R E V I EW A R T I C L E Hepatotoxicity of titanium dioxide nanoparticles Jangrez Khan | Nicholas D. Kim | Collette Bromhead | Penelope Truman | Marlena C. Kruger | Beth L. Mallard School of Health Sciences, Massey University, PO Box 756, Wellington, 6021, New Zealand Correspondence Beth L. Mallard, School of Health Sciences, Massey University, PO Box 756, Wellington 6021, New Zealand. Email: b.mallard@massey.ac.nz Abstract The food additive E171 (titanium dioxide, TiO2), is widely used in foods, pharmaceuti- cals and cosmetics. It is a fine white powder, with at least one third of its particles sized in the nanoparticulate (<100 nm range, TiO2 NPs). The use of E171 is contro- versial as its relevant risk assessment has never been satisfactorily accomplished. In vitro and in vivo studies have shown dose-dependent toxicity in various organs including the liver. TiO2 NPs have been shown to induce inflammation, cell death and structural and functional changes within the liver. The toxicity of TiO2 NPs in experi- mental models varies between organs and according to their physiochemical charac- teristics and parameters such as dosage and route of administration. Among these factors, ingestion is the most significant exposure route, and the liver is a key target organ. The aim of this review is to highlight the reported adverse effects of orally administered TiO2 NPs on the liver and to discuss the controversial state of its toxicity. K E YWORD S E171, liver, nanoparticles, oxidative stress, titanium dioxide 1 | INTRODUCTION Nanotechnology has extensive applications in health and medicine, agriculture, environmental protection and the food industry. In the food industry, nanomaterials are used as coatings in packaging to protect from mechanical damage as well as to minimise microbial contamination. Nanoparticles (NPs) are also added to food to enhance taste, impression, colour, quality and consistency (Kassebaum & Collaborators, 2016; Shen et al., 2017; Von Moos et al., 2017). Ingested NPs can be absorbed from the digestive tract, may accumulate in different organs and can trigger potential health risks. This comprehensive review summarises all available studies focussing on TiO2 NPs and their impact on the liver. The review includes stud- ies reporting various outcomes including toxicity as well as instances of no or minimal effects on the liver. Finally, this review provides a thorough discussion on the evidence of TiO2 NP-induced liver toxicity and its underlying mechanisms from research conducted in rodents and in cultured liver cells. 2 | APPLICATIONS OF TITANIUM DIOXIDE NPs The most common compound of titanium is TiO2, which constitutes almost 95% of all titanium consumed. Global production of TiO2 was 6.1 million tons in 2016 and was expected to reach 7.8 million tons by the end of 2022, while its global market is predicted to increase by 8.9% per annum until 2025 (La Maestra et al., 2021). TiO2 are inorganic materials and are among the most common NPs found in consumer products (Hong et al., 2017). TiO2 has some unique properties in ease of availability, biocompatibility, high specific surface area, long-term photostability, anti-corrosive, strong oxidising Received: 22 February 2024 Revised: 21 April 2024 Accepted: 25 April 2024 DOI: 10.1002/jat.4626 This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. © 2024 The Authors. Journal of Applied Toxicology published by John Wiley & Sons Ltd. J Appl Toxicol. 2024;1–24. wileyonlinelibrary.com/journal/jat 1 https://orcid.org/0000-0001-5523-6385 https://orcid.org/0000-0002-2890-2191 https://orcid.org/0000-0003-1417-1313 https://orcid.org/0000-0002-8646-9672 https://orcid.org/0000-0002-0634-7916 mailto:b.mallard@massey.ac.nz https://doi.org/10.1002/jat.4626 http://creativecommons.org/licenses/by-nc/4.0/ http://wileyonlinelibrary.com/journal/jat http://crossmark.crossref.org/dialog/?doi=10.1002%2Fjat.4626&domain=pdf&date_stamp=2024-05-13 properties and antibacterial properties and are believed to have low toxicity. Due to these characteristics, paints and coatings occupy the biggest market share of total global production of TiO2, that is, 48%. However, other application sectors include plastics (19%), resin (10%), papermaking (8%), fibre (3%), rubber (2%) and others (medicine, food and cosmetics; 10%) (Chen et al., 2020; Kaewklin et al., 2018; Yin et al., 2013). TiO2 is used in a variety of cosmetics as it acts as an effective sunscreen and provides protection against short-wave ultraviolet (UV) radiation. It serves as a clouding agent and is assimilated into dry beverages and tobacco wrappings. Additionally, it is used in the pharmaceutical industry as a component of tablets (Weir et al., 2012). TiO2 is used as an orthopaedic implant biomaterial, particularly for the hip and knee joints, bone plates, dental implants and dental products including crowns, bridges and dentures (Jacobs et al., 1991; Jin et al., 2022; Patri et al., 2009; Sul, 2010). 3 | FOOD In the food industry, TiO2 is known as E171 in the European Union (EU) and INS 171 in the United States (Korotcenkov, 2020b) (for simplicity, this review adopts the EU nomenclature). E171 is manufac- tured by two main processes, namely, a sulfuric acid-based process that yields the predominant crystalline forms of titanium dioxide, that is, anatase, rutile or a mixture of both, or a chlorine-based process that yields rutile only (Gázquez et al., 2014). E171 is commonly used in food and other consumer products such as toothpaste, coatings, preserved fruits, chewing gum, coated candy, carbonated drinks, dairy products, tattoo ink, sauces, icings, beauty creams (day creams, foundations and lip balms) and dressings (Keller et al., 2013; Lim et al., 2018; Lomer et al., 2000; Ortiz & Alster, 2012; Peters et al., 2014; Shi et al., 2013; Weir et al., 2012). Both anatase and rutile have been authorised to be used as a food additive; however, characterisation of food samples from Europe and America have shown that the general population is primarily exposed to anatase (Bischoff et al., 2021; Chen et al., 2013; Dudefoi et al., 2017). In the recent report of the European Food Safety Authority (EFSA) in 2021, it has been shown that the daily dietary intake of E171 in European countries is 11.5 mg/kg bw in children as compared to 6.7 mg/kg bw in adults. This intake does not include the amount of TiO2 NPs coming from other non-food products like toothpastes, mouthwashes, medications and tattoo ink that can greatly impact to overall exposure (Fiordaliso et al., 2022; Younes et al., 2021; Zhang et al., 2018). 4 | ESTIMATED DIETARY INTAKE Earlier studies have reported the estimated dietary intake in countries like the United States and the United Kingdom, where the oral consumption was around 1–2 mg/kg/day in children and 0.2–0.7 mg/kg/day in adults (Weir et al., 2012). It is clear that children are exposed to higher doses, with similar high exposure reported in British children (2.5–4.5 years), where it was above 3 mg/kg/day (Lomer et al., 2000). Similarly, German children estimated consump- tion is reported to be around 2 mg/kg/day (Bachler et al., 2015), and Dutch children up to 2.16 mg/kg/day (Rompelberg et al., 2016) (details of all these studies are reviewed by Bischoff et al., 2021). The daily dietary intake of E171 is variable among different age groups and countries; however, due to lower body mass and disproportionally higher consumption of E171-containing products, children were found to be the most highly exposed group. Most children consume food more likely to contain TiO2 such as candies, chewing gums and jellies (Bischoff et al., 2021; Li et al., 2018; Weir et al., 2012). The maximum concentration of E171 as a food additive is 1% in the United States, while it was quantum satis levels (as much as needed, but not more) in the EU until it was banned in 2022 (Baranowska-Wójcik et al., 2022; Heringa et al., 2016; Rompelberg et al., 2016; Younes et al., 2021). The ideal properties that made it a preferred choice are its high abundance in nature, comparative cheapness, no nutritional value, insolubility in both water and organic solvents and presumed biological inertness (Buettner & Valentine, 2012; FDA, 2014; Korotcenkov, 2020a; Ropers et al., 2017; Zierden & Valentine, 2015). 