Combined exposure to titanium nanoparticles and nanoplastics damages the male reproductive system and sperm activity

Abstract

Plastics (PS) and titanium (Ti) are utilized extensively in industrial, household, and medical applications due to their advantageous physicochemical properties. However, these materials have given rise to growing concerns regarding their biosafety. In this study, we investigated the impact of TiO₂ nanoparticles (TiO₂ NPs) and nanoplastics (nPS) on the reproductive function of male zebrafish (Danio rerio). Following a 30-day exposure period, the accumulation of both TiO₂ NPs and nPS in testicular tissue was observed. Male zebrafish demonstrated significant decreases in secondary sexual characteristics, substantial reductions in sex hormone levels, and impaired sperm quality. While these effects were evident following individual exposures to either TiO₂ NPs or nPS, co-exposure significantly intensified the extent of reproductive toxicity. Furthermore, the offspring of exposed males displayed pronounced developmental toxicity. Subsequent RNA sequencing analysis revealed significant upregulation of apoptosis-related genes in testicular tissue after exposure. Concomitant in vitro investigations employing GC-1 spermatogonial cells substantiated that co-exposure instigated apoptosis, plausibly through the activation of the PPARγ/RIPK3 signaling pathway. Collectively, our findings provide robust evidence that co-exposure to TiO₂ NPs and nPS compromises male zebrafish reproductive function and induces transgenerational developmental toxicity, offering important theoretical insights for the prevention and risk assessment of emerging environmental co-contaminants.

Graphical abstract

Microplastics and titanium dioxide are widely used in daily life. In this study, we demonstrated that nanoscale microplastics and titanium dioxide induced necroptosis in the zebrafish reproductive system to varying degrees, leading to diminished secondary sexual characteristics, hormonal disruption, and developmental malformations in the offspring. Combined exposure further aggravated these effects, providing evidence that co-exposure to nanoscale microplastics and titanium dioxide damages the zebrafish reproductive system. These findings highlight the potential risks these substances may pose to human health.

Highlights

• •Strong adsorption between TiO₂ nanoparticles and nanoplastics leads to persistent hybrid aggregates, intensifying reproductive toxicity in male zebrafish.
• •Co-exposure disrupts androgen homeostasis, impairs sperm quality, and induces testicular necroptosis via the PPARγ/RIPK3 signaling pathway.
• •Transgenerational developmental toxicity in offspring reveals the underestimated ecological and health risks of emerging nanopollutant co-contaminants.

1. Introduction

Titanium holds significant industrial value due to its chemical inertness, low cost, and high availability. This white mineral is utilized extensively in photocatalysis, pharmaceutical manufacturing, and food processing industries [[1], [2], [3]]. Clinically, titanium dioxide (TiO₂) is a key component in implantable materials and is frequently utilized in surgical procedures. For instance, in the treatment of various spinal disorders such as adolescent idiopathic scoliosis—a common condition during puberty characterized by spinal deformity—titanium-based implants are commonly employed for long-segment spinal fixation. Our previous studies have demonstrated that these implants can release nanoscale titanium dioxide particles into surrounding tissues and the circulatory system, which in turn aggravates muscle fibrosis [4]. Furthermore, nanoscale titanium dioxide particles finds extensive application in sunscreen products, food, paint, and a variety of other fields. The particles in question have the potential to enter the human body through inhalation or via water sources. Once inside the body, these particles can accumulate and cause harm. It has been reported that in aqueous environments, TiO₂ nanoparticles (TiO₂ NPs) form stable agglomerates with low sedimentation rates. These agglomerates have been shown to induce genomic instability, compromise DNA integrity, and promote high rates of apoptosis [5,6]. Furthermore, there is evidence that TiO₂ NPs have a detrimental effect on the cardiovascular, digestive, respiratory, and immune systems [[7], [8], [9], [10]]. However, the impact of nanoparticle implantation and long-term retention on reproductive capacity, particularly in adolescent populations undergoing such surgeries, remains an important yet largely overlooked concern.

