The pulmonary effects of nickel-containing nanoparticles: cytotoxicity, genotoxicity, carcinogenicity, and their underlying mechanisms

Abstract

With the exponential growth of the nanotechnology field, the global nanotechnology market is on an upward track with fast-growing jobs. Nickel (Ni)-containing nanoparticles (NPs), an important class of transition metal nanoparticles, have been extensively used in industrial and biomedical fields due to their unique nanostructural, physical, and chemical properties. Millions of people have been/are going to be exposed to Ni-containing NPs in occupational and non-occupational settings. Therefore, there are increasing concerns over the hazardous effects of Ni-containing NPs on health and the environment. The respiratory tract is a major portal of entry for Ni-containing NPs; thus, the adverse effects of Ni-containing NPs on the respiratory system, especially the lungs, have been a focus of scientific study. This review summarized previous studies, published before December 1, 2023, on the cytotoxic, genotoxic, and carcinogenic effects of Ni-containing NPs on humans, lung cells in vitro, and rodent lungs in vivo, and the potential underlying mechanisms were also included. In addition, whether these adverse effects were induced by NPs themselves or Ni ions released from the NPs was also discussed. The extra-pulmonary effects of Ni-containing NPs were briefly mentioned. This review will provide us with a comprehensive view of the pulmonary effects of Ni-containing NPs and their underlying mechanisms, which will shed light on our future studies, including the urgency and necessity to produce engineered Ni-containing NPs with controlled and reduced toxicity, and also provide a scientific basis for developing nanoparticle exposure limits and policies.

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Environmental significance

Nickel-containing nanoparticles (NPs) have been increasingly used in industrial and biomedical fields due to their unique nanostructural and physicochemical properties; thus, the risk of occupational and environmental contamination by Ni-containing NPs is increasing. Previous studies have shown that exposure to Ni-containing NPs caused various toxic effects on cells, animals, and humans. The respiratory system is a major and common point of contact for Ni-containing NPs; therefore, they may cause adverse health effects on the respiratory tract, especially the lungs. This review provided an overview of the cytotoxic, genotoxic, and carcinogenic effects of Ni-containing NPs on lung cells in vitro, rodent lungs in vivo, and humans. The potential underlying molecular mechanisms were also discussed.

1. Introduction

Nanotechnology is one of the most promising new technologies in the world of the 21st century. It directly improves our lives in areas as diverse as materials engineering, information technology, biotechnology, medical sciences, etc.^1,2 Nanomaterials, the building blocks of this new technology, have sizes ranging from 1 to 100 nm and comprise a wide range of different morphologies, including nanotubes, nanowires, nanofibers, nanodots, a range of spherical or aggregated dendritic forms, etc.³ Among them, metal nanoparticles (NPs) have been paid increasing attention because of their unique characteristics, including high mechanical strength, high surface area, high surface energy, low melting point, low burning point, and specific optical and magnetic properties.⁴ Currently, a large number of metal NPs are being developed and produced with new formulations and surface properties to meet various commercial or technological demands.⁴ With the continuous expansion of their production and use, occupational or non-occupational exposure to metal NPs has increased dramatically, and their potential health effects have been a focus of scientific study.

As an important class of transition metal NPs, nickel (Ni)-containing NPs have found a wide range of applications in industrial fields, including catalysts, electrical conductors, sensors, permanent magnets, magnetic fluids, magnetic recording media, solar cells, adsorption of dyes, etc.^4,5 More interestingly, due to their unique nanostructural, chemical, and physical properties, Ni-containing NPs have also received particular interest in biomedical applications. For example, magnetic metal nanoparticles, typically composed of iron, cobalt, and nickel, have been increasingly used for magnetic resonance imaging (MRI), drug delivery, and gene delivery, and in cancer treatments such as magnetic hyperthermia therapy (MHT) and photothermal therapy (PTT) which use the heat that NPs produce when they are placed in an alternating magnetic field to kill cancer cells.^6,7 However, there are increasing concerns over their adverse effects on their portal of entry, such as the respiratory tract, the skin, and the gastrointestinal tract. Since the respiratory tract is the major and most common route for NP exposure, it is by far the most widely studied in nanotoxicology research.

This review aims to summarize the current scientific knowledge concerning the adverse effects of Ni-containing NPs on the respiratory tract, especially the lungs, based on publications before December 1, 2023. The cytotoxicity, genotoxicity, and carcinogenicity of Ni-containing NPs in humans, lung cells in vitro, and rodent lungs in vivo are discussed, and their potential underlying mechanisms are also included. In addition, whether these adverse effects are induced by NPs themselves or Ni ions mobilized from the NPs is also a focus of this review. The extra-pulmonary effects of Ni-containing NPs are briefly mentioned. Finally, considerations and knowledge gaps in the field are highlighted as potential directions for future research.

2. Health effects of Ni-containing NPs on humans

Currently, the literature about the health effects of Ni-containing NPs on humans is limited; there are only two case reports demonstrating the adverse health effects of Ni NPs on humans.^8,9 However, human health effects of standard-sized nickel and nickel compounds have been widely reported.^10–12 Nickel and nickel compounds have a long history of industrial production and applications as well as extensive and ubiquitous distributions in the environment; thus, occupational and non-occupational exposure to them is inevitable. Humans exposed to standard-sized nickel and nickel compounds may develop skin allergies (contact dermatitis), asthma, lung fibrosis, headaches, gastrointestinal or respiratory symptoms, kidney and cardiovascular diseases, etc., as summarized in previous reviews.^10,12 An increased risk of lung and nasal cancers has also been observed among workers involved in a variety of industrial fields related to nickel and nickel compounds.¹¹ In this section, we summarize the adverse human health effects induced by Ni-containing NP exposure.

2.1 Two case reports

A case described that a 38-year-old male worker, who was previously healthy and non-smoking and had no history of respiratory disease, was accidentally exposed to Ni NPs for about 90 min without any protective measures when he operated a metal arc process for spraying nickel onto bushes for turbine bearings. He developed a cough, shortness of breath, and a tight chest the following day. On day 4 after exposure, he was admitted to the hospital, and his chest X-ray showed a picture of bilateral airspace consolidation. His blood gases continued to deteriorate, and he was intubated and ventilated. On day 13 after exposure, he died of adult respiratory distress syndrome (ARDS).⁹ Pathological examination of the lung section by HE staining showed that alveolar spaces filled with fresh blood, fibroblastic plugs, and remnants of hyaline membranes.⁹ In addition, focal areas of acute necrosis were found in the heart, brain, and kidney sections. Lymphocyte deposition was found in the perivascular zones of the spleen.⁹ Ni NPs, less than 20 nm, were found in lung macrophages by transmission electron microscopy, and high levels of nickel were measured in the urine and the kidneys.⁹ The particulate nickel concentration in the vicinity of the operator was 382 mg m⁻³. Most of the particles were determined to be about 50 nm in diameter. It was estimated that during his 90 min of operating the process, the worker would have inhaled about 1 g of Ni NPs.⁹ The findings from this case suggest that inhalation exposure to Ni NPs causes pulmonary and systemic toxic effects on humans.

Another case reported that a 26-year-old non-smoking female formulation chemist developed throat irritation, nasal congestion, post-nasal drip, and facial flushing when she started working with Ni NP powder weighed out and handled on a lab bench without any special respiratory protection or control measures.⁸ In addition, she had skin reactions to earrings and her belt buckle that she was previously able to wear without any reactions. Her T.R.U.E. patch testing showed positive reactions to nickel.⁸ The Ni NP powder she was handling was spherical and of 99.9+% purity, with an aerodynamic particle size of 20 nm and a surface area of 40–60 m² g⁻¹. She weighed Ni NPs on the open lab bench and not in a glove box or fume hood, and she wore latex gloves at that time but no respiratory protection.⁸ This case shows that working with Ni NP powder without protective measures may develop nickel sensitization in humans.

2.2 Effects of metal fume pollution containing nickel NPs on humans

Metal fume has been correlated with metal fume fever, increased susceptibility to infection, decreased lung function, pneumonia, cancers, etc.¹³ A field study in two facilities revealed that nickel concentrations may range from 10–51 μg m⁻³ for both gas metal arc welding (GMAW) and flux-cored arc welding (FCAW) processes of mild or stainless steel. The estimated percentage of the nano-fraction of nickel (smaller than 300 nm) deposited in a welder's respiratory system was 64% of the total nickel.¹⁴

Zelenik et al. reported that in the palatine tonsil tissues of patients with chronic tonsillitis and tonsillar carcinoma who lived in a conurbation with a heavy steel industry and were exposed to the resultant air pollution, mostly from pyrometallurgy, metal elements including iron, chromium, nickel, aluminum, zinc, copper, etc. were detected using a scanning electron microscope with the X-ray microprobe of an energy-dispersive spectroscope (EDS).¹⁵ They believe that these are micro- and nano-sized metallic particles that cause an inflammatory response as well as neoplastic changes in human palatine tonsils, similar to those occurring in the lungs, although the quantitative analysis and chemical form and sizes of these particles need to be determined.¹⁵ The limitations are that these observations, such as chronic tonsillitis and tonsillar carcinoma, cannot be ascribed to Ni NPs alone, thus making it difficult to draw definitive conclusions.

3. Cytotoxic effects of Ni-containing NPs on lung cells in vitro and the underlying mechanisms

In this section, the cytotoxic effects of Ni-containing NPs on human lung cells and non-human mammalian lung cells are summarized. The genotoxic and carcinogenic effects of Ni-containing NPs, reviewed in section 5, are not included here. Most studies have focused on the cytotoxic effects of Ni or NiO NPs on human lung cells; thus, their potential underlying mechanisms or signaling pathways are illustrated in Fig. 1.

Fig. 1 The potential signaling pathways/mechanisms underlying the cytotoxic effects of Ni or NiO NPs on human lung cells in vitro. Note: (1) the pulmonary genotoxic and carcinogenic effects of Ni or NiO NPs are summarized in section 5 and Fig. 3 and not included here. (2) Ni and NiO NPs may have different effects on cells. For example, exposure to Ni NPs caused limited/no ROS generation in human bronchial epithelial BEAS-2B cells, which was robust after NiO NP exposure.²⁴

3.1 Cytotoxic effects of Ni NPs on human lung cells in vitro

See Table 1 and Fig. 1.

Table 1 Cytotoxic effects of Ni NPs on human lung cells in vitro

Cell line	Particle size (diameter)	Exposure dose	Exposure time	Cytotoxicity endpoints	Ref.
Epithelial (A549)	<100 nm (Sigma #577995)	1, 2, 5, 10, 25 μg mL^−1	24, 48 h	Decrease cell viability (MTT assay) and induce LDH leakage; increased ROS generation and membrane lipid peroxidation and depletion of GSH; increased caspase-3 activity	Ahamed 2011 (ref. 16)
Epithelial (HBEC3-kt)	<100 nm (Sigma #577995)	5, 10, 25, 50 μg mL^−1	3, 24 h	No distinct effects of cytotoxicity (alamarBlue assay)	Akerlund et al. 2019 (ref. 22)
Epithelial (BEAS-2B)	<100 nm (Sigma #577995)	1, 5, 10, 25, 50 μg mL^−1	5 min–2 h, 48 h	No significant increase in ROS (10–50 μg mL^−1, 2 h); increased apoptotic cells (10 μg mL^−1) and replication index (1 μg mL^−1) (48 h)	Di Bucchianico et al. 2018 (ref. 24)
Epithelial (A549)	20 nm (NanoAmor)	20, 50, 150 μg mL^−1	4 h	Reduce cell viability; cause protein (methionine) oxidation; suppress MSRA and MSRB3; suppress autophagy (decreased LC3); increased p-ERK1/2	Feng et al. 2015 (ref. 17)
Epithelial (BEAS-2B)	<100 nm (Sigma #577995)	0.5 μg mL^−1	3, 6 weeks	No effects on cell viability/proliferation; NP uptake; cause gene expression changes; several pathways involved	Gliga et al. 2020 (ref. 28)
Epithelial (A549)	<100 nm (Sigma #577995)	20 μg mL^−1	2 h	No changes in ROS (both acellular and cellular)	Latvala et al. 2016 (ref. 19)
		0.1, 1, 5, 10, 20, 40 μg mL^−1	4, 24, 48 h	Decreased colony-forming efficiency (20 or 40 μg mL^−1)
Epithelial (A549)	50 nm (Danyang)	1, 5, 10, 15, 25 μg cm^−2	24 h	Decreased cell viability; increased protein expression of HO-1 and C-myc, but not Nrf2	Magaye et al. 2016 (ref. 18)
Epithelial (A549)	20 nm (NanoAmor)	0.01, 0.1, 1, 10, 100 μg mL^−1	24 h	No measurable effects on membrane integrity and mitochondrial function	Minocha et al. 2012 (ref. 23)
Epithelial (BEAS-2B)	20 nm (InabVacu)	5, 10, 20, 30, 40 μg mL^−1	24 h	Reduce cell viability (30 and 40 μg mL^−1) by the MTS assay; nuclear accumulation of HIF-1α; increased expression of miR-210	Mo et al. 2021 (ref. 21)
Epithelial (H460)	<100 nm (Sigma)	5, 20 μg cm^−2	24, 48, 72 h	HIF-1α stabilization and NDRG1 upregulation; induce cell apoptosis	Pietruska et al. 2011 (ref. 20)
Epithelial (H460, NHBE)		0.63, 1.25, 2.5, 10, 20 μg cm^−2 or 0–0.1 μmol	24, 48, 72 h	Dose- and time-dependent reduction in cell number
Epithelial (BEAS-2B)	∼20 nm (Sun-Inno)	3 μg cm^−2	24 h	Synergistic induction of IL-6 with LPS via STAT3 and C/EBPβ	You et al. 2022 (ref. 27)
Epithelial (BEAS-2B)	20 nm (InabVacu)	10, 20 μg mL^−1	6, 12, 24, 48 h	Nuclear accumulation of HIF-1α; increased expression of HDAC3; histone hypoacetylation; induce EMT via the HIF-1α/HDAC3 pathway	Yuan et al. 2022 (ref. 26)
				Induce autophagy (increased LC3B-2/LC3B-1, Beclin 1, and p62) and apoptosis (upregulation of Bax and cleaved caspase-3 and downregulation of Bcl-2) via HIF-1α/mTOR signaling; autophagy has a protective role against apoptosis	Yuan et al. 2023 (ref. 25)

