Quantification of dietary titanium dioxide (E171) in New Zealand food products to estimate population intake and its role in the pathogenesis of metabolic dysfunction–associated steatotic liver disease (MASLD) : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Health Sciences at Massey University, Wellington, New Zealand

Abstract

Titanium dioxide (TiO₂) is commonly used as a food additive (E171) and has long served as a whitening and opacifying agent in confectionery and processed foods. However, its safety has become the subject of debate, with regulatory bans in several regions following concerns over potential toxicity and health risks associated with nanoparticles. New Zealand (NZ) has not yet imposed bans on E171, and the level of E171 and titanium (Ti) in NZ foods is unknown. TiO₂ has been implicated in liver toxicity, oxidative stress, and metabolic dysfunction; however, its role in metabolic disorders such as metabolic dysfunction–associated steatotic liver disease (MASLD) remains elusive. This thesis presents a representative food survey of Ti intakes/exposure alongside an in vitro study to investigate the potential link between TiO₂ and the pathogenesis of MASLD. A quantitative, comprehensive food survey was conducted to analyse Ti levels in the diet and subsequent population exposure. This survey distinguished between natural Ti intakes and habitual consumption of products containing added TiO₂. A representative range of foods across ten categories was analysed for Ti concentrations using graphite furnace atomic absorption spectroscopy (GFAAS). Using 14-day food intake data from the New Zealand Total Diet Study (NZTDS), the Ti intakes (mg/14 days) were estimated and converted to daily intake doses (mg/kg bw/day) across age, gender, and ethnicity groups. A human hepatoma HepG2 cell line was used in conjunction with oleic acid (OA) treatment to establish a steatotic model and evaluate the effects of TiO₂ (Titanium (IV) oxide nano powder with 21 nm primary particles - P21) and E171 (heterogeneous particle size). Cellular uptake of TiO₂ (E171 and P21) was assessed using a turbidity meter, while cytotoxicity was determined by trypan blue exclusion and neutral red uptake assays. The effects of E171 and P21 on steatosis were investigated using Oil Red O (ORO) staining, complemented by biochemical assays for triglycerides and cholesterol using commercially available kits. Oxidative stress responses were further examined by measuring reactive oxygen species (ROS), total antioxidant capacity (TAC), nitric oxide (NO), and lipid peroxidation (malonaldehyde; MDA). The food survey (43 composites and 180 targeted items) revealed wide variation in Ti concentrations across NZ dietary intakes. Background levels were consistently low in plant and dairy based foods (<0.1–0.7 mg/kg), with natural contributions evident in eggs (1.17 mg/kg), meats (0.765–0.854 mg/kg), dried fruits, berries, and tea leaves (4.77 mg/kg). Among the 180 targeted items, confectionery products displayed a heavy tailed distribution, with several globally recognised brands containing E171 at concentrations exceeding 1000–2200 mg/kg, while chocolate based items ranged from <0.05 to 2076 mg/kg. Toothpaste analysis confirmed that personal care products are the most concentrated point source of TiO₂, with levels up to 6349 mg/kg (0.63% w/w), 2–3 times higher than confectionery and 50–100 times higher than coated chocolates. Overall, the composite survey established a consistent background dietary Ti intake of 500–750 µg/day (0.5–0.75 mg/day), mainly attributable to natural sources, while targeted analysis highlighted residual high dose products and habitual consumption of these products elevates baseline intake and can push exposure beyond upper levels. In vitro, HepG2 steatotic models revealed that E171 and P21 selectively exacerbate cytotoxicity, triglyceride accumulation, and oxidative stress under lipid overload, without significantly altering cholesterol storage or lipid droplet formation. ROS levels increased dose dependently in steatotic cells but remained unchanged in non-steatotic cells. In contrast, NO activity and MDA levels were stable, indicating no progression to inflammatory signalling or lipid peroxidation. Overall, this study demonstrates that TiO₂ exposure in NZ is modest and largely background driven, with only a small number of residual high-dose products contributing to elevated intake. TiO₂ does not universally drive steatosis but selectively perturbs triglyceride metabolism and ROS regulation under metabolic stress. These findings provide a timely baseline for a permitted but declining E171 landscape in NZ and highlight the importance of metabolic context in shaping TiO₂ toxicity.