Influence of Titanium Dioxide Nanoparticles on Human Health and the Environment

Nanotechnology has enabled tremendous breakthroughs in the development of materials and, nowadays, is well established in various economic fields. Among the various nanomaterials, TiO2 nanoparticles (NPs) occupy a special position, as they are distinguished by their high availability, high photocatalytic activity, and favorable price, which make them useful in the production of paints, plastics, paper, cosmetics, food, furniture, etc. In textiles, TiO2 NPs are widely used in chemical finishing processes to impart various protective functional properties to the fibers for the production of high-tech textile products with high added value. Such applications contribute to the overall consumption of TiO2 NPs, which gives rise to reasonable considerations about the impact of TiO2 NPs on human health and the environment, and debates regarding whether the extent of the benefits gained from the use of TiO2 NPs justifies the potential risks. In this study, different TiO2 NPs exposure modes are discussed, and their toxicity mechanisms—evaluated in various in vitro and in vivo studies—are briefly described, considering the molecular interactions with human health and the environment. In addition, in the conclusion of this study, the toxicity and biocompatibility of TiO2 NPs are discussed, along with relevant risk management strategies.


Introduction
TiO 2 is one of the most abundant and widely used metal oxide nanomaterial in the world [1,2]. As an n-type semiconductor, with a band gap energy of 3.2-3.35 eV, depending on its crystal phase, TiO 2 acts as an effective photocatalyst during the photocatalytic process for surface functionalization [3][4][5][6]. It has three crystalline structures: anatase, rutile, and brookite. Anatase is the most common type and rutile is the most stable form, while brookite is the rarest [7]. There is also amorphous TiO 2 , which is a non-crystalline form. TiO 2 exists in various nanostructures, such as nanoparticles, nanotubes, nanorods and nanowires, nanofilms, nanosheets, and nanocoatings, with remarkable photocatalytic activity, which attract scientists to develop potential technological applications in multidisciplinary fields for industrial production [8].
TiO 2 is widely used in food, paints, plastics, printing inks, papers, and biomedical and cosmetic products. Due to its unique properties, TiO 2 is also advantageously used in textiles. For example, when applied to textile fibers, it confers various functionalities such as UV protection, photocatalytic self-cleaning and antimicrobial activity, electrical conductivity, and antistatic properties, as well as increased thermal stability. Moreover, it can also be used for solar energy conversion in the production of electronic textiles or wearable electronics. However, when tailoring its desired functionality, TiO 2 is usually deposited on the surface of textile fibers, but due to its fairly poor adhesion to the fibers, a certain amount of TiO 2 washes away, peels off, or wears off during the product's lifetime. Therefore, such continuous leaching of TiO 2 into the environment results in a potential threat to human health and the ecosystem.

Cytotoxicity
It is well known that ROS (i.e., superoxide (O 2 •− ), hydrogen peroxide (H 2 O 2 ), and the hydroxyl radical (OH − )) are produced by aerobic organisms within the cell and are normally in equilibrium with antioxidant molecules (Figure 1a) [51]. The imbalance between ROS and antioxidants (AOX) caused by the excessive production of ROS or the depletion of antioxidant molecules leads to the occurrence of oxidative stress (Figure 1b,c) [51].

Oral Route
TiO2 NPs can enter the blood via the oral route as they are used in foods, personal care products, sunscreens, and toothpaste. TiO2 NPs remain in the major organs for a long time and are eventually excreted in the stool. Absorbed TiO2 NPs in the liver, spleen, kidney, and lung tissues are responsible for possible nephrotoxicity and liver damage [41]. Even after the prolonged release of NPs, they can affect living organisms in the environment via biological pathways [42].

Dermal Route
Human skin has unique barrier properties that act against the penetration of TiO2 NPs into the skin. Several studies have found that TiO2 NPs cannot penetrate through the human dermis, even when their particle size is smaller than 100 nm [43]. Moreover, other studies have revealed that the TiO2 NPs that do manage to penetrate the skin do not exhibit toxicity under certain conditions [44].

Injection
TiO2 NPs can be developed as a highly efficient photothermal, medicinal, and synergistic agent to repair/remove diseased tissues such as cancer both in vitro and in vivo with negligible toxic properties, tissue damage, and kidney/liver dysfunction [45,46]. After injection of TiO2 NPs, endothelial cells can be disrupted, and they can proliferate and migrate despite the induction of toxicity and immunogenicity. It has been shown that TiO2 NPs injected into the blood during photothermal cancer therapy enrich the cancer tissue by diffusing, engulfing the cancer cells, and eventually killing them completely [47][48][49].

Toxicity of TiO2 NPs
There is a lack of information on the potential toxicity of TiO2 NPs. Indeed, despite numerous studies in this field, it is difficult to find separate studies using the same TiO2 NPs with the same experimental protocol to compare the results obtained. Nevertheless, the existing data support the potential toxicity of TiO2 NPs in humans, model vertebrates and invertebrates, plants, algae, and microorganisms. The mechanism of toxicity of TiO2 NPs to organisms can be outlined as follows: (i) production of reactive oxygen species (ROS) and formation of electron-hole pairs in the presence of light; (ii) binding of TiO2 NPs to the cell membrane via electrostatic interactions, resulting in cell wall damage and peroxidation of lipids in the cell membrane; and (iii) binding of TiO2 NPs to intracellular organelles and biological macromolecules [50].