5 | SAFETY TiO2 NPs were classed as potentially carcinogenic (by inhalation) by the International Agency for Research on Cancer (IARC, 2010). Through the course of time, several studies reported the adverse effects of TiO2 NPs by various modes of administration (Hong et al., 2014; Hu et al., 2015; Ogunsuyi et al., 2022; Talamini et al., 2019). These reports led to the re-evaluation of the safety of TiO2 as a food additive. The EFSA re-evaluated the usage of TiO2 in 2016 and 2018 and concluded that oral intake of E171 has no concerns of genotoxicity and carcinogenicity (EFSA, 2016; Younes et al., 2018). However, in 2020, E171 usage was suspended in France for 1 year, and in 2021, it was declared unsafe to be used as a food additive (reviewed by Boutillier et al., 2021). Recently, the EU has also banned the usage of E171 as a food additive, as they have accepted that TiO2 NPs may pose a risk of genotoxicity (Baranowska-Wójcik et al., 2022; Younes et al., 2021). 6 | ROUTE OF ENTRY, ABSORPTION AND ACCUMULATION OF TIO2 NPs Oral absorption of TiO2 NPs is well studied in animal models (Jovanovi�c, 2015); however, more investigations are needed in humans. Most ingested TiO2 is not absorbed from the gut as several animal studies have confirmed that most of orally administered particles are excreted through the faeces (Cho et al., 2013; Farrell & Magnuson, 2017; Jo et al., 2016; MacNicoll et al., 2015). Orally administered TiO2 NPs in rats are preferentially absorbed by Peyer's patches in the small intestine. Most of the consumed TiO2 NPs are excreted via faeces, while the absorbed particles are translocated 2 KHAN ET AL. 10991263, 0, D ow nloaded from https://analyticalsciencejournals.onlinelibrary.w iley.com /doi/10.1002/jat.4626 by M assey U niversity L ibrary, W iley O nline L ibrary on [12/06/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense across the gastrointestinal (GI) tract and enters the bloodstream (Kreyling et al., 2017; Riedle et al., 2020). It has been shown that TiO2 NPs in mice accumulate mostly in the liver, spleen, kidneys, brain and lungs (Kreyling et al., 2017; Martins et al., 2017; Shinohara et al., 2014; Wang et al., 2007). Similarly, study in humans with a single oral dose of E171 demonstrated that >99% ingested particles were secreted in the stool and <1% were absorbed (Jones et al., 2015). Although TiO2 NPs have shown poor absorption, human autopsies have shown that most accumulation occurs in the liver and spleen (Gilbert et al., 2021; Hamilton, 2013; Heringa et al., 2018; Keller et al., 1995; Lima et al., 2004; Peters et al., 2020; Younes et al., 2021) (see Figure 1). TiO2 NPs have been shown to induce toxicity in other organs including the small intestine (Acar et al., 2015; Coccini et al., 2015; Hu et al., 2011; Jia et al., 2017; Jugan et al., 2012; Orazizadeh et al., 2020; Petkovi�c et al., 2011; Salman et al., 2021; Valdiglesias et al., 2013), lungs (Fukatsu et al., 2018; Hussain et al., 2010; Koltermann-Jülly et al., 2020; Li et al., 2010; Moon et al., 2010; Silva et al., 2013; Zhao et al., 2018), heart (Saber et al., 2013; Yu et al., 2014; Zhao et al., 2018), brain (Wu et al., 2008) (Heidari et al., 2019), kidneys (Hazelhoff et al., 2022) and spleen (Afshari- Kaveh et al., 2021). Several studies have reported the toxicity and adverse mechanisms of TiO2 NPs in the liver via various modes of administration. Recently, an interesting postmortem study has shown F IGURE 1 Route of entry, absorption, distribution, accumulation and excretion of TiO2 nanoparticles (NPs). TiO2 NPs enter the body through the oral cavity. Food elements and NPs get separated in the stomach and a small percentage of the particles are taken up by Peyer's patches in the small intestine from where they enter the bloodstream. Once distributed to the whole body, these particles accumulate in different organs including liver, brain, spleen and kidneys. However, the undistributed particles are excreted through faeces and urine. Created with BioRender. KHAN ET AL. 3 10991263, 0, D ow nloaded from https://analyticalsciencejournals.onlinelibrary.w iley.com /doi/10.1002/jat.4626 by M assey U niversity L ibrary, W iley O nline L ibrary on [12/06/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense the accumulation of TiO2 NPs (24% of total TiO2 particles were less than 100 nm) in the human liver (Heringa et al., 2018). All such results raise concerns about the cytotoxicity and liver impairment by the oral intake of food-grade TiO2 NPs. This review focusses on the adverse effects after oral administration of TiO2 NPs in the liver or studies demonstrating its effects in liver cells (in vitro). Databases including PubMed, Scopus and Web of Science were used with search terms “titanium dioxide OR titanium dioxide nanoparticle* OR nano titanium OR TiO2 nanomaterial OR TiO2 nanoparticle* OR TiO2 food grade OR E171 AND liver OR hepatocyte* AND adverse effects OR toxicity”. Studies demonstrating other routes of administration like intraperito- neal (IP), intravenous (IV), inhalation or intratracheal instillation (IT) and subcutaneous (SC) administration or focusing other organs like lungs, intestine, kidneys, heart, brain and spleen were excluded. Studies used co-administration of TiO2 NPs with any other substance/toxin/biomaterial or nanomaterial were also excluded. The flow diagram of the selection process is summarised in Figure 2. 7 | TIO2 NPs AND LIVER TOXICITY The liver is the most important organ for the detoxification of toxins and xenobiotics. Regardless of the route of administration, TiO2 NPs accumulate in the liver and can have toxic effects (Cui et al., 2010; Cui et al., 2011; Gilbert et al., 2021; Hassanein & El-Amir, 2018; Jia et al., 2017; Nie et al., 2021; Shirdare et al., 2022; Suker & Jasim, 2018; Wang et al., 2013). The reported mechanisms by which TiO2 NPs may cause toxicity in the liver include generation of reactive oxygen species (ROS) (Jia et al., 2017; Shrivastava et al., 2014), oxidative stress (Chen et al., 2019; Chen et al., 2020; Wang, 2014), inflammation (Abbasi-Oshaghi et al., 2019; Hong et al., 2020), ultimately leading to steatosis, histopathological changes (fibrosis and damaged lobular structure) (Azim et al., 2015; Cui et al., 2011; Talamini et al., 2019; Tassinari et al., 2023; Wang et al., 2007; Wang et al., 2013; Zhao et al., 2021), DNA damage, necrosis, apoptosis and cell death (Cui et al., 2010; Jia et al., 2017; Orazizadeh et al., 2020). This review focusses on studies showing toxicological effects of TiO2 NPs. However, there are some studies that have reported very minor or no effects. These studies are shown in Table 1. Overall, these studies report that oral administration of TiO2 NPs result in their accumulation in the liver. TiO2 NP accumulation leads to an increase in the levels of oxidative stress and inflammation, biochemical parameters, ultimately leading to histopathological changes, apoptosis, necrosis and decrease of antioxidants in the liver or its cultured cells. F IGURE 2 Flow chart of study selection process. 