central America and Africa. The aim of the present work was to investigate whether azinphos-methyl causes alterations in the reproduction of B. glabrata. To this end, gastropod pigmented specimens were exposed to various concentrations of the insecticide (0.021, 0.5, 2.5, and 5 mg L⁻¹) for either 2 or 14 d. Along 14 d, several reproduction parameters and cholinesterase (ChE) activity were evaluated. In each group, the number of egg masses, the number of eggs per mass, the number of hatchings, the time to hatching, and the survival of the offspring after one month of treatment was evaluated. The results showed that, depending on the concentration and time of exposure, azinphos-methyl induced alterations in the reproduction of B. glabrata. These alterations were mainly represented by a decrease in the number of egg masses, and, in some cases, by a lower number or even the total absence of hatchings. Thus, the gastropods exposed to 2.5 and 5 mg L⁻¹ of azinphos-methyl for 14 d showed ChE inhibitions higher than 35% along time and completely lost their ability to reproduce. On the other hand, exposure to high acute concentrations or exposure to low concentrations for 14 d resulted in ChE inhibition equal to or lower than 35% between 7 and 14 d of treatment and similar alterations in reproduction. These were represented by a decrease in the number of egg masses. At low pestice levels, the number of egg masses and the number of offspring resulted to be more sensitive biomarkers than ChE inhibition. It is concluded that the insecticide azinphos-methyl can cause a decline in the reproductive performance of B. glabrata.     

Interactive effects of cadmium and hypoxia on metabolic responses and bacterial loads of eastern oysters Crassostrea virginica Gmelin

Pollution by toxic metals including cadmium (Cd) and hypoxia are important stressors in estuaries and coastal waters which may interactively affect sessile benthic organisms, such as oysters. We studied metabolic responses to prolonged hypoxic acclimation (2 weeks at 5% O₂) in control and Cd-exposed (30 d at 50 μg L⁻¹ Cd) oysters Crassostrea virginica, and analyzed the effects of these stressors on abundance of Vibrio spp. in oysters. Hypoxia-acclimated oysters retained normal standard metabolic rates (SMR) at 5% O₂, in contrast to a decline of SMR observed during acute hypoxia. However, oysters spent more time actively ventilating in hypoxia than normoxia resulting in enhanced Cd uptake and 2.7-fold higher tissue Cd burdens in hypoxia. Cd exposure led to a significant decrease in tissue glycogen stores, increase in free glucose levels and elevated activity of glycolytic enzymes (hexokinase and aldolase) indicating a greater dependence on carbohydrate catabolism. A compensatory increase in activities of two key mitochondrial enzymes (citrate synthase and cytochrome c oxidase) was found during prolonged hypoxia in control oysters but suppressed in Cd-exposed ones. Cd exposure also resulted in a significant increase in abundance of Vibrio parahaemolyticus and Vibrio vulnificus levels during normoxia and hypoxia, respectively. Overall, Cd- and hypoxia-induced changes in metabolic profile, Cd accumulation and bacterial flora of oysters indicate that these stressors can synergistically impact energy homeostasis, performance and survival of oysters in polluted estuaries and have significant consequences for transfer of Cd and bacterial pathogens to the higher levels of the food chain.     

Susceptibility of epigeic earthworm Eisenia fetida to agricultural application of six insecticides

Ecotoxicological risks of agricultural application of six insecticides to soil organisms were evaluated by acute toxicity tests under laboratory condition following OECD guidelines using the epigeic earthworm Eisenia fetida as the test organism. The organochlorine insecticide endosulfan (LC₅₀ - 0.002 mg kg⁻¹) and the carbamate insecticides aldicarb (LC₅₀ - 9.42 mg kg⁻¹) and carbaryl (LC₅₀ - 14.81 mg kg⁻¹) were found ecologically most dangerous because LC₅₀ values of these insecticides were lower than the respective recommended agricultural dose (RAD). Although E. fetida was found highly susceptible to the pyrethroid insecticide cypermethrin (LC₅₀ - 0.054 mg kg⁻¹), the value was higher than its RAD. The organophosphate insecticides chlorpyrifos (LC₅₀ - 28.58 mg kg⁻¹), and monocrotophos (LC₅₀ - 39.75 mg kg⁻¹) were found less toxic and ecologically safe because the LC₅₀ values were much higher than their respective RAD.     

Release of silver nanoparticles from outdoor facades

In this study we investigate the release of metallic silver nanoparticles (Ag-NP) from paints used for outdoor applications. A facade panel mounted on a model house was exposed to ambient weather conditions over a period of one year. The runoff volume of individual rain events was determined and the silver and titanium concentrations of 36 out of 65 runoff events were measured. Selected samples were prepared for electron microscopic analysis. A strong leaching of the Ag-NP was observed during the initial runoff events with a maximum concentration of 145 μ Ag/l. After a period of one year, more than 30% of the Ag-NP were released to the environment. Particles were mostly <15 nm and are released as composite colloids attached to the organic binders of the paint. Microscopic results indicate that the Ag-NP are likely transformed to considerably less toxic forms such as Ag₂S.     

Silica nanoparticles and silver-doped silica nanoparticles induce endoplasmatic reticulum stress response and alter cytochrome P4501A activity

Engineered silica nanoparticles (SiO₂-NPs) find widespread application and may lead to exposure of humans and the environment. Here we compare the effects of SiO₂-NPs and SiO₂-NPs doped with silver (SiO₂-Ag-NPs) on survival and cellular function of human liver cells (Huh7) and Pimephales promelas (fathead minnow) fibroblast cells (FMH). In Huh7 cells we investigate effects on the endoplasmatic reticulum (ER), including ER stress, and interactions of nanoparticles (NPs) with metabolizing enzymes and efflux transporters. The NPs formed agglomerates/aggregates in cell culture media as revealed by SEM and TEM. SiO₂ and SiO₂-1% Ag-NPs were taken up into cells as demonstrated by agglomerates occurring in vesicular-like structures or freely dispersed in the cytosol. Cytotoxicity was more pronounced in Huh7 than in FMH cells, and increased with silver content in silver-doped NPs. Dissolved silver was the most significant factor for cytotoxicity. At toxic and non-cytotoxic concentrations SiO₂-NPs and SiO₂-1% Ag-NPs induced perturbations in the function of ER. In Huh7 cells NPs induced the unfolded protein response (UPR), or ER stress response, as demonstrated in induced expression of BiP and splicing of XBP1 mRNA, two selective markers of ER stress. Additionally, SiO₂-1% Ag-NPs and AgNO₃ induced reactive oxygen species. Pre-treatment of Huh7 cells with SiO₂-1% Ag-NPs followed by exposure to the inducer benzo(a)pyrene caused a significant reduced induction of CYP1A activity. NPs did not alter the activity of ABC transporters. These data demonstrate for the first time that SiO₂-NPs and SiO₂-1% Ag-NPs result in perturbations of the ER leading to the ER stress response. This represents a novel and significant cellular signalling pathway contributing to the cytotoxicity of NPs.