Cytotoxicity
It is well known that ROS (i.e., superoxide (O2 •-), hydrogen peroxide (H2O2), and the hydroxyl radical (OH -)) are produced by aerobic organisms within the cell and are normally in equilibrium with antioxidant molecules (Figure 1a) [51]. The imbalance between ROS and antioxidants (AOX) caused by the excessive production of ROS or the depletion of antioxidant molecules leads to the occurrence of oxidative stress (Figure 1b   As for the negative biological effects, oxidative stress is the most important process involved in the formation of TiO 2 NPs-induced ROS [52]. High TiO 2 concentrations cause greater oxidative stress, which correlates with the increase in lipid peroxidation at the cell membrane after TiO 2 NPs adsorption on the cell membrane. TiO 2 NPs-induced ROS Nanomaterials 2021, 11, 2354 4 of 20 and lipid peroxidation damage the integrity of the cell wall and membrane, resulting in increased permeability [53], which allows TiO 2 NPs to enter the cell. In addition to free diffusion, TiO 2 NPs can also enter the cell through the process of endocytosis. While particles larger than 500 nm can be removed by phagocytes [50], smaller particles can be engulfed by a cell membrane vesicle and taken further into the cell. Accordingly, TiO 2 NPs with a size of 25 nm and less have been taken up into human keratinocytes [54], as well as lung cells [52,55], lymphocytes [56], macrophages [57], keratinocytes [58], and hepatocytes [59] after endocytosis. It is also interesting to note the penetration ability of TiO 2 particles with a size of 200 nm into red blood cells, which were chosen as a model for non-phagocytic cells. The results demonstrated the ability of the particles to penetrate the red blood cell membrane by a mechanism other than phagocytosis and endocytosis [60]. In this case, the penetration mechanism remained unexplained. Upon entrance of TiO 2 NP into the cell, the internalized TiO 2 NPs are transported to lysosomes, where they generate lysosomal stress and release cytosol that reacts with cellular components, resulting in DNA damage, DNA rearrangement, altered gene expression, oxidative stress, and inflammation ( Figure 2) [28,56,[61][62][63]. TiO 2 NPs impair micromolecular functions by protein adsorption, blocking signaling pathways, and binding to DNA structure [64,65]. Undoubtedly, such damage affects cell viability [66] and is dose and time dependent [67,68]. As for the negative biological effects, oxidative stress is the most important process involved in the formation of TiO2 NPs-induced ROS [52]. High TiO2 concentrations cause greater oxidative stress, which correlates with the increase in lipid peroxidation at the cell membrane after TiO2 NPs adsorption on the cell membrane. TiO2 NPs-induced ROS and lipid peroxidation damage the integrity of the cell wall and membrane, resulting in increased permeability [53], which allows TiO2 NPs to enter the cell. In addition to free diffusion, TiO2 NPs can also enter the cell through the process of endocytosis. While particles larger than 500 nm can be removed by phagocytes [50], smaller particles can be engulfed by a cell membrane vesicle and taken further into the cell. Accordingly, TiO2 NPs with a size of 25 nm and less have been taken up into human keratinocytes [54], as well as lung cells [52,55], lymphocytes [56], macrophages [57], keratinocytes [58], and hepatocytes [59] after endocytosis. It is also interesting to note the penetration ability of TiO2 particles with a size of 200 nm into red blood cells, which were chosen as a model for non-phagocytic cells. The results demonstrated the ability of the particles to penetrate the red blood cell membrane by a mechanism other than phagocytosis and endocytosis [60]. In this case, the penetration mechanism remained unexplained.
Upon entrance of TiO2 NP into the cell, the internalized TiO2 NPs are transported to lysosomes, where they generate lysosomal stress and release cytosol that reacts with cellular components, resulting in DNA damage, DNA rearrangement, altered gene expression, oxidative stress, and inflammation ( Figure 2) [28,56,[61][62][63]. TiO2 NPs impair micromolecular functions by protein adsorption, blocking signaling pathways, and binding to DNA structure [64,65]. Undoubtedly, such damage affects cell viability [66] and is dose and time dependent [67,68]. It should also be noted that the response of cells in the presence of TiO2 NPs is complex. Thus, the presence of TiO2 NPs can modulate different cell fates, including necrosis and apoptosis, which are regular cell death pathways, or autophagy, which leads to either cytoprotective mechanisms or cell death ( Figure 3) [69]. TiO2 NP-induced autophagy can be exploited in new therapeutic pathway treatment of various diseases (see Section 6). It should also be noted that the response of cells in the presence of TiO 2 NPs is complex. Thus, the presence of TiO 2 NPs can modulate different cell fates, including necrosis and apoptosis, which are regular cell death pathways, or autophagy, which leads to either cytoprotective mechanisms or cell death ( Figure 3) [69]. TiO 2 NP-induced autophagy can be exploited in new therapeutic pathway treatment of various diseases (see Section 6).

Genotoxicity
The genotoxicity of TiO2 NPs has not yet been clarified as scientists do not have sufficient evidence of genotoxicity [70,71]. Genotoxicity refers to the ability of TiO2 NPs to disrupt genetic information by causing breaks, lesions, deletions, mis-segregation, or nondisjunction in the DNA, leading to gene mutations. In vitro testing methods, such as mammalian chromosomal aberration tests, cellular gene mutation tests and bacterial reverse mutation tests, are performed to measure genotoxicity [72,73]. In contrast, the in vivo comet assay and the in vivo micronuclei/chromosome aberration assay evaluate in vivo genotoxicity [74]. The genotoxic effect of TiO2 NPs on cells is mainly studied through the circulatory or respiratory system. Despite the crystallinity of TiO2 NPs, their genotoxicity mostly depends on their particle size. Smaller TiO2 NPs possess a stronger genotoxic effect than larger ones, as they easily penetrate into the nucleus and cytoplasm of the cell [74]. Larger agglomerations of TiO2 NPs cause DNA damage [75]. Several studies have shown the genotoxic and cytotoxic effects of TiO2 NPs on human amniotic epithelial cells [76], human lung fibroblasts [77], human lymphocytes [78], and human hepatoma HepG2 cells [79]. In vitro studies of cell-induced genotoxicity caused by TiO2 NPs due to DNA breakage and gene mutations are shown in Figure 4 [22].

Genotoxicity
The genotoxicity of TiO 2 NPs has not yet been clarified as scientists do not have sufficient evidence of genotoxicity [70,71]. Genotoxicity refers to the ability of TiO 2 NPs to disrupt genetic information by causing breaks, lesions, deletions, mis-segregation, or non-disjunction in the DNA, leading to gene mutations. In vitro testing methods, such as mammalian chromosomal aberration tests, cellular gene mutation tests and bacterial reverse mutation tests, are performed to measure genotoxicity [72,73]. In contrast, the in vivo comet assay and the in vivo micronuclei/chromosome aberration assay evaluate in vivo genotoxicity [74]. The genotoxic effect of TiO 2 NPs on cells is mainly studied through the circulatory or respiratory system. Despite the crystallinity of TiO 2 NPs, their genotoxicity mostly depends on their particle size. Smaller TiO 2 NPs possess a stronger genotoxic effect than larger ones, as they easily penetrate into the nucleus and cytoplasm of the cell [74]. Larger agglomerations of TiO 2 NPs cause DNA damage [75]. Several studies have shown the genotoxic and cytotoxic effects of TiO 2 NPs on human amniotic epithelial cells [76], human lung fibroblasts [77], human lymphocytes [78], and human hepatoma HepG2 cells [79]. In vitro studies of cell-induced genotoxicity caused by TiO 2 NPs due to DNA breakage and gene mutations are shown in Figure 4 [22].