4 KHAN ET AL. 10991263, 0, D ow nloaded from https://analyticalsciencejournals.onlinelibrary.w iley.com /doi/10.1002/jat.4626 by M assey U niversity L ibrary, W iley O nline L ibrary on [12/06/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense TABLE 1 Studies reported no or very minor effects of TiO2 NPs on liver (in vivo). Study model Oral daily dosage (mg/kg/ bw) Treatment timeline Type/particle size General effects Effects on liver References Fischer 344 rats B6C3F1 mice (M, F) 25, 50 103 weeks Anatase/NM No change in body weight, but a few female mice died with in high-dose group No evidence of toxicity Non-significant signs of hepatocellular carcinoma White faeces in high-dose group NCI (1979) Sprague- Dawley rats (M, F) 100, 300, 1000, 5000 28 and 90 days Rutile/rutile and anatase (A/R 79%–21%)/145 and 173 nm No effects on mortality, body weights, organ weights and daily food consumption No change in neurobehavioral parameters, clinical chemistry, urine analysis or any clinical signs No gross or microscopic anatomic pathology or lesions Grey-coloured faeces in high-dose group Warheit et al. (2015) Sprague- Dawley rats (M, F) 500, 1000, 2000 Single dose Anatase/rutile and mix/Uf 1, 2, 3 and Pg 1, 2, 3 Size range: 43, 42, 47, 153, 195, 213 nm No increase of TiO2 in the blood and liver No increase in micronuclei and explicitly negative in the in vivo mammalian erythrocyte micronucleus test Donner et al. (2016) Wistar rats (M) 0.5 45 days/daily Anatase/41.99 nm No change in redox parameters No genotoxic effects No oxidative stress TiO2 accumulated in the liver Martins et al. (2017) Wistar Han IGS rats (M) 0.32, 32, 5 Daily/7 and 100 days E171 No increase in liver to body weight No increase in inflammatory cytokines in plasma, colon or small intestine No histopathological conditions observed in the liver or any other organs No signs of carcinogenicity Blevins et al. (2019) Sprague- Dawley rats (F) 100, 300, 1000 Daily/14 days A/R 80/20%/21 nm No mortality and change in body weight. No significant decrease in food consumption. No change in absolute and relative organ weights including liver, gravid uterine weight No change in caesarean section parameters, foetal weight, placental weight and placental macroscopic observation Titanium concentrations were elevated in maternal liver, maternal brain and placenta at high dose Lee et al. (2019) Sprague- Dawley rats (M, F) 250, 500, 1000 Daily/28 or 90 days A/R 80%–20%/14–21 nm No mortality, no gain or loss in body weight and food consumption. No ocular abnormalities or abnormal clinical signs Heo et al. (2020) (Continues) KHAN ET AL. 5 10991263, 0, D ow nloaded from https://analyticalsciencejournals.onlinelibrary.w iley.com /doi/10.1002/jat.4626 by M assey U niversity L ibrary, W iley O nline L ibrary on [12/06/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense 8 | BIOCHEMICAL PARAMETERS AND HISTOPATHOLOGICAL CHANGES IN THE LIVER TiO2 NPs increase the serum levels of liver injury markers including ALT, AST, LDH and ALP (Abbasi-Oshaghi et al., 2019; Azim et al., 2015; Niu et al., 2017; Sallam, Ahmed, Diab, et al., 2022). These biochemical indicators of injury are echoed by various histopathologi- cal liver changes including focal degeneration of hepatocytes, spotty necrosis, hydropic degeneration, liver oedema, congestion, swelling, vacuolisation, nuclear membrane collapse and overall cell death (Ali et al., 2019; Azim et al., 2015; Hassanein & El-Amir, 2017; Shirdare et al., 2022; Wang et al., 2013). TiO2 NPs cause significant disruption to the liver structure with neutrophilic cell, portal and lobular infiltra- tion by inflammatory cells and congested dilated central veins (Ali et al., 2019). TiO2 NPs also cause necrosis, oedema, Kupffer cell hypertrophy, hydropic degeneration, vacuolisation in hepatocytes and increased infiltration of inflammatory cells (Moradi et al., 2019) (see Tables 1 and 2 for more details). In vitro studies on culture human and rat liver cells have also shown cytotoxic effects of TiO2 NPs (Sha et al., 2011). Similarly, TiO2 NPs induced alterations in cell viability, increased ROS, decreased GSH levels in human hepatocarcinoma cell line (HepG2 cells) and also resulted in distorted cellular morphology in the liver (Abbasi-Oshaghi et al., 2019). In addition, cell growth inhibi- tion, increased apoptosis, cell cycle arrest at G1 stage and induction of ROS-mediated ER stress by activating PERK/ATF6/Bax axis were observed in HepG2 cells (Li et al., 2020). 9 | OXIDATIVE, MITOCHONDRIAL AND ER STRESS IN THE LIVER TiO2 NP toxicity induces oxidative stress, increases in ROS formation and promotes inflammation (Foroozandeh & Aziz, 2015; Liguori TABLE 1 (Continued) Study model Oral daily dosage (mg/kg/ bw) Treatment timeline Type/particle size General effects Effects on liver References No real differences in haematological, biochemical or urinalysis parameters There were no abnormal or gross findings in any of the animals at necropsy including the liver F344 rats (M, F) 10, 100, 300, 1000 Daily/28 or 90 days Anatase/6 nm No mortality and change body weight or even organ weight No change in parameters of urinalysis, haematology, serum biochemistry No genotoxicity (DNA strand breaks and chromosomal aberrations) in the liver No abnormality of colonic crypts TiO2 particles accumulated in the liver, kidneys and spleen Akagi et al. (2023) Sprague- Dawley rats (M, F) 10, 100, 1000 Daily/90 days Anatase/40 nm No mortality or abnormal clinical sign in eyes. No significant change in body weight or food consumption and no systemic toxicity No changes in parameters of haematology, urinalysis, clinical chemistry No abnormal gross findings at necropsy and no significant differences in endocrine-sensitive endpoints except for the absolute pituitary weights No Ti distribution in major tissues/organs No impact on microbiota diversity only a shift of community structure at genus level Lin et al. (2023) 6 KHAN ET AL. 10991263, 0, D ow nloaded from https://analyticalsciencejournals.onlinelibrary.w iley.com /doi/10.1002/jat.4626 by M assey U niversity L ibrary, W iley O nline L ibrary on [12/06/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense TABLE 2 Reported effects of TiO2 NPs on liver (in vivo). Study model Oral daily dosage (mg/kg/bw) Treatment timeline Type/particle size Effects on liver References CD-1 mice (M, F) 5000/single dose 1 day NG/25, 80, and 155 nm "ALT/AST ratios HC: Ti accumulation in the liver, hydropic degeneration, spotty necrosis of hepatocytes Wang et al. (2007) CD-1 mice (F) 62.5, 125, 250 30 days Anatase/5 nm " ALT, ALP, AST, LDH, ChE, TP, TG, TCHO, NO # ALB to GLB (A/G), TBIL, IL-2 HC: Blur hepatocytes, congested interstitial vessels Duan et al. (2010) CD-1 mice (F) 5, 10, 50 60 days Anatase/6–7 nm " H2O2, MDA, NO, O2 ¯, CYP1A # SOD, CAT, GSH-Px, MT, GST, HSP70, p53, TF genes HC: Mitochondrial swelling, apoptotic bodies, chromatin condensation Cui et al. (2010) CD-1 mice (F) 5, 10, 50 60 days Anatase/5 nm " ALT, AST, ALP, LDH, PChE, LAP, TLR2, TLR4, IKK1, IKK2, NF-κB, NF-κBP52, NF-κBP65, TNF-α, NIK # IκB, IL-2, IgM HC: TiO2 accumulation in the liver, fatty degenerations with large vacuoles and congestion, necrosis, inflammatory cell infiltration, mitochondria swelling, apoptotic cell with chromatin condensation Cui et al. (2011) Sprague- Dawley rats (M) 10, 50, 200 30 days Anatase/75 nm " Glu, LDL-C, ALT/AST ratio, BUN, GSH/GSSG # AST, HBDH, CK, TBIL HC: Liver injury, oedema, hepatic cord disarray, peri-lobular cell swelling, hydropic degeneration, inflammatory cell infiltration Wang et al. (2013) Albino rats (M) 1200 9 months Anatase/25–70 nm " GPT, GOT # MDA, GSH HC: Necrosis, hydropic degeneration and dead hepatocytes Attia et al. (2013) Swiss albino mice (M) 