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Exposure to Ni NPs has been reported to cause cytotoxicity in human lung cells. Exposure of human lung epithelial cells A549 to Ni NPs (2–25 μg mL⁻¹) for 24 or 48 h caused reduced cell viability by the MTT assay and induced LDH leakage.¹⁶ The reduced A549 cell viability by Ni NP exposure was also observed by Feng et al. at 150 μg mL⁻¹, but not 20 and 50 μg mL⁻¹, exposure for 4 h¹⁷ and Magaye et al. at 5–25 μg cm⁻² for 24 h.¹⁸ 20 or 40 μg mL⁻¹ Ni NPs also caused decreased A549 colony-forming efficiency.¹⁹ Ni NP exposure also caused cytotoxicity in human large cell lung carcinoma epithelial cells H460²⁰ and human bronchial epithelial cells BEAS-2B (30–40 μg mL⁻¹ for 24 h).²¹ However, others reported that no significant cytotoxicity of Ni NPs was found on human bronchial epithelial cells HBECs,²² and no measurable effects (up to 100 μg mL⁻¹ for 24 h) were found on membrane integrity and mitochondrial function in A549 cells.²³ A low dose (1 μg mL⁻¹) of Ni NP exposure for 48 h was found to increase BEAS-2B cell replication.²⁴

Ni NPs have been shown to cause oxidative stress in human lung cells. Exposure of A549 cells to Ni NPs (2–25 μg mL⁻¹) for 24 or 48 h caused increased reactive oxygen species (ROS) generation, increased membrane lipid peroxidation, and reduced glutathione (GSH) levels.¹⁶ Exposure of A549 cells to Ni NPs also caused increased protein oxidation, such as methionine oxidation, and decreased oxidized protein repair and degradation, as evidenced by downregulation of methionine repairing enzymes, such as methionine sulfoxide reductase A (MSRA) and methionine sulfoxide reductase B3 (MSRB3), and autophagy marker LC3.¹⁷ Increased protein expression of antioxidant response-associated protein HO-1, but not Nrf2, was found in A549 cells exposed to Ni NPs.¹⁸ However, others found no significant increase in ROS in BEAS-2B²⁴ or A549 cells¹⁹ after Ni NP exposure.

In addition, Ni NP exposure (2–25 μg mL⁻¹) for 24 or 48 h induced A549 cell apoptosis; increased caspase-3 activity was observed.¹⁶ Ni NP exposure also caused apoptosis in BEAS-2B^24,25 and H460 cells,²⁰ which was reflected by increased expression of Bax, cleaved caspase-3, cleaved caspase-7, or cleaved PARP and decreased expression of Bcl-2. Ni NP exposure also induced autophagy as evidenced by increased LC3B-2/LC3B-1 ratio and increased expression of Beclin 1 and p62.²⁵ Ni NP-induced apoptosis and autophagy were via HIF-1α/mTOR signaling, and autophagy has a protective role against apoptosis.²⁵

Moreover, multiple signaling pathways may be involved in Ni NP-induced toxic effects. Feng et al. reported that exposure of A549 cells to Ni NPs caused upregulation of p-ERK1/2.¹⁷ HIF-1α stabilization and upregulation of its target NDRG1 (Cap43) after Ni NP exposure have been observed in H460²⁰ and BEAS-2B cells.^21,26 You et al. reported that synergistic induction of IL-6 production in BEAS-2B cells by Ni NPs and lipopolysaccharide (LPS) was mediated by STAT3 and C/EBPβ.²⁷ Yuan et al. reported that Ni NP exposure caused BEAS-2B cells to undergo epithelial-mesenchymal transition (EMT) via nuclear accumulation of HIF-1α and increased expression of HDAC3.²⁶ Long-term low-dose Ni NP exposure also caused changes in gene transcriptional expression, and ERK/MAPK signaling and leukocyte extravasation signaling were involved.²⁸

3.2 Cytotoxic effects of NiO NPs on human lung cells in vitro

See Table 2 and Fig. 1.

Table 2 Cytotoxic effects of NiO NPs on human lung cells in vitro

Cell line	Particle size (diameter)	Exposure dose	Exposure time	Cytotoxicity endpoints	Ref.
Note:a Surface area of nanoparticles (cm^2 mL^−1).
Epithelial (HBEC3-kt)	<50 nm (Sigma #637130)	5, 10, 25, 50 μg Ni mL^−1	3, 24 h	No distinct effects of cytotoxicity	Akerlund et al. 2019 (ref. 22)
		5, 10, 25 μg Ni mL^−1	48 h	Significant cytotoxic effects (25 μg Ni mL^−1)	Vallabani et al. 2022 (ref. 42)
Epithelial (A549)	8–10 nm (lab biosynthesized)	7.8, 15.6, 31.2, 62.5, 125, 250, 500, 1000 μg mL^−1	24 h	Dose-dependent decrease in cell viability	Angel Ezhilarasia et al. 2018 (ref. 29)
Epithelial (A549)	16.1 nm (NanoAmor)	10, 25, 50, 75, 100 μg mL^−1	24, 48 h	Decreased cell viability; cell apoptosis; suppression of cell proliferation; alteration of the cell cycle; increased ROS	Cambre et al. 2020 (ref. 30)
		10, 100 μg mL^−1	12, 24 h	Perturbation of mitochondrial membrane potential
Epithelial (BEAS-2B, A549)	<50 nm (Sigma)	20, 40, 60, 80, 100 μg mL^−1	24 h	Dose-dependent reduction in cell viability; increased number of apoptotic and necrotic cells; IL-6 and IL-8 release through the NF-κB/MAPK pathway; cell cycle alteration	Capasso et al. 2014 (ref. 31)
		60, 100 μg mL^−1	45 min	Increased ROS in BEAS-2B, but not A549
Epithelial (A549)	20 nm (ST-Nano)	12.5, 25, 50, 100, 200 μg mL^−1	6, 12, 24, 36, 48 h	Dose- and time-dependent decrease in cell viability	Chang et al. 2020 (ref. 32)
		25, 50, 100 μg mL^−1	24 h	Increased type I collagen content; induce EMT via the TGF-β1/Smad pathway
Epithelial (A549)	10–20 nm (NanoAmor)	30, 100, 300^a cm^2 mL^−1	24 h	Cause cytotoxicity and IL-8 release (300^a cm^2 mL^−1)	Cho et al. 2012 (ref. 33)
			3, 6 h	No changes in AP-1 and NF-κB activity
Epithelial (BEAS-2B)	<50 nm (Sigma #637130)	10, 25, 50 μg Ni mL^−1	5 min–2 h	Increased ROS	Di Bucchianico et al. 2018 (ref. 24)
		1, 5, 10 μg Ni mL^−1	48 h	Ca^2+-dependent increased number of apoptotic and necrotic cells (5 or 10 μg Ni mL^−1); decreased replication index and mitotic index (10 μg Ni mL^−1)
		5 μg Ni mL^−1	2, 48 h	Increased intracellular calcium
Epithelial (BEAS-2B)	<50 nm (Sigma #637130)	5, 10, 20 μg cm^−2	24, 48 h	Dose-dependent inhibition in cell viability; induce cell apoptosis via p53 hyperacetylation and Bax activation by downregulation of SIRT1	Duan et al. 2015 (ref. 39)
Epithelial (A549)	20 nm (ST-Nano)	25, 50, 100 μg mL^−1	24 h	Cause collagen deposition via lncRNA MEG3 downregulation, Hh pathway activation, and autophagy suppression	Gao et al. 2022 (ref. 49)
Pulmonary artery endothelial (HPAEC)	<50 nm (Sigma #637130)	0.5–150 μg cm^−2	4, 24 h	Decreased cell viability; induce oxidative stress and IL-6 secretion; increased cytosolic calcium (Ca^2+) concentration; mitochondrial dysfunction; cell apoptosis	Germande et al. 2022 (ref. 41)
		0.5–5 μg cm^−2	4, 24 h	Under pathological conditions, ROS and nitrite production, IL-6 secretion, calcium signaling, and mitochondrial impairment increased as compared to physiological conditions	Germande et al. 2022 (ref. 45)
Epithelial (BEAS-2B)	<50 nm (Sigma #637130)	0.5 μg mL^−1	3, 6 weeks	No effects on cell viability/proliferation; NP uptake; cause gene expression changes; several pathways involved	Gliga et al. 2020 (ref. 28)
Epithelial (A549, 16HBE14o)	<20 nm (Sigma)	0.1, 1, 10 μg mL^−1	4, 24 h	Increased HO-1 expression (10 μg mL^−1 × 24 h)	Gutierrez et al. 2015 (ref. 46)
Epithelial (A549)	20 nm (NanoAmor)	106.6 μg mL^−1	2, 6, 12, 24 h	Increased ROS, lipid peroxidation, tHODE level, SP-D (24 h), and HO-1 (6 & 24 h); decreased GSH (24 h)	Horie et al. 2011 (ref. 44)
Epithelial (A549)	<50 nm (Sigma)	1–400 μg mL^−1	48 h	Dose-dependent decrease in cell viability; cytotoxicity increased with increasing secondary particle size due to increasing cellular uptake	Kawakami et al. 2022 (ref. 34)
Epithelial (A549)	<50 nm (Sigma #637130)	20 μg Ni mL^−1	2 h	Increased acellular ROS, but not cellular ROS	Latvala et al. 2016 (ref. 19)
		0.1, 1, 5, 10, 20, 40 μg Ni mL^−1	4, 24, 48 h	Decreased colony forming efficiency (10, 20, 40 μg Ni mL^−1)
Epithelial (BEAS-2B, HPAEpiC)	50 nm (Aladdin #N128916)	100 μg mL^−1	24 h	Decreased cell viability; cell apoptosis and ferroptosis through ATF3 upregulation	Liu et al. 2022 (ref. 40)
Epithelial (A549)	10–20 nm (NanoAmor)	9.4, 18.8, 37.5, 75, 150, 300^a cm^2 mL^−1	24 h	Increased LDH activity in the cell culture medium (300^a cm^2 mL^−1)	Lu et al. 2009 (ref. 43)
Epithelial (A549)	<50 nm (Sigma #637130)	0.5, 10, 20, 40, 60, 80, 100 μg mL^−1	24 h	Decreased cell viability	Mohamed et al. 2018 (ref. 35)
		80, 100 μg mL^−1	24 h	Induce ROS generation; depletion of antioxidants (decreased SOD and CAT activities)
Epithelial (H460, NHBE)	<100 nm (NanoAmor)	0.63, 1.25, 2.5, 10, 20 μg cm^−2 or 0–0.1 μmol Ni	24, 48, 72 h	Dose- and time-dependent reduction in cell number	Pietruska et al. 2011 (ref. 20)
Epithelial (H460)		5, 20 μg cm^−2	2, 4, 6, 12, 24 h	HIF-1α stabilization and nuclear translocation; NDRG1 upregulation
			24, 48 h	Induce cell apoptosis
Fetal lung fibroblasts	<50 nm (Sigma)	0.25, 0.5, 1, 2 μg cm^−2	6, 12, 24, 48 h	Increased DNA synthesis; increased collagen-1 and αSMA expression via upregulation of HIF-1α and TGF-β1	Qian et al. 2015 (ref. 39)
Epithelial (A549)	5.63 nm (lab biosynthesized)	20, 40, 60, 80, 100 μg mL^−1	24 h	Dose-dependent decrease in cell viability	Shwetha et al. 2021 (ref. 36)
Epithelial (HEp-2)	<50 nm (Sigma #637130)	2, 5, 10, 25, 50, 100 μg mL^−1	24 h	Dose-dependent reduction in cell viability and GSH, but increase in ROS and lipid peroxidation; induce cell apoptosis	Siddiqui et al. 2012 (ref. 38)
Epithelial (A549)	20 nm (ST-Nano)	12.5, 25, 50, 100, 200 μg mL^−1	24 h	Dose-dependent decrease in cell viability but increase in LDH release	Tian et al. 2019 (ref. 37)
		25, 50, 100 μg mL^−1	24 h	Induce collagen formation via TGF-β1-mediated MAPK signaling and MMP/TIMP imbalance
Epithelial (A549)	20 nm (ST-Nano)	25, 50, 100 μg mL^−1	24 h	Induce inflammation via downregulation of lncRNA MEG3 and activation of the p38 MAPK pathway	Yang et al. 2022 (ref. 47)
Epithelial (A549)	20 nm (ST-Nano)	25, 50, 100 μg mL^−1	24 h	Induce EMT and collagen deposition via downregulation of lncRNA MEG3 and activation of TGF-β1	Zhan et al. 2021 (ref. 50)
				Induce collagen deposition via downregulation of lncRNA MEG3 and the TGF-β1-mediated PI3K/AKT pathway	Zhan et al. 2021 (ref. 48)
Epithelial (BEAS-2B)	20 nm (ST-Nano)	25, 50, 100 μg mL^−1	24 h	Induce collagen deposition via downregulation of lncRNA HOTAIRM1 and activation of the PRKCB DNA methylation-mediated JNK/c-Jun pathway	Zheng et al. 2022 (ref. 52)
				Induce collagen formation via downregulation of lncRNA AP000487.1, PRKCB DNA hypomethylation, and activation of the TLR4/MyD88/NF-κB pathway	Zheng et al. 2023 (ref. 53)