Genotoxicity
The genotoxicity of TiO2 NPs has not yet been clarified as scientists do not have sufficient evidence of genotoxicity [70,71]. Genotoxicity refers to the ability of TiO2 NPs to disrupt genetic information by causing breaks, lesions, deletions, mis-segregation, or nondisjunction in the DNA, leading to gene mutations. In vitro testing methods, such as mammalian chromosomal aberration tests, cellular gene mutation tests and bacterial reverse mutation tests, are performed to measure genotoxicity [72,73]. In contrast, the in vivo comet assay and the in vivo micronuclei/chromosome aberration assay evaluate in vivo genotoxicity [74]. The genotoxic effect of TiO2 NPs on cells is mainly studied through the circulatory or respiratory system. Despite the crystallinity of TiO2 NPs, their genotoxicity mostly depends on their particle size. Smaller TiO2 NPs possess a stronger genotoxic effect than larger ones, as they easily penetrate into the nucleus and cytoplasm of the cell [74]. Larger agglomerations of TiO2 NPs cause DNA damage [75]. Several studies have shown the genotoxic and cytotoxic effects of TiO2 NPs on human amniotic epithelial cells [76], human lung fibroblasts [77], human lymphocytes [78], and human hepatoma HepG2 cells [79]. In vitro studies of cell-induced genotoxicity caused by TiO2 NPs due to DNA breakage and gene mutations are shown in Figure 4 [22].  Genotoxicity occurs via direct or indirect genotoxicity mechanisms. Namely, TiO 2 NPs can enter the nucleus, inducing direct DNA damage through direct contact with DNA and chromosome, while indirect genotoxicity of TiO 2 NPs results from the increased lysosomal release of DNases, the formation of nanoaggregates that can extrude nucleus or by ROS accumulation. Moreover, TiO 2 NPs can also negatively influence the repair process of DNA. In vitro studies showed the genotoxicity of TiO 2 NPs during short-term exposure, which may be triggered by smaller particle sizes and mixed phases of TiO 2 NPs [22].

Immune System
The immune system defends the body against foreign antigens. If nanoparticles are recognized as foreign substances, they are eliminated by the immune system. If, on the other hand, the foreign substances are not recognized as a threat, they are either ignored or tolerated by the immune system [83]. The effects of TiO 2 NPs on immune function are poorly documented. Underlying molecular mechanism by which TiO 2 NPs influence the immune cell was studied in relation to TLRs, which are a subfamily of pattern recognition receptors, placed strategically on the cell surface and endosome of primary immune cell macrophages [84].TiO 2 -induced immunotoxicity was proven, which occurred through the activation of several receptors, which further activated specific signaling pathways to reduce the antioxidants through the formation of ROS. Upon increased ROS exposure, the mitochondrial membrane potential (∆ψm) reduced, ultimately leading to apoptotic cell death, and inducing immunotoxicity through immune redox imbalance ( Figure 5). However, in vivo experiments are essential to further validate TiO 2 NPs influence on the immune system. Genotoxicity occurs via direct or indirect genotoxicity mechanisms. Namely, TiO2 NPs can enter the nucleus, inducing direct DNA damage through direct contact with DNA and chromosome, while indirect genotoxicity of TiO2 NPs results from the increased lysosomal release of DNases, the formation of nanoaggregates that can extrude nucleus or by ROS accumulation. Moreover, TiO2 NPs can also negatively influence the repair process of DNA. In vitro studies showed the genotoxicity of TiO2 NPs during short-term exposure, which may be triggered by smaller particle sizes and mixed phases of TiO2 NPs [22].

Immune System
The immune system defends the body against foreign antigens. If nanoparticles are recognized as foreign substances, they are eliminated by the immune system. If, on the other hand, the foreign substances are not recognized as a threat, they are either ignored or tolerated by the immune system [83]. The effects of TiO2 NPs on immune function are poorly documented. Underlying molecular mechanism by which TiO2 NPs influence the immune cell was studied in relation to TLRs, which are a subfamily of pattern recognition receptors, placed strategically on the cell surface and endosome of primary immune cell macrophages [84].TiO2-induced immunotoxicity was proven, which occurred through the activation of several receptors, which further activated specific signaling pathways to reduce the antioxidants through the formation of ROS. Upon increased ROS exposure, the mitochondrial membrane potential (Δψm) reduced, ultimately leading to apoptotic cell death, and inducing immunotoxicity through immune redox imbalance ( Figure 5). However, in vivo experiments are essential to further validate TiO2 NPs influence on the immune system.

Neural System
TiO 2 NPs can enter the brain regions by translocation across the blood-brain barrier or thenose-brain barrier, and progeny across the placental barrier, which may be the reason for the dysfunction and potential risks to the central nervous system [32,[85][86][87]. Long-term exposure to TiO 2 NPs can cause damages to the neurons and glial cells (U373), which may subsequently lead to neurotoxicity [32,88], also at low doses. In Figure 6, it is shown that the oral ingestion of TiO 2 NPs can affect brain activities via increased oxidative stress, decreased antioxidant enzyme activity, and increased nitric oxide (NO) and ROS release ( Figure 6) [89]. TiO 2 NPs thus induced a neurotoxic damage accompanied by the increase in degenerated and apoptotic neurons in cerebral cortex.

Neural System
TiO2 NPs can enter the brain regions by translocation across the blood-brain barrier or thenose-brain barrier, and progeny across the placental barrier, which may be the reason for the dysfunction and potential risks to the central nervous system [32,[85][86][87]. Longterm exposure to TiO2 NPs can cause damages to the neurons and glial cells (U373), which may subsequently lead to neurotoxicity [32,88], also at low doses. In Figure 6, it is shown that the oral ingestion of TiO2 NPs can affect brain activities via increased oxidative stress, decreased antioxidant enzyme activity, and increased nitric oxide (NO) and ROS release ( Figure 6) [89]. TiO2 NPs thus induced a neurotoxic damage accompanied by the increase in degenerated and apoptotic neurons in cerebral cortex.