10, 50, 100 14 days Anatase/20–50 nm " ALT/AST ratio, ALT, AST, ALP, MDA, ROS, Hsp60, Hsp70, p53, Bax, caspases 3 and 9 # GSH, Bcl-2 HC: Increased liver weight, accumulation of mononuclear cells near the sinusoidal vesicle, angiectasis Shukla et al. (2014) Wistar rats (M) 300 14 days NG/50–100 nm " AST, ALT, ALP, MDA # GPx, SOD HC: Centrilobular necrosis, congestion, swelling, and vacuolisation, inflammatory cell infiltration, high apoptotic index Orazizadeh et al. (2014) CD-1 mice (M) 2.5, 5, 10 6 months Anatase/5–6 nm " ALT, AST, ALP, LDH, TC, TG, IL-4, IL-5, IL-12, IFN-γ, GATA3, GATA4, T-bet, STAt3, STAT6, eotaxin, MCP-1, MIP-2 genes # STAT1, TBIL HC: TiO2 accumulation in the liver, angiectasis, hyperaemia, infiltration of Hong et al. (2014) (Continues) KHAN ET AL. 7 10991263, 0, D ow nloaded from https://analyticalsciencejournals.onlinelibrary.w iley.com /doi/10.1002/jat.4626 by M assey U niversity L ibrary, W iley O nline L ibrary on [12/06/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense TABLE 2 (Continued) Study model Oral daily dosage (mg/kg/bw) Treatment timeline Type/particle size Effects on liver References inflammatory cells, macrophage aggregation, hepatic tissue crevice, necrosis, apoptosis, mitochondrial swelling, nuclear membrane collapse and chromatin marginalisation Swiss albino mice (M) 500 21 days Anatase/rutile/50– 75 nm " ROS, GSH, TBARS # SOD, CAT, GPX HC: TiO2 particles entrapping in endosomes and Kupffer cells Shrivastava et al. (2014) Sprague- Dawley rats (M) 10, 50, 200 30 days Anatase/75 nm " T-SOD, GSH/GSSG ratio, Rb, ALT, AST, ALP, MDA # Mo, co, Mn, P, GPx, SOD Wang (2014) Wistar rats (M) 300 14 days NG/<100 nm HC: Damaged lobular structures, vacuolisation and congestion, inflammatory cell infiltration Khorsandi et al. (2015) CD-1 mice (M) 64 28 days Anatase/18 and 120 nm " MDA, TNF-α, IL-6 # T-SOD, GSH No effects on plasma glucose and ROS levels HC: Fracture in tissue fibres Gu et al. (2015) Albino mice (M) 150 14 days Anatase/21 nm " ALT, AST, MDA, TNF-α, IL-6, Nrf2, NF-κB, CD68, Bax, caspase-3 # GSH, Bcl-2 HC: Focal degeneration, necrosis, DNA damage, inflammatory cell infiltration Azim et al. (2015) CD-1 mice (M) 13, 64, 320 14 weeks Anatase/25 nm " MDA, JNK1, p38 MAPK, TNF-α, IL-6, IR, plasma glucose # SOD, GSH " TiO2 accumulation in the liver with increasing dose Hu et al. (2015) Albino rats (M) 5000 Every other day for 60 days NG " AST, ALT, LDH, TLR-2, TLR-4, NF- κB/p65, CYP1B1 and CYP2B HC: Aggregation of macrophages, hepatocytes with large nuclei and intracytoplasmic vacuoles mild congestion of the portal blood vessels, hydrophobic degeneration, and lymphocytic aggregations in portal areas Moustafa and Hussein (2016) Sprague- Dawley rats (M) 150 6 weeks NG/21 nm " AST, ALT, LPO, TNF-α # GSH, TBARS HC: Congestion and dilatation, degenerative changes, vacuolar degeneration, necrosis, mononuclear infiltration Hassanein and El-Amir (2017) C57/BL6 mice (M) 250, 500 14 days NG/21 nm " ILIB, TBIL, ALP, TBA, Oapt1, Mrp3, Cyp2b10, 2c37 HC: Number of mitochondria increased and oedema in ER Yang et al. (2017) Kun Ming mice (M, F) 2000 7 days Anatase/25 nm " AST, ALT, TBIL, MDA # SOD, GSH-PX, Nrf2, NQO1, HO-1, GCLC HC: Dilatation of sinusoids, deranged and swollen hepatocytes, vacuoles, necrosis Niu et al. (2017) 8 KHAN ET AL. 10991263, 0, D ow nloaded from https://analyticalsciencejournals.onlinelibrary.w iley.com /doi/10.1002/jat.4626 by M assey U niversity L ibrary, W iley O nline L ibrary on [12/06/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense TABLE 2 (Continued) Study model Oral daily dosage (mg/kg/bw) Treatment timeline Type/particle size Effects on liver References ICR mice (M, F) 5, 10, 50 60 days Anatase/10, 60, 90 nm " ALP, ALT, ALB, LAP, PChe, TBIL, TP, O2�, H2O2, NO, MDA, CYP1A # SOD, CAT, MT, GST, HSP70, p53, TF, GSHPx HC: TiO2 accumulation in the liver, vascular obstruction, dilation, increase basophils, partial ischemia, swelling mitochondria, vacuoles in mitochondria, nucleolus collapse, scattered chromatin, apoptosis Jia et al. (2017) Sprague- Dawley rats (M) 150 28 days Anatase/30–80 nm " ALP, ALT, AST, LPO # CAT, GST HC: Destructed blood vessels, infiltration of neutrophils and apoptosis, damaged and congested vein, vacuolation, haemorrhage, pyknotic nuclei, dilated sinusoids Shakeel et al. (2018) Sprague- Dawley rats (M) 300 14 days NG " AST, ALT, LPO # TBARS HC: Congestion of the central veins, dilatation of the hepatic sinusoids, focal haemorrhagic, coagulative necrosis, proliferation of Kupffer cells, lymphocytic aggregations, vacuolar degeneration Hassanein and El-Amir (2018) Albino rats (M) 100 60 days Anatase/10 nm " ALT, AST, ALP, MDA, Bax # GPx, SOD, GSH, Bcl-2 HC: Hepatic apoptosis, hepatocellular necrosis, steatosis, sinusoidal dilation with leucocytosis, distortion, disorganisation of the hepatic cords Morgan et al. (2018) Wistar rats (M) 100, 200 60 days Anatase/40 nm " ALT, AST, ALP, ALB, MDA # SOD, GPx, CAT, GSH HC: Liver tissue damage, sinusoidal dilation, vacuolisation and leucocyte infiltration Jafari et al. (2018) Wistar albino rats (M) 1000 21 days Anatase 60 nm " ALT, TNF-α, IL-6, CRP, IgG, NO, VEGF, caspase-3, LPO # cytochrome-P450 HC: Severe degeneration, nuclear pyknosis, karyolysis, cytoplasmic vacuolation, increase in collagen, DNA damage Fadda et al. (2018) Wistar albino rats (F) 0.5, 5, 50 14 days NG/21 nm " CAT # GST, SOD, GPX HC: TiO2 accumulation in the liver Canli et al. (2019) Sprague- Dawley rats (M) 2, 10, 50 90 days Anatase/29 nm " TP, ALB, GLB, GSSG, MDA, IL-1α, IL-4, TNF-α # GSH, GSH-Px, SOD HC: Fatty degeneration, fat vacuoles, vacuolation of mitochondria, changes hepatic metabolomics, disrupted energy metabolism Chen et al. (2019) Wistar rats (M) 10, 50, 100 30 days Rutile/30 nm " ALT, AST, LDH, ALP, MDA, TOS, Bax, p53, NLRP3, caspase 1 and 3, IL- 1β, TNF-α, iNOS # SOD, GPX, CAT, TAC, Bcl-2 Abbasi-Oshaghi et al. (2019) (Continues) KHAN ET AL. 9 10991263, 0, D ow nloaded from https://analyticalsciencejournals.onlinelibrary.w iley.com /doi/10.1002/jat.4626 by M assey U niversity L ibrary, W iley O nline L ibrary on [12/06/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense TABLE 2 (Continued) Study model Oral daily dosage (mg/kg/bw) Treatment timeline Type/particle size Effects on liver References HC: Liver cell degeneration, necrosis, congestion of the sinusoids, lymphocytic infiltration, deposition of collagen, apoptosis Wistar rats (M) 300 14 days 80% anatase + 20% rutile/20 nm " ALT, AST, ALP, LDH, MDA, TOS, TNF-α, NF-κB # SOD, GPx, CAT, TAC HC: Dilatation of congested portal vein, hypertrophy of Kupffer cells, hydropic and vacuolar degeneration, oedema and necrosis and inflammatory cell infiltration Moradi et al. (2019) Swiss albino mice (M) 50, 250, 500 5 days NG/ 21 and 80 nm " AST, ALT, MDA, NO, CAT # GSH HC: Chromosomal fragments and dilatations, distorted and loss in lobular architecture, micro regenerating nodules, mild ballooning and infiltration by lymphocytes, mild fibrosis, swelling and degeneration, significant haemorrhage Ali et al. (2019) NFR mice (M) 5 21 days Anatase E171/201 nm " accumulation of TiO2 in the liver, TNF-α, IL-1β, circulatory cytokines (IL-6, SDF-1) # IL-10 HC: Increased necroinflammatory foci infiltrated with F4/80-positive cells, macrophage recruitment Talamini et al. (2019) CD-1 mice (M) 50 8 and 26 weeks NG/25 nm " Cyp2b9, Cyp2c70, Cyp4a14, GRP78, CHOP, PERK, p-eIF2α, GRP78, CHOP, XBP1-s, ATF6, XBP1-s/XBP1-t, Nrf2, Nqo1, (HO-1), NF-κB Activation of MAPK pathways, IR and increase plasma glucose level Hu et al. (2020) Mice 2.5, 5, 10 9 months NG " HIF-1α, Wnt3, Wnt4, NF-κB, TGF- β1, TGF-β1R, Smad-2, ILK, ECM, calpain 2, α-SMA, c-Myc, collagen