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A dose- and time-dependent decrease in cell viability or decrease in colony-forming efficiency after NiO NP exposure has been confirmed in various human lung cells, including lung epithelial A549^19,29–37 and H460,²⁰ airway epithelial HEp-2,³⁸ bronchial epithelial BEAS-2B^31,39 and NHBE,²⁰ alveolar epithelial HPAEpiC,⁴⁰ and pulmonary artery endothelial HPAEC cells.⁴¹ Akerlund et al. reported that exposure of HBECs to 5–50 μg Ni mL⁻¹ of NiO NPs for 3 and 24 h did not cause significant cytotoxicity by the alamarBlue assay,²² but significant cytotoxicity was observed when HBEC cells were exposed to 25 μg Ni mL⁻¹ of NiO NPs for 48 h.⁴² Increased LDH activity was also detected in the cell culture supernatant of A549 cells exposed to NiO NPs.^37,43

Increased ROS generation was observed in BEAS-2B, but not A549, cells exposed to 60 and 100 μg mL⁻¹ NiO NPs for 45 min³¹ or 25 and 50 μg Ni mL⁻¹ of NiO NPs for 5 min to 2 h.^19,24 Others found increased ROS, perturbation of the mitochondrial membrane potential, lipid peroxidation, increased tHODE levels, or depletion of antioxidants, such as decreased GSH, SOD, or CAT, in A549 cells exposed to NiO NPs.^30,35,44 NiO NP exposure also caused ROS and nitrite production and mitochondrial impairment in human pulmonary artery endothelial cells HPAEC,^41,45 as well as upregulation of HO-1 in A549^44,46 and 16HBE14o cells.⁴⁶

NiO NP exposure also caused cell apoptosis, necrosis, or suppression of cell proliferation in A549,^30,31 H460,²⁰ HEp-2,³⁸ BEAS-2B,^24,31,40 HPAEpiC,⁴⁰ and HPAEC cells.⁴¹ Di Bucchianico et al. reported that NiO NP-induced BEAS-2B cell apoptosis and necrosis were calcium-dependent,²⁴ but Duan et al. reported that they were via p53 hyperacetylation and Bax activation by downregulation of SIRT1.³⁹ NiO NP exposure also caused increased cytosolic calcium (Ca²⁺) concentration in human pulmonary artery endothelial cells HPAEC,^41,45 which was responsible for NiO NP-induced cell apoptosis. Liu et al. demonstrated that NiO NP exposure induced apoptosis and ferroptosis in BEAS-2B and HPAEpiC cells via ATF3.⁴⁰ In addition, exposure of BEAS-2B cells to NiO NPs (10 μg Ni mL⁻¹) caused a decreased replication index and mitotic index.²⁴ Exposure of A549 or BEAS-2B cells to NiO NPs also caused alteration of the cell cycle.^30,31

NiO NP exposure has been reported to cause cell inflammation. A549 or BEAS-2B cells released pro-inflammatory cytokines such as IL-6 and IL-8 through the NF-κB/MAPK pathway after NiO NP exposure,^31,33 but no changes were found in AP-1 and NF-κB activity.³³ NiO NP exposure also caused IL-6 production in human pulmonary artery endothelial cells HPAEC.^41,45 Yang et al. reported that NiO NP-induced inflammation was via downregulation of lncRNA MEG3 and activation of the p38 MAPK pathway in A549 cells.⁴⁷

NiO NPs were able to cause human lung cells to undergo epithelial–mesenchymal transition (EMT) and upregulation of fibrosis-associated proteins. The potential underlying mechanisms have been widely studied. It was reported that exposure of A549 cells to NiO NPs caused increased type I collagen contents or EMT via the TGF-β1/Smad pathway,³² TGF-β1-mediated MAPK signaling and MMP/TIMP imbalance,³⁷ TGF-β1-mediated PI3K/AKT pathway,⁴⁸ or lncRNA MEG3 downregulation, Hh pathway activation, and autophagy suppression.^48–50 Increased expression of type 1 collagen and αSMA was also found in fetal lung fibroblasts, which was through NiO NP-induced HIF-1α and TGF-β1 upregulation.⁵¹ Induced collagen deposition was also reported to be via downregulation of lncRNA HOTAIRM1 or AP000487.1 and activation of the PRKCB DNA methylation-mediated JNK/c-Jun pathway or TLR4/MyD88/NF-κB pathway in BEAS-2B cells.^52,53

In addition, NiO NPs can stabilize HIF-1α, leading to its nuclear accumulation and upregulation of its target NDRG1 (Cap43) in human lung epithelial cells H460.²⁰ Long-term low-dose NiO NP exposure also caused alteration of gene transcriptional expression, and several pathways were involved including leukocyte extravasation signaling, the fibrosis pathway, the STAT3 pathway, etc.²⁸

3.3 Cytotoxic effects of other Ni-containing NPs on human lung cells in vitro

See Table 3.

Table 3 Cytotoxic effects of other Ni-containing NPs on human lung cells in vitro

Cell line	Particle size (diameter)	Exposure dose	Exposure time	Cytotoxicity endpoints	Ref.
Epithelial (A549)	26 nm NiFe2O4 (Sigma #637149)	1, 2, 5, 10, 25, 50, 100 μg mL^−1	24 h	Decreased cell viability and increased LDH release (25, 50, 100 μg mL^−1)	Ahamed et al. 2011 (ref. 54)
		100 μg mL^−1	24 h	Increased ROS and decreased GSH; cell apoptosis
Epithelial (A549)	21 nm Sr-doped NiO (lab synthesized)	1, 2, 5, 10, 25, 50, 100 μg mL^−1	24 h	Decreased cell viability (≥10 μg mL^−1); increased ROS (≥25 μg mL^−1)	Ahmad et al. 2022 (ref. 55)
		100 μg mL^−1	24 h	Expression changes of apoptosis-related genes
Epithelial (A549)	15.2 nm Ni(OH)2 (US-Nano)	10, 25, 50, 75, 100 μg mL^−1	24, 48 h	Decreased cell viability; suppression of cell proliferation; cell apoptosis; alteration of the cell cycle; increased ROS	Cambre et al. 2020 (ref. 30)
		10, 100 μg mL^−1	12, 24 h	Perturbation of mitochondrial membrane potential
Epithelial (A549)	20 nm C-coated Ni (NanoAmor)	20, 50, 150 μg mL^−1	4 h	No reduction in cell viability, but increased methionine oxidation and upregulated LC3 and p-ERK1/2	Feng et al. 2015 (ref. 17)
Epithelial (A549)	20 nm C-coated Ni (NanoAmor)	0.01, 0.1, 1, 10, 100 μg mL^−1	24 h	No measurable effects on membrane integrity; 40% decrease (10 μg mL^−1) in mitochondrial function followed by a plateau in the response with increasing dose	Minocha et al. 2012 (ref. 23)
Epithelial (HPAEpiC)	Mostly nanolevel Ni2B (lab synthesized)	0.625, 1.25, 2.5, 5, 10, 20, 40, 80, 160, 320, 640, 1280 μg mL^−1	72 h	Dose-dependent decrease in cell viability; 693 gene expression changes; mainly affected microtubule regulation, centrosome organization, and phosphoprotein synthesis	Turkez et al. 2021 (ref. 57)
Epithelial (BEAS-2B)	42.255 nm Fe–Ni alloy (Sigma)	0.1, 0.5, 1, 2, 4, 8, 16, 32, 64, 128 μg mL^−1	24 h	Decreased cell viability (≥4 μg mL^−1); induce the caspase-dependent apoptotic pathway via ROS generation	Vatan 2022 (ref. 56)

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Besides Ni and NiO NPs, other Ni-containing NPs have also been demonstrated to cause adverse effects on human lung cells in vitro. Exposure of A549 cells to NiFe₂O₄, Sr-doped NiO, or Ni(OH)₂ NPs caused decreased cell viability or increased LDH release.^30,54,55 These NPs also caused increased ROS generation, decreased GSH levels, or perturbation of the mitochondrial membrane potential, as well as induced cell apoptosis or expression changes of apoptosis-associated genes.^30,54,55 Exposure to Ni(OH)₂ NPs also caused cell cycle alteration.³⁰ In addition, Fe–Ni alloy NPs caused a decrease in BEAS-2B cell viability and induced caspase-dependent apoptotic pathway via ROS generation.⁵⁶ Feng et al. reported that exposure of A549 cells to carbon (C)-coated Ni NPs did not cause a reduction in cell viability, but caused increased methionine oxidation and upregulation of LC3 and p-ERK1/2.¹⁷ Minocha et al. found no measurable effects of C-coated Ni NPs on membrane integrity, but they caused a 40% decrease at 10 μg mL⁻¹ in cell mitochondrial function followed by a plateau in the response with increasing dose up to 100 μg mL⁻¹.²³ Ni₂B exposure also caused a dose-dependent decrease in HPAEpC cell viability and changes of 693 gene expression, which mainly affected microtubule regulation, centrosome organization, and phosphoprotein synthesis.⁵⁷

3.4 Cytotoxic effects of Ni-containing NPs on non-human mammalian lung cells in vitro

To investigate the cytotoxic effects of Ni-containing NPs on lung cells in vitro, almost all in vitro studies were performed by using human lung cells; only a couple of studies used non-human mammalian lung cells. Latvala et al. reported that exposure to 0.15 and 0.32 μg cm⁻² of 35–40 nm Ni NPs in the air–liquid interface for 48 h caused decreased cell viability in Chinese hamster lung fibroblasts (V79).⁵⁸ And Zhang et al. reported that exposure of rat alveolar macrophages, isolated by bronchioalveolar lavage (BAL), to 20 nm Ni NPs caused severe macrophage damage, which was reflected in an increase in the relative LDH activity in the supernatant. Furthermore, macrophages from old rats released significantly more TNF-α than macrophages from young rats, suggesting that Ni NP-induced inflammation may be more severe in old individuals.⁵⁹

4. Pro-inflammatory and pro-fibrotic effects of Ni-containing NPs on rodent lungs in vivo and the underlying mechanisms

Previous in vivo studies to investigate the pulmonary effects of Ni-containing NPs were performed solely on mice or rats so far; no other mammals have been used. The following three exposure methods were commonly used: (1) inhalation exposure, (2) intratracheal instillation, and (3) oropharyngeal aspiration. Individual labs established various inhalation exposure systems. For example, it was reported that airborne NiO NPs with minimum agglomeration generated using an ultrasonic nebulizer and diffusion dryers could be used for inhalation exposure tests on animals.⁶⁰ Although inhalation exposure is a natural route for particles to enter the lungs, it cannot always be used due to various reasons. Therefore, intratracheal instillation has been employed in many studies as an alternative exposure procedure due in part to its relative ease and cost efficiency as compared with inhalation exposure. Senoh et al. compared the pulmonary effects of NiO NPs in rats after intratracheal instillation, which was performed by five independent research groups in five different institutions. They found that the histopathological changes induced by intratracheal instillation of NiO NPs, such as degeneration/necrosis of alveolar macrophages, lung inflammation, and proliferation of type II pneumocytes in the lungs, were similar, indicating that intratracheal instillation can be a suitable screening method to detect the pulmonary toxicity of nanoparticles.⁶¹ They also compared the pulmonary toxic responses of NiO NPs in rats by single or multiple intratracheal instillations and found that if given multiple doses, as compared with single intratracheal instillation, stronger pulmonary inflammation and more severe lung injury were only observed on day 3, but not on days 28 and 91, after the last dose, suggesting that single intratracheal instillation can be used to assess the pulmonary toxicity of nanoparticles.⁶² Although oropharyngeal aspiration is also easy to perform, the particle dose explicitly administered in the respiratory tract can be difficult to estimate; thus, intratracheal instillation is superior to it with regard to reproducibility and accuracy.⁶³

4.1 Pulmonary effects of Ni NPs on mice

See Table 4 and Fig. 2.

Table 4 Pulmonary effects of Ni NPs on mice

Strain (exposure route)	Particle size (diameter)	Exposure dose	End time (after exposure)	Endpoints	Ref.
Male C57BL/6 and T-bet^−/− (oropharyngeal aspiration)	∼20 nm (Sun-Inno)	4 mg kg^−1 (once)	Days 1, 21	Airway mucous cell metaplasia; chronic alveolitis; allergic airway inflammation; T-bet-regulated CCL2 has a protective role	Glista-Baker et al. 2014 (ref. 66)
Male C57BL/6J (intratracheal instillation)	20 nm (InabVacu)	10, 20, 50, 100 μg (once)	Day 3	Dose–response increase in acute lung inflammation and injury; increased neutrophil count, CXCL1/KC level, LDH activity, total protein level, and MMP-2/9 protein and activity in the BALF; PMNs infiltration into lung tissues	Mo et al. 2019 (ref. 64)
		50 μg (once)	Days 1, 3, 7, 14, 28, 42	Acute lung inflammation and injury appeared as early as day 1, peaked on day 3, and attenuated on day 7 after exposure; extensive pulmonary fibrosis, proliferation of interstitial cells, and chronic inflammation on days 28 and 42 after exposure
Male C57BL/6J and miR-21^−/− (intratracheal instillation)	20 nm (InabVacu)	50 μg (once)	Days 3, 7, 42	Upregulate miR-21, proinflammatory cytokines (IL-6, TNFα), and profibrotic mediators (TGF-β1, phospho-Smad2, COL1A1, COL3A1); decrease Smad7; extensive pulmonary inflammation and fibrosis in WT, but less severe in miR-21^−/− mice	Mo et al. 2020 (ref. 65)
Male & female C57BL/6J (oropharyngeal aspiration)	∼20 nm (Sun-Inno)	4 mg kg^−1 (once)	24 h	Male mice are more susceptible than female mice to neutrophilic inflammation; produce more CXCL1, pro-inflammatory cytokines such as IL-6, and greater STAT3 activation	You et al. 2020 (ref. 67)
		0.67 mg kg^−1 on day 1, 3, 5, 15, 17, 19 (six times)	Day 24	Cause monocytic lung inflammation and the formation of crystals; greater alveolar inflammatory cell infiltration in male mice; increased CXCL1 and CCL2 protein levels; elevated STAT1 in female mice

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Fig. 2 The acute and chronic effects of Ni or NiO NPs on rodent lungs in vivo and the potential underlying mechanisms. Note: the pulmonary genotoxic and carcinogenic effects of Ni or NiO NPs are summarized in section 5 and Fig. 3 and not included here.