Cardiovascular System
TiO2 NPs of 100 nm significantly reduce mitochondrial 'dehydrogenase activity' in human lymphocyte cells [81]. Mitochondria-mediated apoptosis-based cell death due to DNA damage is induced by TiO2 NPs. When TiO2 NPs react with hemoglobin, they can weaken red blood cell oxygen transport. Kongseng et al. investigated the cytotoxic effects of TiO2 NPs on human blood cells, namely peripheral blood mononuclear cells (PBMCs). TiO2 NPs incubated for 24 h significantly suppressed cell viability and increased the formation of toxic mediators (Figure 7) [10]. At high TiO2 NP concentrations (≥ 25 μg mL −1 ), cell apoptosis and the 'proinflammatory cytokine secretion' of PBMCs increased due to oxidative stress caused by ROS. The effect of TiO2 NPs and bulk material in PBMCs, neopterin formation and tryptophan degradation was studied by Becker et al. [90]. Neopterin production was increased in both unstimulated and stimulated PBMCs, while tryptophan breakdown was suppressed, thus suggesting that the total effect of TiO2 NPs was strongly pro-inflammatory. Namely, in human body fluids, such response is detected in diseases such as infections and cancer, and is also parallel to the course of atherogenesis and neurodegeneration [90].

Cardiovascular System
TiO 2 NPs of 100 nm significantly reduce mitochondrial 'dehydrogenase activity' in human lymphocyte cells [81]. Mitochondria-mediated apoptosis-based cell death due to DNA damage is induced by TiO 2 NPs. When TiO 2 NPs react with hemoglobin, they can weaken red blood cell oxygen transport. Kongseng et al. investigated the cytotoxic effects of TiO 2 NPs on human blood cells, namely peripheral blood mononuclear cells (PBMCs). TiO 2 NPs incubated for 24 h significantly suppressed cell viability and increased the formation of toxic mediators (Figure 7) [10]. At high TiO 2 NP concentrations (≥ 25 µg mL −1 ), cell apoptosis and the 'proinflammatory cytokine secretion' of PBMCs increased due to oxidative stress caused by ROS. The effect of TiO 2 NPs and bulk material in PBMCs, neopterin formation and tryptophan degradation was studied by Becker et al. [90]. Neopterin production was increased in both unstimulated and stimulated PBMCs, while tryptophan breakdown was suppressed, thus suggesting that the total effect of TiO 2 NPs was strongly pro-inflammatory. Namely, in human body fluids, such response is detected in diseases such as infections and cancer, and is also parallel to the course of atherogenesis and neurodegeneration [90].
In contrast, no significant DNA damage caused to human peripheral blood lymphocytes treated with TiO 2 NPs was observed by an alkaline assay. Only extremely higher concentrations (100 µg) can show the genotoxicity of TiO 2 [91].
Very little research has been completed on the effect of TiO 2 NPs on the cardiovascular system. Researchers suspect that deposited TiO 2 NPs in the heart may lead to inflammatory responses, system malfunction, and cardiac damage. Depending on the type and duration of exposure, ultrafine TiO 2 NPs could lead to an increase in heart rate, blood pressure, and cardiac muscle damage [32]. In contrast, no significant DNA damage caused to human peripheral blood lymphocytes treated with TiO2 NPs was observed by an alkaline assay. Only extremely higher concentrations (100 μg) can show the genotoxicity of TiO2 [91].
Very little research has been completed on the effect of TiO2 NPs on the cardiovascular system. Researchers suspect that deposited TiO2 NPs in the heart may lead to inflammatory responses, system malfunction, and cardiac damage. Depending on the type and duration of exposure, ultrafine TiO2 NPs could lead to an increase in heart rate, blood pressure, and cardiac muscle damage [32].

Respiratory System
The lungs are the main target system of environmental air pollution via TiO2 NPs [92]. TiO2-induced human lung epithelial cell injury (A549) and alveolar lung inflammation have been reported after inhalation in several studies [93][94][95]. These phenomena lead to lung dysfunction, as well as irreversible changes to the cells, resulting in fibrosis and tumor development. The lung toxicity and inflammatory effects are related to the properties of TiO2 NPs, such as their size, shape, crystallinity, agglomeration, and surface coating mode [96]. At this point it should be noted that the cytotoxicity of TiO2 NPs increased in the following order: amorphous > anatase > anatase/rutile; thus, amorphous TiO2 NPs possessed greater toxic effect than anatase/rutile TiO2 NPs [95]. Fresegna et al. studied the cellular responses of human alveolar A549 and bronchial BEAS-2B cells to measure the cytotoxic and inflammatory effects of TiO2 NPs in anatase and rutile forms [7]. They found that anatase TiO2 NPs exerted greater cytotoxicity on bronchial cells compared to rutile TiO2 NPs. On the other hand, a higher level of genotoxicity was observed on alveolar and bronchial cells treated with rutile TiO2 compared to anatase TiO2. When TiO2 NPs are preirradiated, they have a greater cytotoxic effect on human lung cells compared to non-irradiated TiO2 NPs [93].

Digestive System
Oral ingestion of TiO2 NPs causes them to enter the digestive system. The blood passes through the stomach and also passes through the liver. Significant concentrations of TiO2 NPs can lead to liver dysfunction, liver cell damage and even liver failure, hepatocyte dysfunction, superficial staining of cytoplasm, and osteoporosis after repeated exposure [40,92]. However, these results, carried out in mice or rats, are controversial, as no toxicity was observed in other studies.