I, p38 MAPK phosphorylation, GSK-3β, β-catenin # cyclin D HC: Hepatic inflammatory cell infiltration, hepatic fibrosis Hong et al. (2020) Sprague- Dawley rats (M) 2, 10, 50 28 days Anatase/25 nm " MDA, TNF-α, SOD HC: Liver is the most sensitive organ to nano-TiO2-induced oxidative/ antioxidant biomarker changes Zhou et al. (2020) Wistar rats (M) 300 14 days NG/50–100 nm " ALT, AST, Bax # Bcl-2 HC: Apoptosis in all lobules Orazizadeh et al. (2020) Sprague- Dawley rats (M) 2, 10, 50 90 days Anatase/29 nm " GSSG, MDA # GSH, GSH/GSSG, SOD, hepatic phosphatidylcholine (PC) HC: High oxidative stress, fatty degeneration of hepatocytes, altered lipid metabolism Chen et al. (2020) Albino rats (M) 500 14 days NG/63–142 nm " ALT, ALP, MDA, TLR-4, NF-κB, NIK, TNF-α Mohammed and Safwat (2020) 10 KHAN ET AL. 10991263, 0, D ow nloaded from https://analyticalsciencejournals.onlinelibrary.w iley.com /doi/10.1002/jat.4626 by M assey U niversity L ibrary, W iley O nline L ibrary on [12/06/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense TABLE 2 (Continued) Study model Oral daily dosage (mg/kg/bw) Treatment timeline Type/particle size Effects on liver References # GSH, CAT, acetylcholinesterase HC: Congested and dilated central vein, vacuolated and degenerated hepatocytes with Kupffer cells, leucocytes infiltration, endothelial hyperplasia, leucocytic aggregates Sprague- Dawley rats (F) 50 21 days NG/100 nm " ALT, AST, T.BIL, D.BIL, creatinine, urea, uric acid, Cho, TG, LDL-Cho, MDA, NO, Bax, TNF-α # TP, Alb, HDL-Cho, CAT, SOD, GPx, Bcl-2 HC: Portal vein dilatation, congestion, periportal necrosis, proliferative bile ducts, aggregation of mononuclear cellular infiltration and fibrous tissues Abdel-Wahhab et al. (2021) Kunming mice (M) 2, 20 8 weeks Anatase/10–25 nm " ALP, TNF-α, IL-1β, IL-6, TLR-4, caspases 3 and 9, VEGFA, TGF-β, IFN-γ, MDA # SOD, CAT, GSH HC: Hepatocyte swelling, fat accumulation, inflammatory infiltration Zhao et al. (2021) Sprague- Dawley rats (F) 150 7 days NG/33 nm " AST, ALT, AST/ALT, ALP # SOD1, SOD2, HO-1, GSH, CAT, GCLC, GCLM HC: Ti accumulation in the liver, disordered arrangement of hepatocytes, ballooning degeneration Nie et al. (2021) Wistar rats (M) 100 30 days NG/21 nm " LDH, ALT, AST, TC, TG, LDL, VLDL, plasma glucose # HDL HC: Congestion and dilatation in the central vein Bakour et al. (2021) Balb/c mice (M) 25 21 days NG/28.9 nm " ALT, AST, TC, TG, LDL, MDA, NO, AFP, TNF-α, CEA # CAT, SOD, TP, Alb, TAC, HDL HC: DNA fragmentation, hepatocytes necrosis, dilated and congested blood vessels, fatty droplets Salman et al. (2021) Sprague- Dawley rats (M) 50 21 days NG/28 nm " AST, ALT, MDA, NO, TG, Chol, LDL, Bax, caspase 3, p53 # HDL, GPx, CAT, Bcl-2 HC: Portal tract dilation, proliferation of bile ducts, necrosis in their epithelial cells, fibrosis, DNA fragmentation in hepatic tissue Sallam, Ahmed, El- Nekeety, et al. (2022) Wistar rats (M) 300 21 days Anatase (80%), rutile (20%)/20 nm " ALT, AST, ALP, LDH, TOS, MDA # TAC, SOD, GPx HC: WBCs infiltration, central vein hyperaemia, enlargement of Kupffer cells, dilation of sinusoids, inflammatory cells accumulation, hepatocyte necrosis Shirdare et al. (2022) Sprague- Dawley rats (M) 50 21 days NG/50 nm " AST, ALT, T.BIL, D.BIL, Cho, TG, LDL-Cho, AFP, CEA, NO, MDA, IL-1β, IL-6, TNF-α # Alb, TP, HDL-Cho, CAT, GPx, SOD, IL-10 Sallam, Ahmed, Diab, et al. (2022) (Continues) KHAN ET AL. 11 10991263, 0, D ow nloaded from https://analyticalsciencejournals.onlinelibrary.w iley.com /doi/10.1002/jat.4626 by M assey U niversity L ibrary, W iley O nline L ibrary on [12/06/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense TABLE 2 (Continued) Study model Oral daily dosage (mg/kg/bw) Treatment timeline Type/particle size Effects on liver References HC: Dilatation in the central vein and hepatic sinusoid, vacuolar degeneration, nuclear degeneration, necrosis, pyknosis, karyolysis, peripheral chromatin clumps, fibrosis, proliferation of bile ducts Sprague- Dawley rats (M) 2, 10, 50 90 days Anatase/29 nm " MDA, imbalance in the liver metabolites, effects on glycerophospholipid metabolism pathway, effect on the liver metabolism # differentially expressed phosphatidylcholine (PCs) Chen et al. (2022) Wistar rats (M) 100 28 days NG " AST, ALT, ALP, TC, TG, MDA, IL-1β, IL-6, TNF-α, IFN-γ, γH2A, Bax, α-SMA, fibronectin #CAT, GPx, SOD, GSH, IL-10, Bcl-2, PI3K/AKT signalling pathway HC: Hepatic fibrosis, inflammatory cell infiltration, hepatic sinusoid congestion, widening liver tissue gap and lamellar tissue necrosis Zhang et al. (2023) ICR mice (M) 50 30 days Anatase/7 nm " AST, ALT, ALP, MDA, Bax, caspase- 3 & 9, p53, p-p38, p-p38/p38 # SOD, GSH-Px, GSH, T-AOC, Bcl-2 HC: Hepatocytes were blurred and disordered, swollen and vacuolated, increased apoptosis, steatosis, congestion and dilation of the central veins and infiltration of inflammatory cells around the blood vessels Chang et al. (2023) Wistar rats (M) 20 14 days Anatase/25 and 150 nm " IL-1β # IL-6 and IFN-γ HC: Increase in liver weight, dilated hepatic vein, loss of native morphology of hepatic lobule and portal triad, structural deformities in RBCs found in liver Ali et al. (2023) Sprague- Dawley (M, F) 1, 2 5 days Anatase/<25 nm " genes like NPY and SPP1 (male rats only) AST and ALT (no effect) HC: Increase in intralobular lymphoid infiltration, focal intralobular necrosis in the middle zone of the liver lobule, hepatocyte vacuolisation/steatosis, congestion in the central vein with enlargement of the sinusoids Tassinari et al. (2023) Wistar rats (M) 100 28 NG " AST, ALT, ALP, TC, TG, MDA, IL-6, TNF-α, IFN-γ, IL-1β, TNF-α, γH2A, Bax, α-SMA, fibronectin # SOD, GPx, GSH, CAT, IL-10, Bcl-2 HC: Hepatic fibrosis, hepatic sinusoid congestion, widening liver tissue gap, inflammatory cell infiltration, lamellar tissue necrosis, and impairment of the PI3K/AKT signalling pathway Zhang et al. (2023) 12 KHAN ET AL. 10991263, 0, D ow nloaded from https://analyticalsciencejournals.onlinelibrary.w iley.com /doi/10.1002/jat.4626 by M assey U niversity L ibrary, W iley O nline L ibrary on [12/06/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense et al., 2018). ROS generation results in the disruption of macromole- cules (lipids, DNA, carbohydrates and proteins) (Abdel-Wahhab et al., 2021; Shukla et al., 2014). Lipid peroxidation causes structural changes in the cell membrane and disturbs the overall functions of the cell. Due to lipid disturbances, free radicals (hydroxyl radicals) are gen- erated that cause oxidative damage by increasing the levels of MDA and NO and decreasing hepatic antioxidant enzymes (GSH, CAT, SOD, GPx) (Chen et al., 2020). TiO2 NPs increase levels of oxidative stress and MDA and a decrease in the expression of antioxidant genes (SOD, CAT, GSH) (Jafari et al., 2018). Oral ingestion of TiO2 NPs in rats induces oxidative stress and causes a slight elemental imbalance in the liver (Chen et al., 2020). Mitochondria are one of the main organelles targeted by TiO2 NPs where they cause alterations in mitochondrial membrane proteins, mitochondrial dynamics and morphology (Hirakawa et al., 2004). TiO2 NPs cause morphological alterations by damaging mitochondrial membranes, swelling and ballooning of mitochondria and overall decrease the activity of mitochondria in hepatic cells (Chen et al., 2019; Jia et al., 2017; Teubl et al., 2015; Yang et al., 2017). TiO2 NPs alter the activity of electron transport chain components, including mitochondrial complex I (nicotinamide adenine dinucleotide [NADH] dehydrogenase) and complex II (succinate dehy- drogenase). Alterations in these enzymes result in defective oxidative metabolism that can lead to mitochondrial cytopathy. Also, changes