C57BL/6J mice exposed to Ni NPs by intratracheal instillation exhibited a dose–response increase in acute lung inflammation and injury, which was reflected by an increased neutrophil count, CXCL1/KC level, LDH activity, total protein level, and MMP-2/9 protein and activity in the BALF as well as infiltration of polymorphonuclear cells (PMNs) and macrophages into lung tissues, which appeared as early as day 1, peaked on day 3, and attenuated on day 7 after Ni NP exposure.⁶⁴ Increased levels of proinflammatory cytokines, such as IL-6 and TNFα, in the BALF were also observed.⁶⁵ Chronic inflammation, extensive interstitial fibrosis, and proliferation of interstitial cells were observed on days 28 and 42 after exposure,^64,65 accompanied by the upregulation of pro-fibrotic mediators such as TGF-β1, phospho-Smad2, etc. and downregulation of the TGF-β signaling inhibitor, Smad7.⁶⁵ Exposure of C57BL/6J mice to Ni NPs by oropharyngeal aspiration also caused airway mucous cell metaplasia, chronic alveolitis, and allergic airway inflammation.⁶⁶ You et al. reported that male mice were more sensitive to Ni NP-induced acute neutrophilic and subchronic monocytic inflammation than female mice through CXCL1, CCL2, and IL-6/STAT3 signaling.⁶⁷ T-bet-regulated CCL2 has a protective role in Ni NP-induced mucous cell metaplasia in the lungs,⁶⁶ and knocking out miR-21 alleviated Ni NP-induced pulmonary inflammation and fibrosis.⁶⁵

4.2 Pulmonary effects of Ni NPs on rats

See Table 5 and Fig. 2.

Table 5 Pulmonary effects of Ni NPs on rats

Strain (exposure route)	Particle size (diameter)	Exposure dose	End time (after exposure)	Endpoints	Ref.
Male SD (intravenous injection)	50 nm (Danyang)	1, 10, 20 mg kg^−1, twice (days 1 & 14)	Day 15	Lung inflammation; lymphocytic and eosinophilic infiltration with thickening of alveolar walls and foamy macrophages	Magaye et al. 2014 (ref. 70)
Male SD (intratracheal instillation)	50 nm (Danyang)	5.6, 12, 25 mg kg^−1 (once)	Day 14	Inflammatory cell infiltration consisting of lymphocytes, neutrophils, and macrophages; decreased protein expression of HO-1 and Nrf2, but not C-myc in the lungs	Magaye et al. 2016 (ref. 18)
Male Wistar (intratracheal instillation)	20 nm (InabVacu)	0.1, 0.5, 1, 5 mg (once)	Day 3	Increased LDH, total protein, total cells, macrophages, and neutrophils in BALF	Zhang et al. 1998 (ref. 68)
		1 mg (once)	Days 1, 3, 7, 15, 30	Epithelial injury and increased permeability; epithelial hyperplasia and persistent inflammation	Zhang et al. 1998 (ref. 71)
Male Wistar (intratracheal instillation)	20 nm (InabVacu)	0.1, 0.5, 1, 5 mg (once)	Day 3	Cause higher levels of LDH, total protein, TNFα, total cells, and differential cell counts in BALF than standard-sized Ni	Zhang et al. 2003 (ref. 69)
		1 mg (once)	Days 1, 3, 7, 15, 30	Induce more severe persistent inflammation than standard-sized Ni

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Wistar rats intratracheally instilled with Ni NPs exhibited increased total cell, macrophage, and neutrophil counts, and increased levels of LDH, total protein, and proinflammatory cytokine TNFα in the BALF on day 3 after exposure.^68,69 Ni NPs caused epithelial injury and hyperplasia, increased permeability, and more severe persistent inflammation in rat lungs than standard-sized Ni.⁶⁹ Two weeks after exposure, besides lung inflammation and neutrophil and macrophage infiltration, lymphocytic and eosinophilic infiltration with thickening of alveolar walls and foamy macrophages was also observed in rats.^18,70 The expression of HO-1 and Nrf2, but not C-myc, was decreased in the Ni NP-instilled SD rats.¹⁸

4.3 Pulmonary effects of NiO NPs on mice

See Table 6 and Fig. 2.

Table 6 Pulmonary effects of NiO NPs on mice

Strain (exposure route)	Particle size (diameter)	Exposure dose	End time (after exposure)	Endpoints	Ref.
Female BALB/c (intratracheal instillation)	20 nm (NanoAmor)	10, 20, 50, 100 μg (once)	24 h	Increased LDH, total protein, and IL-6, but decreased IL-10, in BALF; increased caspase-3 and 8-OHdG in the lungs; lung inflammation; several pathways involved	Bai et al. 2018 (ref. 72)
			Day 29	Sub-acute inflammation and fibrosis; glutathione metabolism and metabolism of xenobiotics by cytochrome P450 pathways involved
			Days 1, 7, 28	Lung or small airway inflammation by SPECT or CT
Male C57BL/6 (intratracheal instillation)	50 nm (Aladdin #N128916)	50, 100, 200 μg for 3 consecutive days	24 h after last dose	Induce lung injury and inflammation; apoptosis and ferroptosis in lung tissues through ATF3 upregulation	Liu et al. 2022 (ref. 40)
Female BALB/cJ (oropharyngeal aspiration)	42 nm, 181 nm (Sigma)	3, 40 μg (once) with/without OVA	Days 1, 10, 19, 29	Induce pulmonary injury and inflammation, which were associated with particle surface area; increased cytokine levels in the lungs; lung eosinophil number and allergen challenge-induced alterations in lung function are related more to particle size	Roach et al. 2019 (ref. 73)
Male C57BL/6 (oropharyngeal aspiration)	<50 nm (Sigma #637130)	20, 40, 80 μg (once)	Days 1, 7	A greater degree of pre-exposure dispersion would cause increased pulmonary inflammation and cytotoxicity, as well as decreases in the integrity of the blood–gas barrier in the lungs	Sager et al. 2016 (ref. 74)
				Cathepsin B release and in turn NLRP3 inflammasome activation generating pro-inflammatory cytokines; act as free radical scavengers, thus ROS is not likely a mechanism of inflammasome activation	Sager et al. 2016 (ref. 75)

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Exposure of female BALB/c mice to NiO NPs by intratracheal instillation caused lung inflammation and injury, which was reflected by increased LDH, total protein, and IL-6 levels, but a decreased IL-10 level, in the BALF.⁷² Increased caspase-3 and 8-OHdG in mouse lungs were also observed.⁷² Several pathways were involved in the NiO NP-induced acute lung inflammation, including focal adhesion, Vibrio cholerae infection, endocytosis, drug metabolism by cytochrome P450, biosynthesis of amino acids, hypertrophic cardiomyopathy, and salmonella infection pathways. Moreover, glutathione metabolism and metabolism of xenobiotics by cytochrome P450 pathways were involved in NiO-NP-induced subacute inflammation and fibrosis.⁷² Liu et al. reported that exposure to NiO NPs induced lung inflammation, apoptosis, and ferroptosis through upregulation of ATF3.⁴⁰ Either surface area⁷³ or pre-exposure dispersion^74,75 of NiO NPs was associated with NiO-NP-induced pulmonary injury and inflammation. Sager et al. reported that NiO NP-induced cathepsin B release, but not ROS, was responsible for NiO NP-induced NLRP3 inflammasome activation, resulting in the production of pro-inflammatory cytokines and lung inflammation since NiO NPs act as free radical scavengers.⁷⁵

4.4 Pulmonary effects of NiO NPs on rats

See Table 7 and Fig. 2.