Respiratory System
The lungs are the main target system of environmental air pollution via TiO 2 NPs [92]. TiO 2 -induced human lung epithelial cell injury (A549) and alveolar lung inflammation have been reported after inhalation in several studies [93][94][95]. These phenomena lead to lung dysfunction, as well as irreversible changes to the cells, resulting in fibrosis and tumor development. The lung toxicity and inflammatory effects are related to the properties of TiO 2 NPs, such as their size, shape, crystallinity, agglomeration, and surface coating mode [96]. At this point it should be noted that the cytotoxicity of TiO 2 NPs increased in the following order: amorphous > anatase > anatase/rutile; thus, amorphous TiO 2 NPs possessed greater toxic effect than anatase/rutile TiO 2 NPs [95]. Fresegna et al. studied the cellular responses of human alveolar A549 and bronchial BEAS-2B cells to measure the cytotoxic and inflammatory effects of TiO 2 NPs in anatase and rutile forms [7]. They found that anatase TiO 2 NPs exerted greater cytotoxicity on bronchial cells compared to rutile TiO 2 NPs. On the other hand, a higher level of genotoxicity was observed on alveolar and bronchial cells treated with rutile TiO 2 compared to anatase TiO 2 . When TiO 2 NPs are pre-irradiated, they have a greater cytotoxic effect on human lung cells compared to non-irradiated TiO 2 NPs [93].

Digestive System
Oral ingestion of TiO 2 NPs causes them to enter the digestive system. The blood passes through the stomach and also passes through the liver. Significant concentrations of TiO 2 NPs can lead to liver dysfunction, liver cell damage and even liver failure, hepatocyte dysfunction, superficial staining of cytoplasm, and osteoporosis after repeated exposure [40,92]. However, these results, carried out in mice or rats, are controversial, as no toxicity was observed in other studies.

Urinary System
The kidneys are one of the major organs that filter the blood, remove metabolic wastes, control the body's extracellular fluid balance and electrolyte composition, and return the purified blood to the body. Chen et al. demonstrated an association between TiO 2 NPs and kidney toxicity, suggesting that the kidney may be a major target or organ of exposure to nano-TiO 2 via various routes into the body [92]. When cultured embryonic kidney cells were incubated with TiO 2 NPs, no significant induction of DNA damage was observed. In this case, only the highest concentration of TiO 2 NPs, equivalent to 100 µg/mL, elicited a significant genotoxic response, but it was concluded that such a high concentration of TiO 2 is not environmentally relevant [91]. However, further studies on renal toxicity in humans due to exposure to TiO 2 NPs need to be conducted.

Reproductive System
Based on the research conducted to date, the toxicity of TiO 2 NPs to the reproductive system such as testes, ovaries, placenta, and fetal tissues in humans is unknown. TiO 2 NPs cause adverse effects on hatching and affect reproduction in zebrafish, as well as pregnancy difficulties such as nanoparticle spread to fetal brain, fetal liver, and placenta in mice [13]. However, the research data are not sufficient to conclude the development of toxicity in the reproductive system of mammals, especially humans.

Dermal System
The penetration of TiO 2 NPs through human skin has not been found in most studies [97][98][99]. TiO 2 NPs do not cause ROS formation, cellular glutathione content, nor apoptosis when applied to human epidermal (A431) and keratinocytic (HaCaT) skin cells [100,101]. TiO 2 NPs cause dermal toxicity only when they pass through healthy or damaged skin after long-term exposure [40]. However, the opposite phenomenon has been observed in several studies. Wright et al. showed that TiO 2 NPs induce superoxide formation, caspase, and cell apoptosis in human keratinocyte cells (HaCaTs) in a dose-dependent manner. This causes cytotoxicity in HaCaT cells at 10 −4 -10 −5 mol/L [100]. In fact, human skin is not only exposed to TiO 2 NPs, but also external chemicals or stressors, such as UV light [102], which also damage human dermal fibroblasts. The Scientific Committee on Consumer Safety (SCCS) therefore suggested that TiO 2 NPs should not be used in sunscreen formulations with high photocatalytic activity [1].

TiO 2 NPs in the Environment (Ecotoxicity)
Ecotoxicity occurs when biological, physical, and chemical stressors affect living organisms in the ecosystem through altered biochemistry, physiology, and cellular interactions. The ecotoxic adverse effects of TiO 2 NPs have been observed in water, aquatic animals, zebrafish gills, food, and aquatic environments [67]. TiO 2 NPs influence the bacterial colonies in soil, reducing microbial biomass and diversity, thereby having a negative effect by changing the bacterial composition of the ecosystem [103,104]. In addition, terrestrial plants collect TiO 2 NPs from the soil and store them in stems, leaves, and fruits, which promotes germination and root expansion [105,106].

TiO 2 NPs in the Plant and Soil Environment
After the use of TiO 2 NPs in various products, this inorganic nanomaterial is mostly abandoned, and a large quantity of TiO 2 NPs is distributed to the environment, reaching the air, soil, water, and living organisms [54]. Figure 8 represents the use, release pathways, distribution, and interaction of TiO 2 NPs to plants and the surrounding environment. TiO 2 NPs used in pigments, food additives, and personal care products are released into the soil (13.8%), water (18.5%), and air (2.2%). Therefore, plants come into direct contact with TiO 2 NPs through the soil, water, and air. After the entry of TiO2 NPs via several pathways, soil properties-such as soil enzymes, microbial communities, nutritional elements, and pH-affect their behavior, mobility, and bioavailability, thereby determining the fate of TiO2 NPs. The interconnected soil properties/factors, such as soil type, pH, ionic strength, and organic matter, affect the transport of TiO2 NPs by changing their zeta potential, aggregation, surface charge, and van der Waals force [108,109]. The existence of microbes in the soil is vitally important for After the entry of TiO 2 NPs via several pathways, soil properties-such as soil enzymes, microbial communities, nutritional elements, and pH-affect their behavior, mobility, and bioavailability, thereby determining the fate of TiO 2 NPs. The interconnected soil properties/factors, such as soil type, pH, ionic strength, and organic matter, affect the transport of TiO 2 NPs by changing their zeta potential, aggregation, surface charge, and van der Waals force [108,109]. The existence of microbes in the soil is vitally important for the decomposition and recycling of organic material. As a result, TiO 2 NPs may alter the microbial population, diversity, and activity by changing the soil properties [103,110]. Low concentrations of TiO 2 NPs also increase urea activity [111]. On the other hand, at extremely higher concentrations (1000 mg/L), TiO 2 NPs decrease urea activity [112,113], affect the level of bacterial nitrogen, and reduce catalase, phosphatase, invertase, and peroxidase activities [103]. Reports suggests that TiO 2 NPs also disrupt the gene expression of bacteria, resulting in decreasing nitrogen fixation and methane oxidation, which are essential for the decomposition of proteins and organic pollutants [81].
The interaction between plants and TiO 2 NPs depends on their particle size, crystal phase, and surface coating [107]. Smaller TiO 2 NPs (less than 30 nm) can enter into plant cells by reducing the size of the pores and the flow of water in corn [114] and wheat [115]. Investigations show that TiO 2 NPs of 12, 22, and 25 nm can be translocated from the roots to the leaves [115]. The antioxidant stress is interrupted by TiO 2 NPs in duckweed [116] and tomato [117]. TiO 2 NPs also interrupt different parameters in raceme elm [118], onion [119], soybean [120], rice, spinach [121], and parsley [122]. As TiO 2 NPs show photocatalytic activity under light irradiation, they can alter photosynthesis, metabolism, and gene expression within plants. Some investigations reported negative effects caused by TiO 2 NPs on plant growth, whereas it was concluded that TiO 2 NPs may cause some sensitive plant growth-promoting bacteria to disappear from soil. Accordingly, such impairment of the soil bacterial community composition may further affect ecosystem functioning [123,124].