in these complexes may result in an aberrant mitochondrial permeability transition pore, further causing uncoupling of oxidative phosphorylation and depletion of ATP (Mehndiratta et al., 2002). Waseem et al. (2022) have shown that TiO2 NPs drop the activity of mitochondrial complex I and complex II mitochondrial dehydrogenase (complex III) in the rat liver. Also, TiO2 NPs aggravated membrane peroxidation, decreased the activity of Mn-SOD (which is the first line of defence against the toxic oxyradicals), reduced the levels of GSH (crucial for maintaining mitochondrial functionality) and reduced mitochondrial GST (responsible for inactivating the cytotoxic effects of oxidative stress). The endoplasmic reticulum (ER) controls the accurate folding of proteins. Accumulation of misfolded proteins in the ER causes ER stress, which activates an unfolded protein response (UPR). The UPR either maintains homeostasis or triggers cell death to inhibit accumulation of damaged cells (Cao et al., 2017). The UPR has three signal transducers: protein kinase RNA-like ER kinase (PERK); inositol-requiring enzyme 1 alpha/beta (IRE1α/β); and activating transcription factor 6 (ATF6). During ER stress, PERK (ER-resident protein) mediates signal transduction and, together with ATF6 and IRE1, triggers either ROS-ER stress-mediated apoptosis or autophagy (Li et al., 2022; Liu et al., 2015). Nanoparticle-induced ER stress is one of the early biomarkers for the evaluation of nanotoxicity. TiO2 NPs increase the expression of PERK, Bax and ATF6 in HepG2 cell lines. TiO2 NPs induce ER stress that activates PERK/ATF6 signalling pathways contributing to the TiO2-mediated apoptosis of the liver cancer cells (Li et al., 2020). The PERK– eIF2α–ATF4 pathway up-regulates the UPR target genes and induces the folding and excretion of proapoptotic protein C/EBP homologous protein (CHOP), which regulates both lipogenesis and hepatic steatosis (Hernández-Gea et al., 2013). Thus, TiO2 NPs can induce ER stress in the liver cells; however, further studies are needed to elucidate the role of TiO2 NPs in mediating ER stress, apoptosis and autophagy in the liver cells. 10 | INFLAMMATION, APOPTOSIS AND NECROSIS IN THE LIVER Oral administration of TiO2 NPs causes chromatin condensation, nuclear fragmentation and apoptosis in hepatocytes (Cui et al., 2010; Zhu et al., 2012). TiO2 NPs increase the expression of pro-apoptotic TABLE 2 (Continued) Study model Oral daily dosage (mg/kg/bw) Treatment timeline Type/particle size Effects on liver References Sprague- Dawley rats (M) 250 28 Anatase/15 nm " TOS, MDA #TAS, CAT HC: Mononuclear cell infiltration, hyperaemia, glycogen accumulation, fibrosis proliferation, hepatocellular hypertrophy Ogut et al. (2024) Albino rats (M) 500 60 Anatase/72.1 nm " CYP1A1 and NBN Moselhy et al. (2024) Swiss Webster mice (M) 50 5 Anatase and rutile mixture/NG " damage to genomic DNA, p53, MDA # SOD, Gpx HC: Diffusion and degeneration of hepatocytes. Mohamed et al. (2024) C57BL/6 mice (F) 10 28 NG/25–70 nm " body weight, liver weigh, Ti content in liver, AST, ALT, AKP, MDA, keap-1 # GSH, CAT, SOD, Nrf2, Gclc, Gclm Jia et al. (2024) KHAN ET AL. 13 10991263, 0, D ow nloaded from https://analyticalsciencejournals.onlinelibrary.w iley.com /doi/10.1002/jat.4626 by M assey U niversity L ibrary, W iley O nline L ibrary on [12/06/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense genes like p53, Bax, Cyto-c, Apaf-1, caspase-9 and caspase-3, and reduce the expression of anti-apoptotic genes like Bcl-2, indicating that TiO2 NPs mediate apoptosis via the caspase-dependent signalling pathway. This pathway has been demonstrated in vitro in HepG2 cells (Shukla et al., 2013), while in vivo it was confirmed in the liver of rats using the TUNEL assay (Abbasi-Oshaghi et al., 2019). Similar studies using immunoblot analysis also revealed that TiO2 NPs activate the intrinsic pathway of apoptosis by increasing the expression of pro-apoptotic proteins and decreasing the levels of anti-apoptotic protein (Sallam, Ahmed, Diab, et al., 2022; Sallam, Ahmed, El-Nekeety, et al., 2022; Shukla et al., 2014). TEM studies have shown that TiO2 NPs cause the breakdown of nucleolus, dispersed chromatin, apopto- sis and apoptotic cell bodies in the liver cells of mice (Jia et al., 2017). This shows that TiO2 NPs induce liver apoptosis as well as structural damage in the liver. Another study reported that TiO2 NPs increase pro-apoptotic fac- tors (Bax, caspase-3 and p53) and decrease anti-apoptotic factor (Bcl-2) in the rat liver (Abbasi-Oshaghi et al., 2019). The caspases and Bcl-2 proteins are the main regulators of the apoptotic pathway. Translocation of Bax into mitochondria alters the permeability of cytochrome C and further stimulates post-mitochondrial caspases that lead to apoptotic cell death (Jin et al., 2017; Wang, 2014). Necrosis is one of the primary features of liver injury and up-regulates the expression of apoptotic genes (p53, p38, TNF-α, caspase-3, caspase-8, caspase-9). This suggests the role of TiO2 NPs in causing cell necrosis through ROS (Abbasi-Oshaghi et al., 2019; Moradi et al., 2019; Morgan et al., 2018; Sallam, Ahmed, Diab, et al., 2022). TiO2 NPs activate MAPK and nuclear factor kappa B (NF-κB) inflammatory signalling cascades, leading to transient liver inflamma- tion due to increased levels of pro-inflammatory cytokine tumour necrosis factor α (TNF-α) and decreased expression of NF-κB inhibitor A20 (anti-inflammatory gene) and decreased cell viability (Chen et al., 2016). In addition, oral administration of TiO2 NPs causes infil- tration of white blood cells, inflammation with infiltration of white blood cells, Kupffer cell enlargement, sinusoidal dilation with the accu- mulation of inflammatory cells and hepatocyte necrosis (Shirdare et al., 2022). Abbasi-Oshaghi et al. (2019) have reported that TiO2 NPs induce inflammatory responses by increasing the expression of TNF-α (a pro-inflammatory cytokine) and iNOS (major source of reactive nitrogen species) levels dose-dependently in the liver. Zhao et al. (2021) have shown that high dose oral intake of TiO2 NPs causes severe oxidative stress, serious hepatic inflammation, fibrosis and apoptosis in the liver of mice, and these effects were exacerbated with fructose-induced metabolic syndrome. These results suggest that some populations, such as those with metabolic syndrome, may be more at risk of adverse health outcomes associated with TiO2 NPs. Overall, these studies suggest that TiO2 NPs induce necrosis, apo- ptosis, inflammation and other liver impairments via ROS generation, DNA damage, ER stress and mitochondrial stress (Mohammadinejad et al., 2019). The molecular mechanisms by which TiO2 NPs induce apoptosis, necrosis and inflammation have been summarised in Figures 3 and 4. However, the role of TiO2 NPs in molecular signalling pathways towards autophagy in the liver cells remains elusive. 11 | GENOTOXICITY IN THE LIVER Genotoxicity is defined as the ability of biological or chemical agents (harmful substances) to damage the genetic information in the cells F IGURE 3 Mechanisms of hepatotoxicity induced by TiO2 nanoparticles (NPs). TiO2 NPs activate Toll-like receptors (TLRs) and enhance NF-κB-inducible kinase (NIK), leading to nuclear factor-kappa B (NF-κB) response to cause cellular inflammation. TiO2 NPs induce oxidative stress, generate ROS species and increase the expression of stress protein Hsp (heat shock protein) and activate MAPK (mitogen-activated protein kinases) signalling pathway. TiO2 NPs increase the expression of p53 that is involved in increasing the expression of Bax and Bcl2 subsequently increasing the expression of caspases 3 and 9, resulting in apoptosis. TiO2 NPs also induce mitochondrial and ER stress mediated by apoptotic protease activating factor 1 (APAF1) and caspases (caspases 12, 6 and 7). Created with BioRender. 