Table 7 Pulmonary effects of NiO NPs on rats

Strain (exposure route)	Particle size (diameter)	Exposure dose	End time (after exposure)	Endpoints	Ref.
Note:a Surface area of nanoparticles (cm^2).
Male SD (intratracheal instillation)	<50 nm (Sigma)	0.8 mg (once)	Days 3, 7, 28	Induce pulmonary inflammation, NLRP3 inflammasome activation, and cytokine release; require phagocytosis and ROS production	Cao et al. 2016 (ref. 93)
Male Wistar (intratracheal instillation)	20 nm (ST-Nano)	0.015, 0.06, 0.24 mg kg^−1 (twice a week for 6 weeks)	At the end of the exposure	Abnormal changes in indicators of nitrative stress (NO, TNOS, and iNOS), inflammatory cytokines (TNFα, IL-2, and IL-10), and cytokine-induced neutrophil chemoattractants (CINC-1, CINC-2αβ, and CINC-3) in lung tissues; NF-κB activation and Th1/Th2 imbalance	Chang et al. 2017 (ref. 98)
				Widespread lung fibrosis and increased content of hydroxyproline, collagen types I and III in lung tissues; upregulate TGF-β1, Smad2, Smad4, MMP-9, TIMP-1, and CTGF	Chang et al. 2017 (ref. 99)
Female Wistar (intratracheal instillation)	10–20 nm (NanoAmor)	50, 150^a cm^2 (once)	Days 1, 28	Inflammogenic to the lungs (150^a cm^2); cause neutrophilic inflammation and cytotoxicity (24 h); induce neutrophilic/lymphocytic inflammation and alveolar lipoproteinosis (day 28)	Cho et al. 2010 (ref. 100)
		150^a cm^2 (once)	Days 1, 28	Induce chronic interstitial inflammation and pro-inflammatory Th1 and Th17 immune responses characterized by increases in the cytokines MCP-1/CCL2, IL-12 p40, IFN-γ, and IL-17A; induce pulmonary alveolar proteinosis due to over-production of surfactant by proliferation of type II cells and impaired clearance of surfactant by macrophages	Cho et al. 2012 (ref. 101)
				Increased total cells, PMNs, lymphocytes, LDH, total protein, IL-1β, MIP-2, and IFN-γ, but not eosinophils, in BALF	Cho et al. 2012 (ref. 33)
Male Wistar (inhalation)	59 nm (NanoAmor #4210SD)	0.2 mg m^−3 (6 h d^−1, 5 d per week for 6 weeks)	Day 3, 1 m	Upregulation of genes associated with chemokines, oxidative stress, and MMP-12; acute lung inflammation; damaged tissues were repaired in the post-exposure period	Fujita et al. 2009 (ref. 94)
Male Wistar (intratracheal instillation)	20 nm (NanoAmor)	0.2 mg (once)	1 h, 4 h, days 1, 3, 7	Increased LDH, SP-D, tHODE (lipid peroxidation), and HO-1 in BALF	Horie et al. 2011 (ref. 44)
			1 h, days 1, 3, 7	Increased LDH, tHODE (lipid peroxidation), HO-1, SP-D, and α-tocopherol in BALF; Ni^2+ release from NiO NPs is an important factor in oxidative stress-related toxicity	Horie et al. 2012 (ref. 102)
Male F344 (intratracheal instillation)	15–35 nm (US-Nano #US3355)	0.2, 1.0 mg (once)	Day 3, 1 m, 3 m, 6 m	Induce pulmonary oxidative stress (upregulate HO-1 and increase 8-iso-PGF2, thioredoxin, and iNOS); increased MPO	Horie et al. 2016 (ref. 95)
(Inhalation)		0.32–1.65 mg m^−3 (6 h d^−1, 5 d per week for 4 weeks)		Inhalation causes milder oxidative stress than that caused by intratracheal instillation, even if the amount of NiO NPs in the lungs was similar
Female Wistar (oropharyngeal aspiration)	5.3 nm (NanoAmor)	98.1 μg (once)	Days 1, 28	Severe neutrophilic inflammation on day 1 and lymphocytic inflammation with pulmonary alveolar proteinosis on day 28 after exposure	Jeong et al. 2016 (ref. 76)
Female Wistar (intratracheal instillation)	5.3 nm (NanoAmor)	50, 150^a cm^2 (once)	Days 1, 28	Cause a neutrophilic and lymphocytic inflammatory response; alter microbial composition; more Burkholderiales than in the control group at day 1 after instillation	Jeong et al. 2022 (ref. 97)
Male Wistar (inhalation)	54 nm (NanoAmor)	0.2 mg m^−3 (6 h d^−1, 5 d per week for 4 weeks)	Day 3, 1 m, 3 m	Increased neutrophil counts, phospholipids, total protein, and SP-D in BALF; decreased BALF surface tension	Kadoya et al. 2016 (ref. 103)
White female (own breeding colony) (nose-only inhalation)	23 nm (lab generated)	2.4 μg m^−3 (4 h d^−1, 5 d per week for 2 weeks, 4 weeks, 3 m, 6 m)	24 h after last exposure	This kind of exposure can be considered as close to the LOAEL (lowest observed adverse effect level), or even to the NOAEL (no observed adverse effect level), but increased counts of total cells, neutrophils, and AMs were observed in BALF	Katsnelson et al. 2021 (ref. 79)
Male F344 (intratracheal instillation)	20 nm (A) (US-Nano #US3352)	0.67, 2, 6 mg kg^−1 (once)	Days 3, 28, 91	The most soluble product (B) caused the most severe toxicity, but the response was transient. It has the highest pulmonary clearance rate constant. The second-most-soluble material (D) and the third one (A) caused evident pulmonary inflammation, and the responses persisted for at least 91 days with collagen proliferation. In contrast, the most insoluble NiO (C) induced barely detectable inflammation and no marked histopathological changes. These results indicate that the early phase toxic potential of NiO products, but not the persistence of pulmonary inflammation, is associated with their solubility	Kobayashi et al. 2021 (ref. 77)
	29 nm (B) NovaWireNi01 (Novarials)				Shinohara et al. 2017 (ref. 106)
	140 nm (C) I small particle (Kusaka)
	39 nm (D) (Sigma #637130)
Female Wistar (intratracheal instillation)	5.3 nm (91.8 m^2 g^−1) (NanoAmor)	54.5, 109, 218 μg (once)	Days 1, 2, 3, 4	Acute neutrophilic inflammation; recruited eosinophils on days 3 and 4, but not related to total IgE or anaphylatoxins; high level of eotaxin in AMs and lung tissues	Lee et al. 2016 (ref. 105)
		120, 180, 360^a cm^2 (once)	24 h	Acute pulmonary inflammation; additive effect on the neutrophil counts and ROS generation with CuO NPs; antagonistic effect on the neutrophil counts with carbon black NPs	Lee et al. 2021 (ref. 96)
Female Wistar (intratracheal instillation)	10–20 nm (NanoAmor)	250^a cm^2 (once)	24 h	Increased number of PMNs in BALF	Lu et al. 2009 (ref. 43)
Male & female albino-derived SD (nose-only inhalation)	20 nm (Nanoshel #NS6130-03-337)	5 mg L^−1 for 4 h	Day 14	No mortality; reduced body weight during the first 7 days, then recovered; irregular respiration and hypoactivity; slight to moderate lung discoloration	Lyons-Darden et al. 2023 (ref. 82)
Male Wistar (intratracheal instillation)	26 nm (DLS) (1) from 10–20 nm (2) (NanoAmor)	0.1, 0.2, 1, 2 mg (once)	Day 3, 1 m	The dose-dependent increased number of PMNs in BALF was based on the surface area doses, but not the mass doses; when the same NiO NPs were tested, the amount of pulmonary deposition of the sample after 4-week inhalation and an intratracheally instilled dose of about ten-times higher induced similar PMN responses on day 3 after exposure	Mizuguchi et al. 2013 (ref. 81)
	NiO (3) (VacuMeta)
	NiO (4) (Nakalai)
(Inhalation)	26 nm (DLS) (1) from 10–20 nm (2) (NanoAmor)	9.2 × 10^4 particles per cm^3 (6 h d^−1, 5 d per week for 4 weeks)	Day 3, 1 m, 3 m after 4 week exposure
Male Wistar (intratracheal instillation)	20 nm (NanoAmor)	1 mg (once)	Day 3, 1 week, 1 m, 3 m, 6 m	NiO NP agglomerates caused increased counts of total cells and neutrophils in BALF persistently; neutrophil and macrophage infiltration and elevated CINC-2αβ in lung tissues	Morimoto et al. 2011 (ref. 80)
				Infiltration of neutrophils and AMs; increased CINC-1, CINC-2αβ, and CINC-3	Morimoto et al. 2014 (ref. 88)
Male Wistar (inhalation)	20 nm (NanoAmor)	1 × 10^5 particles per cm^3 (6 h d^−1, 5 d per week for 4 weeks)	Day 4, 1 m, 3 m after 4 week exposure	Minimum inflammation only at day 4; no changes in mRNA expression of MMP-2, TIMP-2, and type I collagen	Morimoto et al. 2011 (ref. 83)
				Increased total cells in BALF at day 4, but not at 1 m. Histopathological change was not severe. The deposited amount of NiO NPs in the lungs at day 4 was 29 ± 4 μg. The retained particle amount in the lungs after inhalation exponentially decreased and the calculated biological half-time was 62 days	Ogami et al. 2009 (ref. 84)
					Oyabu et al. 2007 (ref. 60)
Male F344 (intratracheal instillation)	19 nm (US-Nano #US3355)	0.2, 1 mg (once)	Day 3, 1 week, 1 m, 3 m, 6 m	Induce persistent inflammation and upregulation of CINC-1, CINC-2, and HO-1, suggesting that intratracheal instillation studies may be useful for studying the harmful effects of NPs	Morimoto et al. 2016 (ref. 89)
(Inhalation)		1.65 mg m^−3 (6 h d^−1, 5 d per week for 4 weeks)	Day 3, 1 m, 3 m after 4 week exposure	Increased neutrophil counts and the levels of CINC-1, CINC-2, and HO-1 in BALF
Male Wistar (intratracheal instillation)	20 nm (NanoAmor)	0.1, 0.2 mg (once)	Day 3, 1 week, 1 m, 3 m, 6 m	Cause a persistent inflammatory effect; MIP-1α showed a continued increase in lung tissues and BALF; IL-1α and IL-1β in lung tissues and MCP-1 in BALF showed transient increases	Morimoto et al. 2010 (ref. 87)
				Increased CINC-1, CINC-2αβ (day 3–6 m), and CINC-3 (day 3) in lungs and BALF; increased counts of total cells and neutrophils in BALF (day 3–3 m); infiltration of neutrophils and AMs in lung tissues (day 3–6 m)	Nishi et al. 2009 (ref. 90)
				More toxic at day 3 to 1 m; take about 1 week to dissolve NiO NPs in ALF, but 1 m or more in vivo; increased proteins and phospholipids in BALF	Nishi et al. 2020 (ref. 104)
Male Wistar (intratracheal instillation)	27 nm [lab prepared from NiO (VacuMeta)]	2 mg (once)	Day 3, 1 week, 1 m, 3 m, 6 m	Increased numbers of total cells and PMNs in BALF; infiltration of macrophages or PMNs and alveolitis; pulmonary inflammation and fibrosis	Ogami et al. 2009 (ref. 78)
Male F344 (inhalation)	19 nm (US-Nano #US3355)	0.32, 1.65 mg m^−3 (6 h d^−1, 5 d per week for 4 weeks)	Day 3, 1 m, 3 m after 4 weeks exposure	Biological half-times were 2.9 and 5.2 m. NPs were phagocytized by macrophages and many PMNs in BALF at day 3	Oyabu et al. 2017 (ref. 86)
(Intratracheal instillation)		0.2, 1 mg (once)	Day 3, 1 week, 1 m, 3 m, 6 m	Biological half times were 4.9 and 9.5 m
Male SD (intratracheal instillation)	<50 nm (Sigma)	0.8 mg (once)	Days 28, 60	HIF-1α and TGF-β1 act in synergy to foster lung fibrosis and increase αSMA expression	Qian et al. 2015 (ref. 51)
Male F344 (intratracheal instillation)	20 nm (US-Nano #US3352)	1 dose of 1 or 2 mg kg^−1, 3 doses of 0.67 mg kg^−1, 4 doses of 0.5 mg kg^−1	Days 3, 28, 91	Pulmonary injury and inflammation (increased total cell, macrophage, and neutrophil counts and total protein, LDH, γ-GTP in BALF); phagocytosis of NPs by AMs; degeneration and necrosis of AMs	Senoh et al. 2017 (ref. 62)
Male Crl:CD (SD) (intratracheal instillation)	18 nm (US-Nano #US3352)	0.2, 0.67, 2 mg kg^−1 (once)	Day 3	Degeneration and necrosis of AMs; lung inflammation (infiltration of inflammatory cells and AMs); proliferation of type II pneumocytes. The pulmonary lesions tend to be spread over a wider area when using an aerosolizer	Senoh et al. 2020 (ref. 61)
White female (own breeding colony) (inhalation)	23 nm (lab generated)	0.23 mg m^−3 (4 h d^−1, 5 d per week for 3, 6, 10 m)	24 h after last exposure	Increased number of total cells, neutrophils, and AMs in BALF; increased γ-glutamyl transferase, amylase, LDH, alkaline phosphatase, and aspartate aminotransferase in BALF; little pronounced pulmonary pathology; a rather low chronic retention of NPs in the lungs	Sutunkova et al. 2019 (ref. 85)
Male F344 (intratracheal instillation)	19 nm (US-Nano #US3355)	0.2, 1 mg (once)	Day 3, 1 week, 1 m, 3 m, 6 m	Persistent increase in the neutrophil counts in BALF; persistent lung inflammation; increased MPO; the concentration of MPO correlated with the number of total cells and neutrophils, the concentration of CINC-1 and HO-1, and the activity of released LDH in BALF	Tomonaga et al. 2018 (ref. 91)
(Inhalation)		0.32, 1.65 mg m^−3 (6 h d^−1 for 4 weeks)	Day 3, 1 m, 3 m after 4 week exposure	Increased neutrophil counts and MPO in BALF; lung inflammation at high dose
Male Wistar (intratracheal instillation)	20 nm (ST-Nano)	0.015, 0.06, 0.24 mg kg^−1 (twice a week for 9 weeks)	24 h after last dose	Collagen deposition and fibrosis in lungs via lncRNA MEG3 downregulation, Hh pathway activation, and autophagy suppression	Gao et al. 2022 (ref. 49)
				Widen alveolar septum; decreased size of alveolar cavity; increased protein in lungs; lung inflammation; activate the p38 MAPK pathway; downregulate MEG3	Yang et al. 2022 (ref. 47)
				Induce EMT and lung fibrosis; downregulate lncRNA MEG3	Zhan et al. 2021 (ref. 50)
				Induce fibrosis; downregulate MEG3; increased TGF-β1 and phosphorylation of PI3K, AKT, and mTOR	Zhan et al. 2021 (ref. 48)
				Induce fibrosis; downregulate lncRNA HOTAIRM1; hypomethylation; upregulate PRKCB2; JNK/c-Jun pathway activation	Zheng et al. 2022 (ref. 52)
Male Wistar (intratracheal instillation)	20 nm (ST-Nano)	0.015, 0.06, 0.24 mg kg^−1 (twice a week for 6 weeks)	At the end of exposure	Increased levels of ·OH, lipid peroxidation, and 8-OHdG; decreased levels of CAT, GSH-Px, and T-AOC; upregulation of HO-1 and downregulation of MT-1 in lung tissues	Zhu et al. 2017 (ref. 92)

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Exposure of rats to NiO NPs caused acute lung injury, neutrophilic lung inflammation, and degeneration and necrosis of alveolar macrophages (AMs) at a short time after exposure,^43,61,62,76 and caused chronic inflammation and pulmonary fibrosis at a long time after exposure.^77,78 Even the exposure close to the LOAEL (lowest observed adverse effect level) or NOAEL (no observed adverse effect level) also caused increases in the number of total cells, polymorphonuclear cells (PMNs), and AMs in the BALF, suggesting that these effects may have no threshold at all.⁷⁹ NiO NP agglomerates also can induce a persistent inflammatory response.⁸⁰ In addition, Mizuguchi et al. reported that the amount of pulmonary deposition of the sample after 4-week inhalation, and an intratracheally instilled dose about ten-times higher, induced similar PMN responses on day 3 after termination of inhalation and instillation.⁸¹ However, others reported that inhalation exposure to NiO NPs caused no mortality, reduced body weight only during the first 7 days, which then recovered, and slight to moderate lung discoloration.⁸² In addition, NiO NP inhalation only caused minimum inflammation and no changes in mRNA expression of MMP-2, TIMP-2, and type I collagen.^83,84 Low-dose long-term inhalation exposure to NiO NPs only caused mild to moderate chronic toxicity.⁸⁵ The calculated biological half-times were 2.9 and 5.2 months when the rats were exposed to 0.32 and 1.65 mg m⁻³ NiO NPs for 6 h d⁻¹ and 5 d per week for 4 weeks, which were 4.9 and 9.5 months when the rats were intratracheally instilled with 0.2 and 1 mg of NiO NPs,^60,86 suggesting that biopersistence is a good indicator of the hazards of nanoparticles.

NiO NP exposure caused cytokine and ROS production in rats. NiO NP exposure induced a continued increase in inflammation-related cytokines such as MIP-1α in lung tissues and the BALF, but IL-1α and IL-1β in lung tissues and MCP-1 in the BALF showed transient increases.⁸⁷ An increased level of CINC-1, CINC-2αβ, CINC-3, or HO-1 was also observed in the BALF or rat lungs.^88–90 In addition, the concentration of MPO in the BALF could be used as a biomarker for the ranking of pulmonary toxicity of nanoparticles since it was closely correlated with the number of total cells and neutrophils, the concentration of CINC-1 and HO-1, and the activity of released LDH in the BALF.⁹¹ Moreover, increased levels of ·OH, LPO, and 8-OHdG and decreased levels of CAT, GSH-Px, and T-AOC as well as upregulation of HO-1 and downregulation of MT-1 mRNA expression in rat lung tissues were observed after NiO NP exposure.⁹² Cao et al. reported that NiO NP-induced pulmonary inflammation, NLRP3 inflammasome activation, and cytokine release were due to phagocytosis of the particles by the cells and ROS production.⁹³ High expression of genes associated with chemokines, oxidative stress, and MMP-12 was observed, indicating that NiO NP exposure caused acute inflammation and damaged tissues were repaired in the post-exposure period.⁹⁴ In addition, inhalation caused milder oxidative stress than that caused by intratracheal instillation, even if the amount of NiO NPs in the lungs was similar.⁹⁵ NiO NPs caused additive effects on the neutrophil counts and ROS generation with CuO NPs, implying that the physicochemical properties of each type of nanoparticle are not influenced by the other type. NiO NPs also caused an antagonistic effect on the neutrophil counts with carbon black NPs due to their scavenging activity of ROS generated by NiO rather than the competition in cellular uptake to target cells (i.e. alveolar macrophages).⁹⁶ Further, Jeong et al. reported that dysbiosis in the lung microbiome was thought to be associated with acute lung inflammation since NiO NP exposure altered the lung microbial composition.⁹⁷

Many signaling pathways may be involved in NiO NP-induced inflammation and fibrosis. Chang et al. reported that NF-κB activation and Th1/Th2 imbalance were responsible for NiO NP-induced nitrative stress and inflammation,⁹⁸ and TGF-β1/Smad signaling was responsible for NiO NP-induced pulmonary fibrosis.⁹⁹ LncRNA MEG3 ameliorates NiO NP-induced pulmonary inflammatory damage by suppressing the p38 MAPK pathway.⁴⁷ Lung fibrosis after NiO NP exposure was also reported via MEG3 downregulation, Hh pathway activation, autophagy suppression, the TGF-β1-mediated PI3K/AKT pathway, or EMT development.^48–50 LncRNA HOTAIRM1 was involved in NiO NP-induced pulmonary fibrosis via regulating the PRKCB DNA methylation-mediated JNK/c-Jun pathway.⁵² HIF-1α and TGF-β1 may act in synergy to foster NiO NP-induced pulmonary fibrosis.⁵¹

Pulmonary alveolar lipoproteinosis was observed at four weeks after NiO NP instillation, which could be explained by the over-production of surfactant by the proliferation of type II pneumocytes and impaired clearance of surfactant by macrophages.^76,100,101 Increased levels of SP-D, total protein, and phospholipids and decreased surface tension were detected in the BALF after NiO NP exposure.^44,102–104

NiO NP exposure also caused a delayed-type hypersensitivity (DTH) response in rat lungs.¹⁰¹ And NiO NPs can recruit eosinophils in the lungs of rats by the direct release of intracellular eotaxin, but it was not related to the levels of total IgE and anaphylatoxins.¹⁰⁵

4.5 Pulmonary effects of other Ni-containing NPs on mice

See Table 8.