TiO 2 NPs in the Aquatic Environment
The significantly increased leakage of TiO 2 NPs into surface and marine water environments has a great impact on aquatic ecosystems. The related studies have been mainly concentrated in determining the behavior of TiO 2 NP in marine environments, focusing mostly on marine plankton and benthos, with comparable contribution of papers, i.e., 42.1% and 44.7%, respectively, as well as in marine fish, with the smallest share of the research, i.e., 13.2% [125]. As TiO 2 NPs are very reactive, they follow different transformation processes when they are released into the aquatic ecosystem. These transformation processes involve physical (agglomeration, aggregation, and sedimentation) interactions with TiO 2 (adsorption), chemical (photochemical) interactions, and biological (biomodification) interactions. The ability of TiO 2 NPs to enter in the aquatic organisms is worrying, as it leads to bioaccumulation in their cellular tissue. Accordingly, NPs negatively affect environmental food webs in three different major ecosystems-freshwater, marine, and terrestrial [24,126]. Bearing in mind that mammalians are at the top of the ecological food chain, the ecotoxicity induced by TiO 2 NPs in the environment is easily converted into cytotoxicity in humans (Figure 9) [24].
Phytoplankton is the most dominant factor in marine ecosystems and the food web [127]. The interaction of TiO 2 NPs with marine phytoplankton has therefore been investigated [127,128]. In an aquatic environment, TiO 2 NPs may be adsorbed or diffused by the phytoplankton surface. TiO 2 NP-mediated ROS may be diffused by the cell wall when the TiO 2 -plankton complex generates 'ligand-to-metal charge transfer reactions' [126]. After the aggregation and settling of TiO 2 in marine environments, there is still a small fraction of TiO 2 NP nanoparticles in the water column that may be hazardous to the living organisms in that water column. Thus, at higher TiO 2 concentrations (>/20 mg/L), TiO 2 NPs can significantly reduce P. tricornutum growth, one of the most widely used model organisms used in marine ecotoxicology studies [127]. Miller et al. demonstrated that, at low UV levels, TiO 2 NPs' photocatalytic activity can induce toxicity in marine phytoplankton [128]. However, in the absence of UV light, no effects were observed. processes involve physical (agglomeration, aggregation, and sedimentation) interactions with TiO2 (adsorption), chemical (photochemical) interactions, and biological (biomodification) interactions. The ability of TiO2 NPs to enter in the aquatic organisms is worrying, as it leads to bioaccumulation in their cellular tissue. Accordingly, NPs negatively affect environmental food webs in three different major ecosystems-freshwater, marine, and terrestrial [24,126]. Bearing in mind that mammalians are at the top of the ecological food chain, the ecotoxicity induced by TiO2 NPs in the environment is easily converted into cytotoxicity in humans (Figure 9) [24].

Figure 9.
Environmental and biological effects of the TiO2 NPs. Adapted with permission from [24]. Copyright, 2019 MDPI AG Phytoplankton is the most dominant factor in marine ecosystems and the food web [127]. The interaction of TiO2 NPs with marine phytoplankton has therefore been investigated [127,128]. In an aquatic environment, TiO2 NPs may be adsorbed or diffused by the phytoplankton surface. TiO2 NP-mediated ROS may be diffused by the cell wall when the TiO2-plankton complex generates 'ligand-to-metal charge transfer reactions' [126]. After the aggregation and settling of TiO2 in marine environments, there is still a small fraction of TiO2 NP nanoparticles in the water column that may be hazardous to the living organisms in that water column. Thus, at higher TiO2 concentrations (>/20 mg/L), TiO2 NPs can significantly reduce P. tricornutum growth, one of the most widely used model organisms used in marine ecotoxicology studies [127]. Miller et al. demonstrated that, at low UV levels, TiO2 NPs' photocatalytic activity can induce toxicity in marine phytoplankton [128]. However, in the absence of UV light, no effects were observed.
The toxicity of TiO2 NPs in marine mussels has been investigated in several studies [38,[129][130][131][132][133][134]. Due to the filtrating behavior and bioaccumulation tendency of bivalve mollusks, they accumulate various pollutants, such as microalgae, sediments, bacteria, and contaminants, within their tissues [38]. However, no acute TiO2 NP toxicity was found in marine abalone at TiO2 NP concentrations from 0.1 to 10.0 mg/L [135]. In spite of the absence of toxicity, minor oxidative stress was induced. In another study, the combined effect of TiO2 NPs and ocean acidification was assessed in mussels [136]. A low pH increases the toxicity of TiO2 NPs and the impairment of feeding and metabolism was observed in mussels at different pH and concentrations.
In a different study, the TiO2 NP-fish interaction was examined at different TiO2 NP concentrations and exposure conditions [137]. TiO2 NPs of 0, 1, 10, and 100 mg/L were applied to fish for 96 h, resulting in no mortality or sublethal effects. Very few studies associated with TiO2 NPs have been conducted on fish. As such, more studies need to be conducted in order to understand the potentially cytotoxic effects of TiO2 NPs on fish. The toxicity of TiO 2 NPs in marine mussels has been investigated in several studies [38,[129][130][131][132][133][134]. Due to the filtrating behavior and bioaccumulation tendency of bivalve mollusks, they accumulate various pollutants, such as microalgae, sediments, bacteria, and contaminants, within their tissues [38]. However, no acute TiO 2 NP toxicity was found in marine abalone at TiO 2 NP concentrations from 0.1 to 10.0 mg/L [135]. In spite of the absence of toxicity, minor oxidative stress was induced. In another study, the combined effect of TiO 2 NPs and ocean acidification was assessed in mussels [136]. A low pH increases the toxicity of TiO 2 NPs and the impairment of feeding and metabolism was observed in mussels at different pH and concentrations.
In a different study, the TiO 2 NP-fish interaction was examined at different TiO 2 NP concentrations and exposure conditions [137]. TiO 2 NPs of 0, 1, 10, and 100 mg/L were applied to fish for 96 h, resulting in no mortality or sublethal effects. Very few studies associated with TiO 2 NPs have been conducted on fish. As such, more studies need to be conducted in order to understand the potentially cytotoxic effects of TiO 2 NPs on fish.
Moreover, in aquatic environments, TiO 2 NPs can interact with heavy metals (Cu, Zn, Cd, As, etc.) and toxic organics, resulting in the formation of a harmful environment that can alter the bioavailability of aquatic organisms [38,138,139]. If TiO 2 NPs are exposed to arsenic (As), this can increase arsenic accumulation in aquatic animals and the human food web [139]. However, TiO 2 NP-Cd interactions do not result in toxicity in Mediterranean mussels [140]. The immunotoxicity, genotoxicity, and neurotoxicity of TiO 2 NPs in marine living organisms were reported by several researchers, with varying results depending on the particle size, exposure duration, exposure type, and stress factors [38,134,[141][142][143][144][145].