14 KHAN ET AL. 10991263, 0, D ow nloaded from https://analyticalsciencejournals.onlinelibrary.w iley.com /doi/10.1002/jat.4626 by M assey U niversity L ibrary, W iley O nline L ibrary on [12/06/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense (directly or indirectly) triggering genomic instability and mutations that may cause various diseases including cancer (Phillips & Arlt, 2009). Oral exposure to TiO2 NPs causes DNA damage, increases the expres- sion of pro-apoptotic proteins (Bax) and p53 and decreases the expression of anti-apoptotic proteins (Bcl-2), further leading to apo- ptosis and distorted liver function (Abdel-Wahhab et al., 2021; Orazizadeh et al., 2020; Sallam, Ahmed, El-Nekeety, et al., 2022; Shukla et al., 2014). In addition, TiO2 NPs also induced chromosome breaks and polyploidy. In vivo studies have confirmed that TiO2 NPs cause chromosomal aberrations and DNA breaks and can induce gen- otoxicity in the liver, spleen and thymus cells (Ali et al., 2019; Chakrabarti et al., 2019; Manivannan et al., 2020). For more details on TiO2 NP-induced genotoxicity, see Wani and Shadab (2020). 12 | CHALLENGES AND PERSPECTIVES E171 is a widely used additive in food and pharmaceutical formula- tions. Recently, the usage of E171 in food and pharmaceutical prod- ucts was banned in the EU due to its potentially genotoxic effects (Younes et al., 2021). Subsequently, the use of E171 in food was banned in several other countries, including Jordan, Saudi Arabia, Yemen, Qatar, Turkey and Israel. However, other countries including the United States, the United Kingdom, Canada, Japan, Australia and New Zealand are still using E171, as the Food and Drug Administra- tion (FDA), the UK Food Standards Agency (FSA), Health Canada, Japanese National Institute of Health Sciences (NIHS) and Food Stan- dards Australia New Zealand (FSANZ) determined that there is no conclusive scientific evidence that E171 is harmful to human health (FSANZ, 2022; TDMA, 2023). The different regulatory responses to the same evidence are presumably due to different risk tolerance thresholds. For example, France took precautionary regulatory action and banned the usage of E171 in 2019, because the risk to public health could not be conclusively ruled out. Later, following a similar approach, the use of E171 as a food additive was banned in the EU. The EFSA concluded that risk associated with E171, primarily genotoxicity, could not be ruled out. In contrast, other countries have not implemented any restrictions or bans because their regulatory agencies require more conclusive evidence of risk to human health before adding restrictions to products already in use. Orally consumed TiO2 NPs are mostly excreted but some get absorbed via the small intestine and accumulate in various organs including liver, which is the primary detoxification system of the body (Gilbert et al., 2021; Hamilton, 2013; Heringa et al., 2018; Keller F IGURE 4 TiO2 NP-induced oxidative stress triggers inflammation. TiO2 NPs induce oxidative stress and cause hepatic inflammation by changing the histopathology of the liver, generation of protein adducts, lipid peroxidation, necrosis, hepatocyte death, mitochondrial dysfunction and DNA damage. Created with BioRender. KHAN ET AL. 15 10991263, 0, D ow nloaded from https://analyticalsciencejournals.onlinelibrary.w iley.com /doi/10.1002/jat.4626 by M assey U niversity L ibrary, W iley O nline L ibrary on [12/06/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense TABLE 3 Effects of TiO2 NPs on liver cells (in vitro). Cell type Dosage (μg/ml) Treatment timeline (h) Type/size Effects on liver cells References HepG2 1, 10, 100, 250 4, 24, 48 Anatase/<25 nm Rutile/<100 nm Anatase: 2-fold increase in ROS Persistent DNA stranded breaks " Fpg-sensitive sites, p53, mdm2, p21, gadd45α Rutile: 1.4-fold increase in ROS, DNA- stranded breaks but not persistent " Fpg-sensitive sites, p53, mdm2, p21, gadd45α Petkovi�c et al. (2011) SMMC-7721 HL-7702 0.1, 0.5, 1, 5, 10, 50, 100 12, 36, 24, 48 NG/3.7 nm " cell shrinkage, nuclear condensation, ROS, cytotoxicity # GSH Sha et al. (2011) C3A 0.5–256 4, 6, 24 Rutile/7 and 10 nm " DNA damage, ROS, oxidative stress, genotoxicity, IL8 # GSH (Kermanizadeh et al., 2012) HepG2 1, 10, 20, 40, 80 6, 24, 48 Anatase/30–70 nm " cellular uptake, cytotoxicity, oxidative DNA damage, apoptosis, micro nucleated cells, ROS, Hsp60, Hsp70, p53, Bax, Cyto-c, Apaf-1, caspases 3 and 9, hydroperoxide # MSD, GSH, MMP, Bcl-2 Shukla et al. (2013) HepG2 100 24 Anatase 86% Rutile 14%/sP25 (21 nm) " DNA damage, MN frequencies, NF- κB activity No significant transcriptional activation of AP1 Prasad et al. (2014) C3A 10–100 4 Rutile/30.5 nm " ROS Limited effect on glycogen breakdown, glucose, LP release and metabolism Filippi et al. (2015) HepG2 5, 20, 80, 160, 320 1, 9, 12, 16, 24 Anatase/10 nm Rutile/50 nm " cytotoxicity, immunogenicity, TNF-α, MAPK, NF-κB pathway, p38, ERK1/2 phosphorylation, inflammation, Young's modulus, and adhesion force.# A20 Chen et al. (2016) QGY 40, 80 72 NG/21 nm " H2O2, chromosome instability, ROS " telomere length, TRF1, TRF2, POT1, Nrf-2 Wang et al. (2018) HepG2 2.5, 5, 7.5, 10 12, 24, 48 Anatase/10 nm " G1 phase, caspases 3 and 7, apoptosis, VDAC1, Cyt c, αENaC, SIRT3, ADP/ATP, depolarisation and disruption of mitochondria # cell growth and proliferation, S phase, ACSS1, ATP Xia et al. (2018) HepG2 100, 150, 200, 300 24 Rutile/30 nm " LDH, AST, ALT, ROS, apoptosis # cell viability, GSH Abbasi-Oshaghi et al. (2019) HepG2 100 24, 72 P25/21 nm " promoter methylation in CDKN1A, DNAJC15, GADD45A, GDF15, INSIG1, SCARA3, TP53, BNIP3 # global methylation, DNMT3a, DNMT3b, MBD2, UHRF-1 Pogribna et al. (2020) HepG2 10, 50, 100, 200 3, 24 Anatase (80%), rutile (20%)/25 nm " NP uptake No micronuclei expression Brandão et al. (2020) HepG2 2.5, 5, 7.5, 10 48 NG " apoptosis, cell cycle arrest at G1 stage, ROS, ER stress, PERK, ATF6, Bax # cell growth Li et al. (2020) HepG2 4, 8, 12, 50 24 NG/21 nm NG/125 nm " cytotoxicity, oxidative stress, TiO2 uptake # fatty acid oxidation Zhang et al. (2021) 16 KHAN ET AL. 10991263, 0, D ow nloaded from https://analyticalsciencejournals.onlinelibrary.w iley.com /doi/10.1002/jat.4626 by M assey U niversity L ibrary, W iley O nline L ibrary on [12/06/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense et al., 1995; Lima et al., 2004). Considering the potential effects of TiO2 NPs on human liver health, all relevant literature (to date) report- ing toxicity of TiO2 NPs in the liver after oral administration in rodents and humans or in cultured liver cells has been summarised in this review. Briefly, most studies have shown that TiO2 NPs can cause liver toxicity by various mechanisms including increasing oxidative stress, disturbing the antioxidant system, inflammation, apoptosis, necrosis and changing the expression levels of protective genes (Chen et al., 2020; Nie et al., 2021; Sallam, Ahmed, El-Nekeety, et al., 2022). Similarly, orally administered TiO2 NPs can induce toxicity in the liver of rodents from low doses, which are more relevant to daily human exposure, that is, 2–10 mg/kg/bw. Other studies have shown liver toxicity using doses well above normal human intakes, with doses as high as 50–2000 mg/kg/bw. In addition to dose ranges, a variety of particle sizes from 5 to 200 nm or even bulk particles can induce tox- icity in the liver. These studies have also demonstrated that higher doses and smaller particle size are capable of causing more hepatotox- icity