Table 8 Pulmonary effects of other Ni-containing NPs on mice

Strain (exposure route)	Particle size (diameter)	Exposure dose	End time (after exposure)	Endpoints	Ref.
Male C57BL/6 (whole-body inhalation)	∼40 nm Ni(OH)2 (lab generated)	65, 358, 763 μg Ni m^−3 for 4 h	0.5, 24, 48 h	Lung injury and inflammation: PMN infiltration and protein leakage into the lungs; increased mRNA expression of inflammatory cytokines and chemokines (TNFα, MIP-2, and CCL2)	Gillespie et al. 2010 (ref. 107)
		∼79 μg Ni m^−3 for 5 h d^−1, 5 d per week for 1 week, 3 m, or 5 m	24 h after last exposure	Increased counts of neutrophils and lymphocytes; increased protein leakage into lungs; upregulate inflammatory cytokines and chemokines (IL-1α, TNFα, MIP-2, and CCL2); lung inflammation histologically
Male ApoE^−/− (whole-body inhalation)	∼40 nm Ni(OH)2 (lab generated)	∼79 μg Ni m^−3 for 5 h d^−1, 5 d per week for 1 week or 5 m	24 h after last exposure	Lung inflammation; increased total cells, neutrophils, and total protein in BALF; increased mRNA expression of antioxidant enzymes (HO-1) and proinflammatory cytokines (CCL2, IL-6, and TNFα)	Kang et al. 2011 (ref. 108)
Male C57BL/6 (whole-body inhalation)	∼40 nm Ni(OH)2 & NiSO4 (lab generated)	361, 775 μg Ni m^−3 for Ni(OH)2 & 833 μg Ni m^−3 for NiSO4 for 4 h	0.5, 24 h after exposure	Ni(OH)2 NPs have stronger inflammogenic potential than NiSO4 NPs. Pulmonary effects are chemical-specific and deposited dose and solubility are key factors	Kang et al. 2011 (ref. 109)
Female C57BL/6 (intratracheal instillation)	20–30 nm NiFe2O4, 10–30 nm NiZnFe4O8 (NanoAmor)	14, 43, 128 μg (once)	Days 1, 3, 28	Increased neutrophil counts in BALF for NiFe2O4 (days 1, 3, 28) and NiZnFe4O8 (day 28)	Hadrup et al. 2020 (ref. 110)
Male C57BL/6 (intratracheal instillation)	20 nm passivated or C-coated Ni NPs (US-Nano)	50 μg (once)	Days 3, 42	Much milder acute and chronic lung injury, inflammation, and fibrosis as compared with Ni NPs	Mo et al. 2019 (ref. 64)

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The adverse pulmonary effects of other Ni-containing NPs have been explored on mice but not on rats. Either short-term or long-term whole-body inhalation exposure of C57BL/6 mice to Ni(OH)₂ NPs resulted in lung injury and inflammation; PMN infiltration and protein leakage into the lungs and increased mRNA expression of proinflammatory cytokines and chemokines (TNFα, MIP-2, CCL2, or IL-1α) were observed.¹⁰⁷ Exposure of ApoE^−/− mice to Ni(OH)₂ NPs also caused lung inflammation and increased mRNA expression of antioxidant enzyme HO-1.¹⁰⁸ Slightly soluble Ni(OH)₂ NPs have stronger inflammogenic potential than readily soluble NiSO₄ NPs.¹⁰⁹ However, passivated or carbon-coated Ni NPs caused less acute and chronic lung inflammation, injury, and fibrosis as compared with Ni NPs.⁶⁴ Intratracheal instillation of NiFe₂O₄ and NiZnFe₄O₈ NPs into mice caused increased neutrophil counts in BALF for NiFe₂O₄ NPs on days 1, 3, and 28 and for NiZnFe₄O₈ NPs on day 28 after exposure.¹¹⁰

Taken together, exposure of mice or rats to Ni-containing NPs may cause acute lung injury and neutrophilic inflammation, which appear as early as day one after exposure. Other acute lung effects are also observed within one week after exposure, including increased lung permeability, allergic inflammation, degeneration and necrosis of alveolar macrophages, apoptosis and ferroptosis of lung epithelial cells, perturbation of lung microbiota, oxidative stress, etc. The acute responses progress to subchronic and chronic lung injury over time, which is reflected by chronic inflammation, the proliferation of lung interstitial cells and pulmonary fibrosis, delayed-type hypersensitivity (DTH), airway mucous cell metaplasia, autoimmune inflammation with increased lymphocyte counts, pulmonary alveolar proteinosis or lipoproteinosis, etc.Fig. 2 summarizes the acute and chronic effects of Ni-containing NPs on rodent lungs in vivo and the potential underlying mechanisms. The genotoxic and carcinogenic effects of Ni-containing NPs are reviewed in section 5 below and not included in Fig. 2.

5. Pulmonary genotoxicity and carcinogenicity of Ni-containing NPs and the underlying mechanisms

Standard-sized nickel and nickel compounds have genotoxic and carcinogenic effects. According to the International Agency for Research on Cancer (IARC) monographs, nickel compounds are listed as group 1 carcinogens (carcinogenic to humans), and metallic nickel and nickel alloys are group 2B (possibly carcinogenic to humans).¹¹ An increased risk of lung and nasal cancers has been observed in workers exposed to nickel and nickel compounds.¹¹ In this section, the genotoxic and carcinogenic effects of Ni-containing NPs on lung cells in vitro and rodent lungs in vivo and the potential underlying mechanisms are summarized (Fig. 3).

Fig. 3 The potential mechanisms involved in pulmonary genotoxic and carcinogenic effects of Ni-containing NPs.

5.1 Genotoxic and carcinogenic effects of Ni NPs on lung cells in vitro and mouse lungs in vivo

See Table 9.

Table 9 Genotoxic and carcinogenic effects of Ni NPs on lung cells in vitro and mouse lungs in vivo

Cell line or animal strain	Particle size (diameter)	Exposure dose	Exposure time or end time	Endpoints	Ref.
Human lung cells in vitro
Epithelial (HBEC3-kt)	<100 nm (Sigma #577995)	5, 10, 25 μg mL^−1	24 h	Induce DNA strand breaks (comet assay); lack of induction of γH2AX foci	Akerlund et al. 2018 (ref. 111)
		10, 50 μg mL^−1	THP-1*: 3, 18 h; HBEC: 3 h	Direct exposure to, co-culture of, or conditioned media from THP-1* macrophages exposed to Ni NPs caused genotoxicity in HBEC cells (comet assay)	Akerlund et al. 2019 (ref. 22)
Epithelial (BEAS-2B)	<100 nm (Sigma #577995)	1, 5, 10 μg mL^−1	48 h	Cause DNA damage (comet assay); chromosomal damage and rearrangements	Di Bucchianico et al. 2018 (ref. 24)
		0.5 μg mL^−1	42 d	Cause DNA strand breaks (comet assay), but no induction of micronuclei and hypodiploid nuclei; no clear changes in cell transformation or cell motility	Gliga et al. 2020 (ref. 28)
Epithelial (BEAS-2B)	20 nm (InabVacu)	5, 10, 20, 30 μg mL^−1	24 h	Cause DNA damage and DNA damage response (upregulation of p-ATM, ATM, p-p53, γH2AX); DNA repair defect (downregulation of Rad52 via upregulation of HIF-1α and miR-210)	Mo et al. 2021 (ref. 21)
		0.25, 0.5 μg mL^−1	21 cycles (150 d)	Cause DNA damage and DNA damage response (upregulation of p-p53, γH2AX); DNA repair defect (downregulation of Rad52); cause cell transformation
Epithelial (A549)	<100 nm (Sigma #577995)	20 μg cm^−2	4, 24 h	Increase but not significantly in DNA damage (comet assay)	Latvala et al. 2016 (ref. 19)
Epithelial (A549)	50 nm (Danyang)	1, 5, 10, 15, 25 μg cm^−2	24 h	Significant increase in DNA damage (10, 15, and 25 μg cm^−2) (comet assay)	Magaye et al. 2016 (ref. 18)

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Non-human mammalian lung cells in vitro
Chinese hamster lung fibroblasts (V79-4)	<100 nm (Sigma #577995)	1, 5, 10 μg mL^−1	48 h	No clear conclusions about HPRT mutations can be drawn due to the large variation between the experiments	Akerlund et al. 2018 (ref. 111)
Chinese hamster lung fibroblasts (V79)	35–40 nm aerosol (lab generated)	~0.32 μg cm^−2	2 h	Increased amount of DNA strand breaks (alkaline DNA unwinding technique)	Latvala et al. 2017 (ref. 58)
		0.05, 0.15, 0.32 μg cm^−2	48 h	No induction of HPRT mutations

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Mouse lungs in vivo
Male C57BL/6J (intratracheal instillation)	20 nm (InabVacu)	50 μg (once)	Day 7 after exposure	DNA damage (upregulation of γH2AX); DNA repair defect (downregulation of Rad52)	Mo et al. 2021 (ref. 21)
			Days 7, 42 after exposure	Cell proliferation (increased PCNA- or Ki-67-positive cells)
Male and female gpt delta transgenic (intratracheal instillation)			4 m after exposure	No changes in the mutant frequency (MF) and mutant spectrum of genomic DNA in mouse lungs

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Direct exposure to Ni NPs caused DNA strand breaks detected by the comet assay or alkaline DNA unwinding technique in either human lung epithelial cells such as HBECs,^22,111 BEAS-2B,^24,28 and A549^18,19 or Chinese hamster lung fibroblasts V79.⁵⁸ Exposure to Ni NPs induced DNA damage and DNA damage response in BEAS-2B cells, which was reflected by upregulation of p-ATM, ATM, p-p53, and γH2AX.²¹ Ni NP exposure also caused DNA repair defects by downregulation of homologous recombination (HR) repair gene Rad52 via nuclear accumulation of HIF-1α and upregulation of miR-210.²¹ Exposure of BEAS-2B cells to 1, 5, and 10 μg mL⁻¹ Ni NPs for 48 h caused chromosomal damage and rearrangements as reflected by increased micronuclei and increased frequencies of nucleoplasmic bridges and nuclear buds,²⁴ although low-dose (0.5 μg mL⁻¹) Ni NP exposure for 6 weeks did not induce micronuclei or hypodiploid nuclei in the cells.²⁸ In addition, indirect exposure to Ni NPs also caused DNA damage. Culture of HBECs with conditioned media from Ni NP-exposed THP-1* cells or transwell co-culture of Ni NP-exposed THP-1* and HBECs caused an increased DNA amount in the tail of HBEC nuclei by the comet assay, suggesting that Ni NPs could induce secondary (inflammation-driven) genotoxicity.²² Moreover, Ni NP-induced DNA damage was also observed in mouse lungs; increased expression of the DNA damage marker, H2AX, and decreased expression of the DNA HR repair gene, Rad52, were observed in the lungs of Ni NP-exposed mice.²¹

The carcinogenic effect of Ni NPs has been evidenced. Exposure to low doses (0.25 and 0.5 μg mL⁻¹) of Ni NPs for a long time (150 days) caused normal human bronchial epithelial cells BEAS-2B to undergo malignant transformation,²¹ although no apparent changes in cell transformation or cell motility were observed after 6-week exposure.²⁸ Epithelial cell proliferation, as indicated by increased PCNA- or Ki-67-positive cells, was also observed in Ni NP-exposed mouse lungs.²¹ However, no induction of HPRT mutations was detected in Chinese hamster lung fibroblasts V79 after Ni NP exposure.^58,111 And no increased mutant frequency (MF) and no significant changes of the mutant spectrum in the genomic DNA of Ni NP-exposed mouse lungs were observed by using gpt delta transgenic mice.²¹

5.2 Genotoxic and carcinogenic effects of NiO NPs on lung cells in vitro

See Table 10.