Biocompatibility of TiO 2 NPs
The potential toxicity of the TiO 2 NPs discussed above does not suggest that they are unsafe for humans or the environment. Depending on the size and shape of the nanoparticles, TiO 2 NPs may be safe, due to their very low toxicity. When it comes to the issue of skin permeation, long-term skin exposure to TiO 2 NPs can be harmful to humans if they overdose, which is almost impossible in everyday life [146]. Biocompatibility refers to the testing of TiO 2 NPs for cytotoxicity, genotoxicity, immunotoxicity, systemic toxicity, hemocompatibility, pyrogenicity, and implantation, evaluated by ISO, ISO/TR 10993-22:2017 via in vitro and in vivo studies [47]. The evaluation of the toxicity and biocompatibility of TiO 2 NPs is crucial to understanding the deleterious biological responses of the properties of TiO 2 NPs, their functionalities, and their contact surfaces (Figure 10a).
The potential toxicity of the TiO2 NPs discussed above does not suggest that they are unsafe for humans or the environment. Depending on the size and shape of the nanoparticles, TiO2 NPs may be safe, due to their very low toxicity. When it comes to the issue of skin permeation, long-term skin exposure to TiO2 NPs can be harmful to humans if they overdose, which is almost impossible in everyday life [146]. Biocompatibility refers to the testing of TiO2 NPs for cytotoxicity, genotoxicity, immunotoxicity, systemic toxicity, hemocompatibility, pyrogenicity, and implantation, evaluated by ISO, ISO/TR 10993-22:2017 via in vitro and in vivo studies [47]. The evaluation of the toxicity and biocompatibility of TiO2 NPs is crucial to understanding the deleterious biological responses of the properties of TiO2 NPs, their functionalities, and their contact surfaces (Figure 10a). In terms of the biocompatibility of TiO2 NPs, they are used in drug carrier biosensing, implants, and antibacterial activity [148]. Due to the spherical shape of TiO2, TiO2-cell interactions, TiO2′s biocompatibility, excellent drug release properties, and lower toxicity (than Al2O3 and SiO2) [148][149][150], TiO2 NPs are used in antitumor treatments (Figure 10b). Due to the cytotoxic ROS (O2 − , OH − , H2O2)-based interaction between TiO2 NPs and specific cancer cell membranes, surface-functionalized biocompatible TiO2 NPs have recently been used for targeted cancer therapy [48]. TiO2 NPs have also been used in the biomedical field in photothermal therapy (PTT), photodynamic therapy (PDT), and sonodynamic therapy (SDT) for cancer treatment via the targeted, controlled, stimulus-driven delivery and release of cytotoxic anti-cancer agents [148]. Due to their low phototoxicity, biocompatibility and stable structure, TiO2 NPs have potential applications in phototherapy for the treatment of cancer cells [49,151]. When TiO2 NPs are exposed to light, an oxidative radical (ROS) is generated, which subsequently destroys the cell and cellular components, such as the lipids, proteins, carbohydrates, and nucleic acids in cancer cells [147,152].
Furthermore, several authors have reported that the toxicity of TiO2 NPs arises due to a 'particle effect' rather than a 'chemical effect' [40]. However, further studies are required to assess the nature, mechanisms, and effects of the toxicity of TiO2 NPs on humans and the surrounding environment. In terms of the biocompatibility of TiO 2 NPs, they are used in drug carrier biosensing, implants, and antibacterial activity [148]. Due to the spherical shape of TiO 2 , TiO 2 -cell interactions, TiO 2 s biocompatibility, excellent drug release properties, and lower toxicity (than Al 2 O 3 and SiO 2 ) [148][149][150], TiO 2 NPs are used in antitumor treatments (Figure 10b). Due to the cytotoxic ROS (O 2 − , OH − , H 2 O 2 )-based interaction between TiO 2 NPs and specific cancer cell membranes, surface-functionalized biocompatible TiO 2 NPs have recently been used for targeted cancer therapy [48]. TiO 2 NPs have also been used in the biomedical field in photothermal therapy (PTT), photodynamic therapy (PDT), and sonodynamic therapy (SDT) for cancer treatment via the targeted, controlled, stimulus-driven delivery and release of cytotoxic anti-cancer agents [148]. Due to their low phototoxicity, biocompatibility and stable structure, TiO 2 NPs have potential applications in phototherapy for the treatment of cancer cells [49,151]. When TiO 2 NPs are exposed to light, an oxidative radical (ROS) is generated, which subsequently destroys the cell and cellular components, such as the lipids, proteins, carbohydrates, and nucleic acids in cancer cells [147,152].
Furthermore, several authors have reported that the toxicity of TiO 2 NPs arises due to a 'particle effect' rather than a 'chemical effect' [40]. However, further studies are required to assess the nature, mechanisms, and effects of the toxicity of TiO 2 NPs on humans and the surrounding environment.