According to recent statistics, global plastic production reached 414 million tons in 2023. Despite increasing efforts to promote biodegradable bioplastics, these materials account for only 0.7 % of total plastic production, approximately 3 million tons. The vast majority of plastic waste remains non-degradable or non-recyclable, leading to accumulation in agricultural lands, rivers, and oceans [11]. Over time, this accumulated plastic waste can degrade into nanoplastics (nPS) through biological degradation, ultraviolet radiation, and mechanical abrasion. nPS have now been detected in freshwater and marine ecosystems worldwide [12]. The study reported the presence of microplastics in both human testicular tissue and semen, with an average concentration of 0.23 ± 0.45 particles/mL in semen and 11.60 ± 15.52 particles/g in testicular tissue [13]. Notably, polystyrene (PS) accounted for 67.7 % of the microplastics detected in testicular samples, while polyethylene (PE) and polyvinyl chloride (PVC) were the predominant types found in semen. Similarly, it has been demonstrated that microplastic exposure significantly impairs sperm motility [[14], [15], [16]]. These findings collectively suggest that microplastic pollution poses a considerable threat to human reproductive health, although the underlying mechanisms remain poorly understood.

The high adsorption capacity of plastics has led to an increased focus on the interactions between plastics and metal particles. Research has demonstrated that nanoplastics (nPS) in aquatic environments possess the capacity to adsorb a range of heavy metals, including Pb, Fe, Al, Mn, Cu, Zn, and Ag. This process has been shown to result in alterations to bioavailability and an enhancement of toxicity to organisms [17]. The aging of nPS has been shown to promote the adsorption process by facilitating the formation of new functional groups [18]. Interestingly, research by Ula Rozman et al. revealed that, owing to their tendency to form multilayer aggregates, nPS exhibit a high adsorption affinity for TiO₂ NPs, with adsorption rates reaching up to 67 %—significantly higher than for other metals such as Zn. Moreover, once adsorbed, TiO₂ NPs demonstrate a low desorption rate and are released only in limited quantities in nanoparticulate form, independent of pH conditions [19]. These findings suggest that TiO₂ NPs may be transported over long distances by nPS and persist in biological tissues and circulatory systems. A number of studies have examined the toxicity of co-exposure to metals and microplastics. Zhang et al. reported that microplastics (MPs) and cadmium (Cd) exhibit antagonistic toxicity toward zebrafish embryos at low concentrations, while high concentrations of MPs result in synergistic sublethal toxicity [20]. In contrast, Hui Yang et al. found that MPs can enhance the toxicity of low-dose Cd in zebrafish, leading to reduced antioxidant enzyme activity, elevated reactive oxygen species (ROS) levels, more severe tissue damage, growth inhibition, and decreased survival rates [21]. Similarly, Santos et al. observed synergistic toxicity between MPs and copper (Cu) in zebrafish embryos [22], and Zike Li et al. reported comparable findings in Yangtze finless porpoises [23]. The inconsistent results obtained from this study suggest that the combined effects of MPs and heavy metals remain inconclusive and require further investigation.

Despite the extensive exploration of the toxic effects of nPS and TiO₂ NPs on various organs and systems in zebrafish in previous studies, there is a paucity of knowledge regarding their impacts on the reproductive system or potential transgenerational effects. In the present study, we employed zebrafish as a model to investigate the reproductive toxicity of nPS and TiO₂ NPs, with a particular focus on whether a synergistic interaction exists between these two pollutants.

2. Results and discussion

2.1. Altered physicochemical properties of nanoparticle mixtures

The morphology of TiO₂ NPs, nPS, and their mixtures was characterized by scanning electron microscopy (SEM) (Fig. 1a). The particle size of TiO₂ NPs ranged from 91 to 341 nm (nm), while the particle size of nPS particles ranged from 78 to 164 nm. In contrast, the mixed TiO₂+PS particles exhibited a narrower size distribution, with measurements ranging from 164 to 196 nm (Fig. 1b). Zeta potential measurements revealed that TiO₂ NPs, carried a surface charge of −22.69 mV, whereas nPS particles had a zeta potential of −8.23 mV. Subsequent to the mixing process, the zeta potential of the TiO₂ + PS composite was −19.98 mV, suggesting an alteration in surface electrostatic properties upon interaction (Fig. 1c).

Fig. 1.

Characteristics of TiO₂ NPs and nPS. a. Electron microscopy images of TiO₂ NPs (left), nPS (middle), and their mixture (right). b. Particle size distribution of TiO₂ NPs (left), nPS (middle), and their mixture (right). c. Statistical analysis of zeta potential for TiO₂ NPs (left), nPS (middle), and their mixture (right). TiO₂: TiO₂ NPs group; PS: nPS group; TiO₂+PS: combined TiO₂ NPs and nPS group.

These results indicate strong adsorption of TiO₂ onto nPS, consistent with previous studies showing a high adsorption affinity between plastics and metal nanoparticles [17]. Such interactions have been demonstrated to promote the persistence of hybrid aggregates in biological tissues and may exacerbate toxicity in comparison with individual exposures.