Table 10 Genotoxic and carcinogenic effects of NiO NPs on lung cells in vitro

Cell line	Particle size (diameter)	Exposure dose	Exposure time or end time	Endpoints	Ref.
Human lung cells in vitro
Epithelial (HBEC3-kt)	<50 nm (Sigma #637130)	5, 10, 25 μg Ni mL^−1	24 h	Induce DNA strand breaks (comet assay); lack of induction of γH2AX foci	Akerlund et al. 2018 (ref. 111)
		10, 50 μg Ni mL^−1	THP-1*: 3, 18 h; HBEC: 3 h	Direct exposure to, co-culture of, or conditioned media from THP-1* macrophages exposed to NiO NPs caused DNA damage in HBEC cells (comet assay)	Akerlund et al. 2019 (ref. 22)
Epithelial (BEAS-2B, A549)	<50 nm (Sigma)	100 μg mL^−1	2 h	Nuclear translocation of p-ATM and p-ATR (IHC & WB)	Capasso et al. 2014 (ref. 31)
		20, 40, 60, 80, 100 μg mL^−1	24 h	A549: decreased cell population in G1, increased cell population in G2/M, and no change in the S phase
				BEAS-2B: increased cell population in G1, but decreased cell population in G2/M and S phases
Epithelial (BEAS-2B)	<50 nm (Sigma #637130)	1, 5, 10 μg Ni mL^−1	48 h	Cause DNA damage (comet assay); induce chromosomal damage and rearrangements	Di Bucchianico et al. 2018 (ref. 24)
		0.5 μg Ni mL^−1	42 d	Cause DNA strand breaks (comet assay), but no induction of micronuclei and hypodiploid nuclei; no clear changes in cell transformation or cell motility	Gliga et al. 2020 (ref. 28)
Epithelial (BEAS-2B, A549)	<50 nm (Sigma)	20, 40 μg cm^−2	4 h	Induce DNA breaks (comet assay)	Kain et al. 2012 (ref. 112)
Epithelial (A549)	<50 nm (Sigma #637130)	20 μg Ni cm^−2	4, 24 h	Cause DNA damage (comet assay)	Latvala et al. 2016 (ref. 19)
Epithelial (HBEC3-kt)	<50 nm (Sigma #637130)	5, 10, 25 μg Ni mL^−1	48 h	No increased micronucleus formation	Vallabani et al. 2022 (ref. 42)
		5, 25 μg Ni mL^−1	Co-culture: THP-1*: 24, 48 h	Induce DNA strand breaks (25 μg Ni mL^−1) (comet assay), but no increased micronucleus formation

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Non-human mammalian lung cells in vitro
Chinese hamster lung fibroblasts (V79-4)	<50 nm (Sigma #637130)	1, 5, 10 μg Ni mL^−1	48 h	No clear conclusions about HPRT mutations can be drawn due to the large variation between the experiments	Akerlund et al. 2018 (ref. 111)
Chinese hamster lung fibroblasts (V79)	32.75 ± 24.65 nm (lab synthesized)	125, 250, 500 μg mL^−1	4, 24 h	Increased binucleated micronucleated (BNMN) cells (CBMN assay)	De Carli et al. 2018 (ref. 113)
		62, 125, 250, 500 μg mL^−1	4 h	Induce DNA breaks (comet assay)

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Only in vitro experiments have been performed to study the pulmonary genotoxic and carcinogenic effects of NiO NPs. It was reported that exposure to NiO NPs caused DNA strand breaks in human bronchial epithelial cells HBECs¹¹¹ and BEAS-2B,^24,28,112 human lung epithelial cells A549,^19,112 and Chinese hamster lung fibroblasts V79.¹¹³ Direct exposure to, co-culture of, or conditioned media from THP-1* macrophages exposed to NiO NPs all caused DNA damage in HBEC cells by the comet assay, suggesting that NiO NPs appear to cause both primary and secondary (inflammation-driven) genotoxicity.^22,42 NiO NP exposure also caused chromosomal damage and rearrangements in BEAS-2B cells.²⁴ In addition, exposure of BEAS-2B or A549 cells to NiO NPs caused a significant nuclear translocation of DNA damage response proteins, p-ATM and p-ATR.³¹ However, a lack of γH2AX focus induction or micronucleus formation was observed when HBEC cells were exposed to NiO NPs.^42,111

Capasso et al. reported that exposure of BEAS-2B or A549 cells to NiO NPs caused cell cycle alterations, which were reflected by decreased cell population in the G1 phase, increased cell population in the G2/M phase, and no change in the S phase in A549 cells, and increased cell population in the G1 phase and decreased cell population in both G2/M and S phases in BEAS-2B cells.³¹

Gliga et al. showed that exposure of BEAS-2B cells to 0.5 μg Ni mL⁻¹ NiO NPs for six weeks did not cause induction of micronuclei and hypodiploid nuclei, and no apparent changes in cell transformation or cell motility were observed.²⁸ No clear conclusions about HPRT mutations can be drawn after exposure of Chinese hamster lung fibroblasts V79 to NiO NPs.¹¹¹ However, De Carli et al. found increased binucleated micronucleated (BNMN) V79 cells after NiO NP exposure.¹¹³

5.3 Genotoxic and carcinogenic effects of other Ni-containing NPs on lung cells in vitro and mouse lungs in vivo

See Table 11.

Table 11 Genotoxic and carcinogenic effects of other Ni-containing NPs on human lung cells in vitro and mouse lungs in vivo

Cell line or animal strain	Particle size (diameter)	Exposure dose	Exposure time or end time	Endpoints	Ref.
Human lung cells in vitro
Epithelial (BEAS-2B)	42.255 nm Fe–Ni alloy (Sigma)	9.5, 19, 38, 57 μg mL^−1	24 h	Induce DNA breaks (comet assay); increased amount of DNA DSBs (γH2AX) at 57 μg mL^−1	Vatan 2022 (ref. 56)

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Mouse lungs in vivo
Female C57BL/6 (intratracheal instillation)	20–30 nm NiFe2O4, 10–30 nm NiZnFe4O8 (NanoAmor)	14, 43, 128 μg (once)	Days 1, 3, 28	No increases in DNA strand breaks in BAL cells (comet assay)	Hadrup et al. 2020 (ref. 110)

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Exposure of human bronchial epithelial cells BEAS-2B to Fe–Ni alloy NPs induced DNA breaks, detected by the comet assay, and an increased amount of DNA DSBs as reflected by increased γH2AX-expressing cells after exposure.⁵⁶ However, Hadrup et al. did not observe increased DNA strand breaks in bronchoalveolar lavage (BAL) cells by the comet assay after female C57BL/6 mice were intratracheally instilled with NiFe₂O₄ or NiZnFe₄O₈ NPs.¹¹⁰

6. Extra-pulmonary effects of Ni-containing NPs

There are three major routes for Ni-containing NPs to enter the body: inhalation, skin contact, and digestion. Inhalation of Ni-containing NPs will cause adverse effects on the respiratory tract, which are the most widely and deeply studied, as summarized above. Ni-containing NPs have also been reported to cause skin toxicity. For example, Ni NPs, when applied on the human skin surface, caused increased nickel content into the skin and a significant permeation flux through the skin as compared with bulk nickel.¹¹⁴ Exposure to Ni NPs in the presence of lipopolysaccharide (LPS) has been reported to cause skin sensitization in mice, which is also called metal allergy, a type of allergic contact dermatitis.¹¹⁵ Ni NPs also perturbed tight junction-associated proteins via the HIF-1α/miR-29b/MMP pathway in human epidermal keratinocytes (HaCaT).¹¹⁶ ROS-mediated DNA damage and apoptosis were observed in human skin epidermal cells (A431) exposed to Ni NPs.¹¹⁷ Ni NPs induced mouse epidermal JB6 cell apoptosis through a caspase-8/AIF mediated cytochrome c-independent pathway.¹¹⁸ A 6 kDa nickel-binding molecule, a tumor-related polypeptide, was found to be synthesized by human epidermal keratinocytes (HEKa) exposed to Ni NPs.¹¹⁹

In addition, exposure of liver or gastrointestinal cells to Ni-containing NPs caused toxicity. For example, exposure of human liver cells (HepG2) to Ni, NiO, or NiFe₂O₄ NPs caused ROS generation, cell cycle arrest, apoptosis,^120–122 or transcriptomic alterations,^123,124 but the cytotoxicity in HepG2 cells was much milder as compared with that in human lung epithelial cells (A549).³⁰ Cytotoxic, genotoxic, and apoptotic effects of NiO NPs were also observed in human intestinal epithelial cells (Caco-2).¹²⁵ Moreover, Ni-containing NPs can also cause cytotoxicity in other types of cells including monocytes,^126–128 neutrophils,¹²⁹ lymphocytes,¹³⁰ RBCs,¹³¹ endothelial progenitor cells,^132,133 endothelial cells,^134,135 spermatogonial cells (GC-1 spg),¹³⁶ trophoblasts (HTR-8/SVneo),^137,138 Sertoli-germ cells,¹³⁹ mesenchymal stem cells,¹⁴⁰ adipose stem cells,¹⁴¹ pleural mesothelial 2 cells (NRM2),¹⁴² kidney cells (NRK-52E),¹⁴³ skeletal myoblasts (L-6),¹⁴⁴etc.

Different from standard-sized particles, nanoparticles, because of their small sizes, can enter the circulation from their portal of entry and translocate to other organs, causing toxicity to other tissues or systemic toxicity. For example, oral administration of Ni or NiO NPs caused toxicity in the rat liver, kidneys, thymus, brain, immune system, RBCs, reproductive organs, etc.^145–153 and also induced significant increases in chromosomal aberrations, micronucleus formation, and DNA damage in the rat liver.¹⁵⁴ Oropharyngeal aspiration of Ni NPs induced increased mRNA expression of IL-6 (acute) and CCL-2 (chronic) in the mouse liver.⁶⁷ Inhaled Ni NPs by mice caused vascular reactivity¹⁵⁵ and rapid doubling of Alzheimer's amyloid-beta40 and 42 levels in brains.¹⁵⁶ Inhalation exposure of rats to NiO NPs caused systemic toxicity.⁸⁵ Intratracheal instillation of Ni or NiO NPs induced activation of peripheral blood monocytes and neutrophils,^126,129 and induced liver toxicity,¹⁵⁷ hepatocyte apoptosis,¹⁵⁸ hepatic fibrosis,^159,160 disturbance of bile acid metabolism,¹⁶¹ and reproductive toxicity.¹⁶² Intraperitoneal injection of Ni or NiO NPs caused toxicity in the rodent liver,¹⁶³ kidneys,¹⁶⁴ brain,^165,166 spleen,¹⁶⁷ heart,¹⁶⁸ and reproductive system.¹⁶⁹ In addition, time- and dose-dependent increased translocation of NiO NPs from the lungs to the thoracic lymph nodes has been reported.¹⁰⁶ Oral gavage of Ni NPs caused significantly higher concentrations of nickel in the female rat ovary as compared with control (saline) and nickel microparticle-treated groups.¹⁵³ However, whether these extra-portal-of-entry effects are induced by direct or indirect contact of Ni-containing NPs needs further investigation, since their ions, cytokines, chemokines, etc., produced at their portal of entry, may also enter the circulation to cause toxicity in other organs or tissues. For example, Kang et al. found that inhalation of Ni(OH)₂ caused increased serum amyloid P (SAP), which induced systemic inflammation.¹⁷⁰

7. The main culprit of the pulmonary effects of Ni-containing NPs: NPs or ions?

Many factors may influence the pulmonary toxic effects of Ni-containing NPs, including the size, shape, and dose of the NPs,^{19,34,78,81,106,109} the chemical toxicity of the native material,^107,109 the different sensitivity of the cells to the particles,^20,31 the pulmonary clearance rate of the particles,¹⁰⁶etc. However, whether the observed pulmonary effects were through NP–cell interactions or induced by soluble ions released from the NPs is still argued.

Many studies have shown that it is the NPs themselves that play a crucial role in their pulmonary toxicity, which is mainly based on the following reasons. (1) The solubility of studied Ni-containing NPs, such as Ni or NiO NPs, is low. It was about 16 ppm (∼1.6%) in 1xPBS and 81 ppm (∼8.1%) in the cell culture medium (RPMI-1640) after 1 mg mL⁻¹ Ni NPs were incubated at 37 °C for 48 h.¹²⁷ Only approximately 1–3% (wt%) of Ni was released into the cell culture medium (DMEM) following 4 and 24 h incubation of 10 mg Ni mL⁻¹ Ni or NiO NP suspension at 37 °C.¹⁹ Akerlund et al. showed that, on average, approximately 2% (Ni NPs, wt%) and 6% (NiO NPs, wt%) of Ni were released in the medium following 18 h incubation of NPs in serum-free cell culture medium (RPMI/LHC-9) at 37 °C.¹¹¹ In the conditioned media collected from THP-1* cells exposed to Ni or NiO NPs for 3 h and 18 h, the percentage of Ni released after 3 h was 1.4% (Ni NPs) and 2.3% (NiO NPs), and this increased to approximately double that after 18 h (2.3% for Ni NPs and 5.5% for NiO NPs).²² Capasso et al. reported that the Ni²⁺ release from NiO NPs was negligible in the cell-free systems since the ion concentration was always lower than 0.04 μg mL⁻¹. The amount of Ni²⁺ released into cell culture supernatants, collected after 24 h exposure to 100 μg mL⁻¹ NiO NPs, was 2.8 μg mL⁻¹ (2.8%) in A549 and 4.2 μg mL⁻¹ (4.2%) in BEAS-2B cells.³¹ (2) Some NP-induced effects were not observed in cells treated with easily soluble NiCl₂. For example, both Ni and NiO NPs caused DNA damage in HBECs, but NiCl₂ did not.¹¹¹ (3) Some effects could be induced by NPs, but not by their aqueous extracts (AEs). Cho et al. reported that exposure to 300 cm² mL⁻¹ NiO NPs, but not their NiO AEs, caused cytotoxicity and increased IL-8 release in A549 cells. NiO NPs also recruited neutrophils in rat lungs and caused significant increases in LDH, total protein, MIP-2, and IL-1β in the BALF, but NiO AEs did not.³³ (4) Human lung cells can efficiently take up Ni-containing NPs, but not extracellular Ni ions,²⁴ and the lungs can rapidly clear water-soluble Ni species.¹⁷¹