Risk Following Oral Exposure
Considering the published studies, TiO 2 NPs have no acute toxic effects after oral exposure. There are insufficient data on the repeated-dose toxicity of TiO 2 NPs, so there is not believed to be any significant risk from oral exposure.

Risk Following Dermal Exposure
Based on the current data, short-term dermal exposure to TiO 2 NPs has very little or no toxic effect on healthy skin. Further studies on long-term dermal exposure, as well as on damaged skin, are required in order to evaluate TiO 2 NPs' nanotoxicity on the skin [40].

Risk Following Inhalation
It is well established that smaller TiO 2 NPs are more toxic than comparatively coarser TiO 2 NPs. Anatase TiO 2 is more toxic than rutile TiO 2 , with its crystalline structure. In terms of inhalation, particle size, surface area, crystalline structure, agglomeration, and exposure, time plays a crucial role in the toxicity of TiO 2 NPs. Inhalation is a major problem in workplaces due to high concentrations of TiO 2 NPs, as well as the fact that TiO 2 NPs enter the body mainly through respiration.
The establishment of risk management strategies is therefore crucial (Figure 11), particularly in relation to short-/long-term exposure, as well as the frequency and level of exposure; however, such strategies have not yet been developed. Regulations and legalization for controlling TiO 2 NPs and other engineered NPs (ENPs) are unsure, living the interpretation of the scientific data challenging to the government, agencies, industry, and consumers. Recently, suggestions for the safe handling of TiO 2 NPs and other engineered NPs (ENPs) were proposed by Besha et al. [153], highlighting the mitigation approaches to curtail the possible hazard effects of ENPs.

Risk Following Inhalation
It is well established that smaller TiO2 NPs are more toxic than comparatively coarser TiO2 NPs. Anatase TiO2 is more toxic than rutile TiO2, with its crystalline structure. In terms of inhalation, particle size, surface area, crystalline structure, agglomeration, and exposure, time plays a crucial role in the toxicity of TiO2 NPs. Inhalation is a major problem in workplaces due to high concentrations of TiO2 NPs, as well as the fact that TiO2 NPs enter the body mainly through respiration.
The establishment of risk management strategies is therefore crucial (Figure 11), particularly in relation to short-/long-term exposure, as well as the frequency and level of exposure; however, such strategies have not yet been developed. Regulations and legalization for controlling TiO2 NPs and other engineered NPs (ENPs) are unsure, living the interpretation of the scientific data challenging to the government, agencies, industry, and consumers. Recently, suggestions for the safe handling of TiO2 NPs and other engineered NPs (ENPs) were proposed by Besha et al. [153], highlighting the mitigation approaches to curtail the possible hazard effects of ENPs. Figure 11. Risk management strategies for human exposure to TiO2 NPs via different exposure routes.

Conclusions and Future of TiO2 NPs in Textile Applications
TiO2 NPs are important photocatalysts that are implemented in various fields of application. The toxicity of TiO2 NPs to human health and the environment is still controversial. TiO2 NPs are classified as possibly carcinogenic to humans by the International Agency for Research on Cancer (IARC), and as a chemical risk to humans by the Workplace Hazardous Materials Information System (WHMIS) (in group D2A). At lower TiO2 NP concentrations, recent studies have found that TiO2 NPs are almost non-toxic, as no remarkable toxicity was observed [99,154,155]. However, TiO2/textile composites, for example, distinguished by remarkable functional properties [156], have no adverse effects on human skin, as superoxide and hydroxyl radicals are not able to damage human cells from the outside as they cannot enter the inner layer of the human skin [155,157]. As Figure 11. Risk management strategies for human exposure to TiO 2 NPs via different exposure routes.

Conclusions and Future of TiO 2 NPs in Textile Applications
TiO 2 NPs are important photocatalysts that are implemented in various fields of application. The toxicity of TiO 2 NPs to human health and the environment is still controversial. TiO 2 NPs are classified as possibly carcinogenic to humans by the International Agency for Research on Cancer (IARC), and as a chemical risk to humans by the Workplace Hazardous Materials Information System (WHMIS) (in group D2A). At lower TiO 2 NP concentrations, recent studies have found that TiO 2 NPs are almost non-toxic, as no remarkable toxicity was observed [99,154,155]. However, TiO 2 /textile composites, for example, distinguished by remarkable functional properties [156], have no adverse effects on human skin, as superoxide and hydroxyl radicals are not able to damage human cells from the outside as they cannot enter the inner layer of the human skin [155,157]. As TiO 2 /rGO-coated cotton fabric is biocompatible, it does not cause cell membrane damage and cell proliferation, as textiles coated with TiO 2 NPs do not exhibit cytotoxicity over 24 h of incubation [158]. With the exception of the other previously mentioned study, to the best of our current knowledge, no experiments have been conducted with TiO 2 NP-modified textiles on human skin. Considering the toxicity and ecotoxic pathways described in in vivo and in vitro studies, TiO 2 NPs are the safer option when compared to other metal oxide nanomaterials [148]. The nanotoxicity and contradictive biocompatibility of TiO 2 NPs should be further investigated by researchers, whereas studies on long-term or accelerated effects of TiO 2 need to be carried out in order to estimate precise implications of TiO 2 NPs for human health and the surrounding environment. Therefore, careful consideration should be given to the benefits of TiO 2 , and the associated potential risks based on its intended use. Moreover, mitigating the toxicity of TiO 2 NPs transferred from consumer products to the environment requires appropriate strategies and regulatory frameworks to protect humans and the environment. Accordingly, the establishment of international standard methods for the exact evaluation of risk-benefit assessments is a prerequisite to allowing the safe use of TiO 2 -functionalized materials.

Conflicts of Interest:
The authors declare no conflict of interest.