2.2. Suppression of male secondary sexual characteristics and endocrine disruption by nanoparticle mixtures

In adult male zebrafish, brightly colored and enlarged pectoral and caudal fins represent key secondary sexual characteristics [24]. To evaluate the effects of TiO₂ NPs and nPS on male reproductive traits, we exposed zebrafish to TiO₂ NPs, nPS, or their combination (TiO₂+PS group) in aquatic systems. Exposure to either TiO₂ NPs or nPS resulted in a significant decrease in male secondary sexual traits, as indicated by alterations in fin pigmentation (Fig. 2a). Quantitative analysis revealed a significant decline in pigment intensity in the pectoral and caudal fins of exposed groups. Compared to control males (pectoral: 4316 ± 1283 units; caudal: 4678 ± 1625 units), the TiO₂+PS group displayed markedly diminished pigmentation (pectoral: 962 ± 328; caudal: 1014 ± 491), even lower than untreated females’ group. The TiO₂ group exhibited moderate reductions (pectoral: 1887 ± 1041; caudal: 3269 ± 850), as did the PS group (pectoral: 2444 ± 794; caudal: 3138 ± 902), both showing statistically significant declines compared to the control group (Fig. 2b and c). This phenotypic change resembles that of cyp17a1 knockout zebrafish, in which cyp17a1—an enzyme with 17α-hydroxylase and 17,20-lyase activity—is critical for steroidogenesis pathways producing androgens and estrogens [25].

Fig. 2.

TiO₂ NPs, nPS treatment induced the decline of secondary sexual characteristics and hormone disorder in male zebrafish. a. Combined treatment of TiO₂ NPs and nPS aggravated pigment loss in male zebrafish’s anal and caudal fins. b. Quantification of pigmentation in zebrafish pectoral fins (n=10). c. Quantification of pigmentation in zebrafish caudal fins (n=10). d. Measurement of 11-ketotestosterone concentration in zebrafish gonadal tissue (n=10). e. Measurement of 11β-hydroxyandrostenedione concentration in zebrafish gonadal tissue (n=10). f. Measurement of testosterone concentration in zebrafish gonadal tissue (n=10). g. Measurement of 17β-estradiol concentration in zebrafish gonadal tissue. CT male: untreated male zebrafish; TiO₂: TiO₂ NPs treatment group; PS: nPS treatment group (n=10); TiO₂+PS: combined TiO₂ NPs and nPS treatment group; CT female: untreated female zebrafish. Symbols: # P < 0.05 compared to CT male group; $ P < 0.01 compared to CT male group; & P < 0.001 compared to CT male group; ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.0001.

11-Ketotestosterone (11-KT), a key androgen in zebrafish, plays essential roles in sex differentiation, spermatogenesis, male behavior, and maintenance of secondary sexual traits [26]. In the TiO₂+PS group, 11-KT levels were dramatically reduced to 0.0017 ± 0.00045 pmol/mg, representing only ∼8 % of that in the control group (0.0059 ± 0.0017 pmol/mg). Similarly, TiO₂ NPs and nPS-exposed zebrafish exhibited moderately a modest decline in 11-KT levels (0.0034 ± 0.0013 and 0.0031 ± 0.0011 pmol/mg, respectively. Fig. 2d), respectively. Other key steroid hormones—including testosterone and its precursor 11β-hydroxytestosterone—were also significantly reduced in the TiO₂ + nPS group (0.058 ± 0.046 pmol/mg and 0.0040 ± 0.0019 pmol/mg, respectively), marking the lowest levels among all groups (Fig. 2e–g).

The marked decrease of 11-ketotestosterone and its precursor 11β-hydroxylation suggests that TiO₂ NPs and nPS exposure severely disrupts androgen homeostasis in male zebrafish, which likely underlies the observed attenuation of secondary sexual traits [27]. However, it is important to note that the concentrations utilized in the experimental setting may not accurately reflect the concentrations that would be encountered under real-world conditions. Specifically, the exposure levels of TiO2 nanoparticles and nPS in human studies require further validation.

2.3. Nanoparticle mixtures induced testicular damage and histopathological changes

Following 30 days of exposure, statistical analysis revealed that the combination of TiO₂ NPs and nPS significantly inhibited zebrafish development. Male zebrafish in the TiO₂ + PS group exhibited a marked reduction in body weight (0.24 ± 0.072 g) compared to the control group (0.35 ± 0.060 g. Fig. 3a). Similarly, average body length was significantly decreased in the TiO₂ + PS group (2.88 ± 0.40 cm) compared to the control (3.43 ± 0.26 cm. Fig. 3b). Conversely, zebrafish subjected to TiO₂ or PS exhibited no substantial alterations in body size.