in rodents. Adverse effects have been reported following expo- sure to TiO2 NPs in acute treatments for as low as 5 days and in chronic treatments for up to 9 months. The use of both crystalline forms of TiO2, that is, anatase or rutile separately or a mixture of both, can elicit adverse effects on the liver (for details, see Tables 2 and 3). In contrast, there are many studies that have reported very minor or no toxic effects of TiO2 NPs in rodents (Akagi et al., 2023; Blevins et al., 2019; Han et al., 2021; Heo et al., 2020; Lin et al., 2023; Younes et al., 2021) (for details see Table 1). Research supporting the safety of TiO2 raises some shortcomings in evidence suggesting adverse effects of TiO2 NP administration. For example, the level or extent of toxicity of TiO2 NPs is highly dependent on particle characterisation (size, form, purity, surface charge, particle distribution and stability in the experimental medium) and other factors including duration of exposure, its dose range, dosage relevancy to human, type of experi- mental model and sample size (Ali et al., 2019; Kassama & Liu, 2017; Violatto et al., 2023). Firstly, in terms of dose, most animal experi- ments have used doses higher than those encountered in typical human exposure scenarios to observe potential effects of TiO2 NPs more quickly. Some studies have used doses of up to 2000– 5000 mg/kg/bw, which are several orders of magnitude higher than the average daily intake in humans, and therefore, care must be taken in the extrapolation of these results to humans. Conversely, some studies have also shown adverse effects in the dose range of 1– 5 mg/kg body weight, which is within the range of normal human exposure (0.2–3 mg/kg/bw) (Bischoff et al., 2021). This use of high dose range may not intend to represent realistic human exposure levels but rather to provide a safety buffer in risk assessments. There is a difficulty in modelling human TiO2 consumption in ani- mal studies. Studies in rats and mice most commonly deliver TiO2 in a daily bolus dose via oral gavage. In contrast, human consumption of TiO2 NPs occurs over time and is typically spread out across multiple meals and snacks, as individuals ingest products containing TiO2 NPs as part of their daily diet. Delivery of substances via gavage is known to effect the absorption, bioavailability and metabolism compared to other oral routes (Mohammadparast & Mallard, 2023). Furthermore, the use of gavage can induce stress responses, which may alter the effect of a given chemical (Vandenberg et al., 2014). This difference in exposure patterns between animal models and humans should be con- sidered when interpreting study results and extrapolating findings to human risk assessments. Although it is hard to achieve human eating pattern in rodents yet, instead of a single bolus, TiO2 NPs can be administered in food to mimic human consumption pattern. All other factors like study designs, type (nano or bulk particles) and crystalline form of TiO2 NPs, exposure duration, methods of administration and endpoints assessed can impact the consistency and reproducibility of results. For instance, as discussed earlier, TiO2 NPs can be in rutile or anatase or in a mixture form (% proportion vari- ability) and may have diverse surface coatings and can significantly influence their biological interactions and toxicity. Standardisation can be achieved by using only food-grade E171, removing variation in crystalline form and mixture composition. Similarly, duration of expo- sure time should be maximised, alongside preference needs to be given to oral administration of TiO2 NPs rather than other administra- tion routes. Studies have assessed different toxicity endpoints, such as inflammation, oxidative stress, histopathological changes or alter- ations in enzyme activity, and hence, the choice of endpoints can con- tribute to variability in reported outcomes. It is important to consider the totality of evidence and recognise that the effects of TiO2 NPs may depend on specific conditions and contexts. Epidemiological studies are needed to investigate potential asso- ciations between exposure to TiO2 NPs and adverse health effects in human populations. Long-term observational studies can provide valu- able insights into chronic effects on liver and help establish causation. Furthermore, dietary intervention studies in murine models (metabolic associated disease models) based on estimated human intake levels of titanium could clarify the potential risk of E171 in liver health and metabolic disorders. In vivo studies with wider dose ranges via oral administration from different suppliers of E171 (characterising the TABLE 3 (Continued) Cell type Dosage (μg/ml) Treatment timeline (h) Type/size Effects on liver cells References HepG2 1.56, 3.13, 6.25, 12.5, 25, 50, 100, 200 48 Anatase/25 nm " cytotoxicity, epigenetic changes # HADHB, BRIP1, ZNF562 Shi et al. (2023) KHAN ET AL. 17 10991263, 0, D ow nloaded from https://analyticalsciencejournals.onlinelibrary.w iley.com /doi/10.1002/jat.4626 by M assey U niversity L ibrary, W iley O nline L ibrary on [12/06/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense physicochemical properties of E171 obtained from local and interna- tional vendors) to understand variability of effects can be helpful. While standardising the variables of experimental protocols (such as species, dose, duration of experiment and route of exposure), as may occur for regulatory requirements, may lessen the degree of variability we have noted, these studies are prohibitively expensive. In the absence of this standardisation, we must continue to be cautious with the extrapolation of the results of animal studies to understand the risks of TiO2 NPs to human liver health. Addressing these challenges requires a collaborative effort within the scientific community. Some of the challenges discussed could be addressed through data sharing and the use of systematic reviews and meta-analyses. Systematic reviews and meta-analyses that aggregate data from multiple studies can help provide a more comprehensive understanding of the poten- tial risks associated with TiO2 NPs and their impact on liver health. In conclusion, further research is needed to enhance our understanding of the potential risks associated with TiO2 NPs and to develop guide- lines for their safe use in various applications. CRediT STATEMENT Jangrez Khan: Conceptualisation; investigation; visualisation; method- ology; writing original draft. Nick Kim: Supervision; review and edit- ing. Collette Bromhead: Supervision; review and editing; resources. Penelope Truman: Supervision; review and editing. Marlena Kruger: Supervision; review and editing. Beth Mallard: Supervision; concep- tualisation; visualisation; writing—review and editing. ACKNOWLEDGEMENTS This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. Open access pub- lishing facilitated by Massey University, as part of the Wiley–Massey University agreement via the Council of Australian University Librar- ians. Open access publishing facilitated by Massey University, as part of the Wiley - Massey University agreement via the Council of Austra- lian University Librarians. CONFLICT OF INTEREST STATEMENT The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influ- ence the work reported in this paper. DATA AVAILABILITY STATEMENT Data sharing is not applicable to this article as no new data were cre- ated or analyzed in this study. ORCID Nicholas D. Kim https://orcid.org/0000-0001-5523-6385 Collette Bromhead https://orcid.org/0000-0002-2890-2191 Penelope Truman https://orcid.org/0000-0003-1417-1313 Marlena C. 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