However, others reported that ionic nickel mobilized from Ni-containing NPs may mediate or contribute to their toxicity. An earlier study by Pietruska et al. showed that approximately 50% of the nickel in NiO NPs was mobilized within 24 h incubation in the cell culture medium (identical RPMI-1640 as for cell culture).²⁰ Similar to soluble NiCl₂, NiO NPs induced stabilization and nuclear translocation of HIF-1α, and were equally toxic to human lung epithelial cells, suggesting that ionic nickel mobilized from NiO NPs activates the HIF-1α pathway.²⁰ However, Ni NPs, from which only approximately 1–3% of the nickel was mobilized, caused HIF-1α pathway activation that was stronger than that induced by NiCl₂.²⁰ Thus, it is still premature to conclude that the NiO NP-induced HIF-1α pathway activation is induced by mobilized Ni²⁺. Gliga et al. reported that both Ni or NiO NPs and soluble NiCl₂ caused changes in gene transcriptional expression and signaling pathways in human bronchial epithelial cells BEAS-2B, although cells hardly take up nickel ions, suggesting that these effects may be mediated via cell membrane receptors and downstream signaling.²⁸ In addition, mobilized Ni²⁺ has also been shown to play an essential role in adverse effects induced by Ni-containing NPs in vivo. Nishi et al. found that it took about one week to dissolve NiO NPs in artificial lysosomal fluid (ALF) (pH 4.5) but one month or more in vivo, suggesting that NiO NPs dissolve slowly in the phagolysosomes of rat alveolar macrophages (AMs) when intratracheally instilled, and the resulting Ni²⁺ causes the AMs to transform into foamy cells and induces a change in inflammatory response over time.¹⁰⁴ Horie et al. demonstrated that NiO NPs, but not insoluble nano and fine TiO₂ particles, caused rat lung injury and oxidative stress after intratracheal instillation, suggesting that Ni²⁺ release is an important factor in the NiO NP-induced lung injury and oxidative stress.¹⁰²

Furthermore, some pulmonary effects may be induced by NPs and some by their dissociated components, i.e., nickel(II) ions. For example, the pulmonary inflammation induced by four kinds of NiO NPs with different solubility was compared after intratracheal instillation into rats. The results showed that wire-like NiO NPs (B) with 100% solubility in artificial lysosomal fluid (ALF) within 24 h caused the most severe pulmonary inflammation, but the response was transient. The second-most-soluble material (D) and the third one (A) caused evident pulmonary inflammation, and the responses persisted for at least 91 days with collagen proliferation. In contrast, the most insoluble NiO (C) induced barely detectable inflammation and no marked histopathological changes. These results indicate that the early phase toxic potential of NiO products, but not the persistence of pulmonary inflammation, is associated with their solubility (see Table 7).^77,106

Taken together, it seems that both the interactions between Ni-containing NPs and cells and the nickel ions mobilized from NPs may play roles in various NP-induced biological effects. Different effects may be mediated by either NPs or ions, or both. Further studies are needed to clarify this issue.

8. Conclusions and perspectives

In summary, previous in vitro studies have shown that exposure of human or non-human mammalian lung cells to Ni-containing NPs may cause multiple cytotoxicity (Fig. 1), including (1) decreased cell viability and increased cell necrosis, apoptosis, ferroptosis, or autophagy, (2) suppression of cell proliferation, a decreased replication index and mitotic index, and alteration of the cell cycle, (3) inflammation, (4) lung epithelial cells to undergo epithelial-to-mesenchymal transition (EMT) and increased collagen expression, (5) genotoxicity including DNA damage, DNA repair defects, chromosomal damage and rearrangements, and (6) lung epithelial cells from normal to malignant transformation. The following mechanisms may be involved in Ni-containing NP-induced adverse effects: (1) oxidative or nitrative stress; (2) increased cytosolic calcium (Ca²⁺) concentration or cathepsin B; (3) alteration of signaling pathways including HIF-1α, TGF-β1/Smad, PI3K/AKT, MAPKs, STAT3, C/EBPβ, NF-κB, SIRT1, ATF3, Hh, JNK/c-Jun, ATM/ATR, leukocyte extravasation, etc.; and (4) epigenetic changes such as upregulation of miR-21 and miR-210, histone hypoacetylation, PRKCB DNA hypomethylation, downregulation of lncRNA MEG3, HOTAIRM1, or AP000487.1, etc. (Fig. 1 and 3).

The results of in vitro studies well reflected many symptoms and effects observed in in vivo studies (Fig. 2 and 3) or in humans after exposure to Ni-containing NPs, including cell injury, inflammation, fibrosis, genotoxicity, etc. Thus, using in vitro models to study the signaling pathways or mechanisms involved in these effects is recommended in order to replace, reduce, and refine (3Rs) the use of in vivo experimentation, which is especially important and necessary in the field of nanomaterials.¹⁷² The transformation of lung epithelial cells from normal to malignant phenotypes after Ni NP exposure was observed in in vitro studies but has not been observed in animal studies or in humans. However, the increased risk of lung and nasal cancers by exposure to standard-sized Ni has been confirmed by epidemiological studies.¹¹ In contrast, the allergic airway inflammation or delayed-type hypersensitivity (DTH) response after Ni-containing NP exposure was observed in in vivo animal studies and in humans but not in in vitro studies, due mainly to the lack of simple and appropriate in vitro experimental models to accurately reflect the human in vivo system. Thus, testing the allergenicity of Ni-containing NPs relies heavily on the use of animals. Although the adverse effects of Ni-containing NPs have been widely investigated not only on the respiratory system, but also on the skin, digestive tract, reproductive tract, systemic, etc., there are still knowledge gaps in this field. The mechanisms underlying various Ni-containing NP-induced adverse effects are still obscure and need further investigation.

In addition, as compared to bulk particles, Ni-containing NPs have been confirmed to cause much more severe toxicity, as evidenced by both in vitro and in vivo studies. For example, Ni NP exposure caused rapid and prolonged activation of the HIF-1α pathway in human lung epithelial cells (H460), but Ni microparticles did not.²⁰ Crosera et al. showed that human skin absorption of Ni NPs was higher compared to bulk nickel,¹¹⁴ and Ni NPs caused higher cytotoxicity and apoptotic induction than fine particles in mouse epidermal JB6 cells.¹¹⁸ Magaye et al. also showed that Ni NPs were more potent in causing cell toxicity and genotoxicity in vitro than fine particles.¹⁸ Moreover, intratracheal instillation of Ni or NiO NPs caused a much more toxic effect in rat lungs than fine particles.^69,102 On the other hand, Ni-containing NPs, because of their small sizes, can enter the circulation from their portal of entry and translocate to other organs or tissues, causing extra-portal-of-entry toxicity, as described above in Section 6. Thus, preventive measures are needed when Ni-containing NPs are produced and used due to their higher potential to enter the body than bulk particles. However, occupational exposure limits for Ni-containing NPs are still undefined. Furthermore, the toxic effects of Ni-containing NPs and the specific aspects of engineering controls to produce NPs with controlled or reduced toxicity need to continue to be explored. The general guide to safe practices when working with Ni-containing NPs in either worksite or lab environments also needs to continue to be researched.

Abbreviations

(1) Company abbreviations

Abbreviation	Full name	Address
Aladdin	Shanghai Aladdin Bio-Chem Technology Co., Ltd.	Shanghai, China
Danyang	Danyang City Alloy and Steel Refinery Co., Ltd.	Jiangsu, China
InabVacu	Inabata & Co., Ltd., Vacuum Metallurgical Co., Ltd.	Osaka, Japan
Kusaka	Kusaka Rare Metal Products Co., Ltd.	Tokyo, Japan
Nakalai	Nakalai Chemicals Ltd.	Kyoto, Japan
NanoAmor	Nanostructured & Amorphous Materials, Inc.	Houston, TX, USA
Nanoshel	Nanoshel, LLC	County Cavan, Ireland
Novarials	Novarials Corporation	Woburn, MA, USA
Sigma	Sigma-Aldrich	St. Louis, MO, USA
ST-Nano	ST-Nano Science and Technology Co., Ltd.	Shanghai, China
Sun-Inno	Sun Innovations, Inc.	Fremont, CA, USA
US-Nano	US Research Nanomaterials, Inc.	Houston, TX, USA
VacuMeta	Vacuum Metallurgical Co., Ltd.	Chiba, Japan

(2) Other abbreviations

ALF	Artificial lysosomal fluid (pH 4.5)
AM	Alveolar macrophage
AP-1	Activator protein 1
ARDS	Adult respiratory distress syndrome
αSMA	α-Smooth muscle actin
ATF3	Activating transcription factor 3
ATM	Ataxia telangiectasia mutated
ATR	RAD3-related
BAL	Bronchoalveolar lavage
BALF	Bronchoalveolar lavage fluid
Bax	Bcl-2-associated X protein
BNMN	Binucleated micronucleated
CAT	Catalase
CBMN assay	Cytokinesis-block micronucleus assay
CCL2 (MCP-1)	Chemokine ligand 2
C/EBPβ	CCAAT/enhancer-binding protein beta
CINC-1/2αβ/3	Cytokine-induced neutrophil chemoattractant-1/2αβ/3 (CXCL-1/3/2)
C-myc	Cellular myc
COL1A1/3A1	Collagen, type I, alpha 1/ type III, alpha1
CTGF	Connective tissue growth factor
CuO	Copper oxide
CXCL-1 (KC)	C-X-C motif ligand 1 (keratinocyte-derived cytokine)
DSBs	(DNA) double strand breaks
EMT	Epithelial-mesenchymal transition
ERK1/2	Extracellular signal-regulated kinase 1/2
GSH	Glutathione
HDAC3	Histone deacetylase 3
Hh pathway	Hedgehog pathway
HIF-1α	Hypoxia-inducible factor-1 subunit alpha
HO-1	Heme oxygenase-1
HOTAIRM1	HOXA transcript antisense RNA myeloid-specific 1
HPRT	Hypoxanthine phosphoribosyltransferase 1
IARC	International Agency for Research in Cancer
IFN-γ	Interferon-gamma
IgE	Immunoglobulin E
IHC	Immunohistochemistry
IL	Interleukin
iNOS	Inducible nitric oxide synthase
JNK/c-Jun	c-Jun N-terminal kinase/c-Jun
LC3	Microtubule-associated protein light-chain 3
LDH	Lactate dehydrogenase
lncRNA	Long noncoding RNA
LOAEL	Lowest observed adverse effect level
LPS	Lipopolysaccharide
MAPK	Mitogen activated protein kinase
MCP-1 (CCL2)	Monocyte chemoattractant protein-1
MEG3	Maternally expressed gene 3
MF	Mutant frequency
MIP-2/1α	Macrophage inflammatory protein-2/1α (MIP-2 = CXCL-2)
miR-21/210	microRNA-21/210
MMP	Matrix metalloproteinase
MPO	Myeloperoxidase
MRI	Magnetic resonance imaging
MSRA	Methionine sulfoxide reductase A
MSRB3	Methionine sulfoxide reductase B3
MT-1	Metallothionein-1
mTOR	Mammalian target of rapamycin
MTS assay	CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay
MTT	3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
NDRG1	N-myc downstream-regulated gene 1
NF-κB	Nuclear factor kappa-light-chain-enhancer of activated B cells
NLRP3	NOD-, LRR- and pyrin domain-containing protein 3
Ni	Nickel
NiO	Nickel oxide
Ni(OH)2	Nickel(II) hydroxide
NiSO4	Nickel sulfate
NO	Nitric oxide
NOAEL	No observed adverse effect level
NPs	Nanoparticles
Nrf2	Nuclear factor-erythroid factor 2-related factor 2
8-OHdG	8-Hydroxy-2′-deoxyguanosine
OVA	Ovalbumin
PCNA	Proliferating cell nuclear antigen
PGF2	Prostaglandin F2
PI3K/AKT	Phosphoinositide 3 kinase (PI3K)/protein kinase B
PMNs	Polymorphonuclear leukocytes
PRKCB	Protein kinase C beta type
Rad52	Radiation sensitive 52
ROS	Reactive oxygen species
SD rat	Sprague Dawley rat
SIRT1	Sirtuin 1
Smad	Suppressor of mothers against decapentaplegic
SOD	Superoxide dismutase
SP-D	Surfactant protein D
STAT1/3	Signal transducer and activator of transcription 1/3
T-AOC	Total antioxidant capacity
T-bet	T-box transcription factor Tbx21
TGF-β1	Transforming growth factor-β1
Th1/2/17	T-helper type 1/2/17
tHODE	Total hydroxyoctadecanoic acid
TIMP-1	Tissue inhibitor of matrix metalloproteinase 1
TNFα	Tumor necrosis factor-alpha
TNOS	Total nitric oxide synthases
γ-GTP	γ-Glutamyl transpeptidase
γH2AX	Phosphorylated H2A histone family member X
WB	Western blot

Author contributions

Conceptualization: Y. M., Y. Z. and Q. Z.; writing – original draft: Y. M. and Y. Z.; writing – review & editing: Y. M., Y. Z. and Q. Z.; funding acquisition: Q. Z.; supervision: Q. Z.

Conflicts of interest

The authors report no conflicts of interest.

Acknowledgements

This work was partly supported by grants from NIH (ES023693, ES028911 and HL147856), KSEF-148-RED-502-16-381, and the Kentucky Lung Cancer Research Program to Dr. Qunwei Zhang. This work was also partly supported by P30ES030283 from the National Institute for Environmental Health Sciences.

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