Fig. 3.

TiO₂ NPs and nPS exposure inhibited testis development in male zebrafish. a. Body weight of zebrafish in control, TiO₂, PS, and TiO₂+PS groups (n=10). b. Body length of zebrafish in control, TiO₂, PS, and TiO₂+PS groups (n=10). c. Gonadosomatic index (GSI) of zebrafish in control, TiO₂, PS, and TiO₂+PS groups (n=10). d. H&E staining of zebrafish testes from control, TiO₂, PS, and TiO₂+PS groups. e. Electron microscopy images of zebrafish testicular cells in control, TiO₂, PS, and TiO₂+PS groups. CT: untreated male zebrafish group; TiO₂: TiO₂ NPs treatment group; PS: nPS treatment group; TiO₂+PS: combined TiO₂ NPs and nPS treatment group. Symbols: $ P < 0.01 compared to CT group; & P < 0.001 compared to CT group; ∗ P < 0.05; ∗∗ P < 0.01.

To further assess the impact on reproductive function, the gonadosomatic index (GSI) was evaluated. The GSI of the TiO₂ + PS group was significantly lower (10.0 ± 2.02 %) than that of the control group (13.6 ± 1.05 %), indicating impaired testicular development (Fig. 3c).

Histological analysis revealed substantial structural alterations in the testes of the TiO₂ + PS group. These included reduced density of seminiferous tubules, enlarged intercellular spaces, and varying degrees of vacuolization. Although groups exposed to TiO₂ NP and nPS also exhibited signs of testicular atrophy, no significant vacuolization was observed (Fig. 3d).

Regarding the male reproductive system, TiO₂ NPs has been reported to penetrate the blood-testis barrier, directly damaging testicular tissue by activating the MAPK signaling pathway, inducing oxidative stress and inflammatory responses, and ultimately causing germ cell apoptosis and impaired spermatogenesis [28]. In accordance with these findings, we observed that zebrafish exposed to TiO₂ NPs and nPS demonstrated retarded development in comparison to the control group, as indicated by a substantial decrease in body weight and length. Notably, the gonadosomatic index (GSI) was significantly decreased in the TiO₂+PS group relative to controls, indicating marked reproductive toxicity. We conducted istological examination, which revealed evidence of developmental impairment of the testes. This impairment was characterized by sparse seminiferous tubules, widened interstitial spaces, and varying degrees of vacuolization.

TEM further confirmed testicular damage in the TiO₂ + PS group. Aberrant intracellular structures, apoptotic bodies, and swollen mitochondria were observed, along with the presence of unidentified particulate matter between cells. Given the strong adsorption interactions between nPS and TiO₂ NPs observed in our physicochemical analyses, we speculate these deposits correspond to complexes formed by these nanoparticles. These findings suggest that TiO₂ NP and nPS co-exposure increases apoptotic activity in testicular cells, contributing to developmental impairment of the zebrafish reproductive system (Fig. 3e). However, it is imperative to acknowledge the inherent limitations of zebrafish toxicity testing. Zebrafish inhabit aquatic environments, where the types and concentrations of toxins encountered differ significantly from those in the human environment, making it impossible to fully replicate human conditions. Additionally, the mechanisms by which zebrafish metabolize toxic substances in their liver and kidneys exhibit certain differences when compared to those observed in humans. Consequently, a more comprehensive interpretation of toxicity necessitates further research in mammals.

2.4. Nanoparticle mixtures impaired sperm quality

To evaluate the effect of TiO₂ NPs and nPS on sperm quality, a computer-assisted sperm analysis (CASA) system was employed. The sperm concentration in the TiO₂ + PS group was significantly reduced (0.60 ± 0.32 × 10⁷/mL) compared to the control group (3.41 ± 0.83 × 10⁷/mL. Fig. 4a). Furthermore, a decline in sperm concentrations was observed in both the TiO₂ group and the PS group, with concentrations measuring 1.66 ± 1.14 × 10⁷/mL and 1.45 ± 0.52 × 10⁷/mL, respectively.

Fig. 4.

TiO₂ NPs and nPS exposure impaired sperm quality in male zebrafish. a. Sperm concentration in male zebrafish under different treatments (n=10). b. Sperm deformity rate in male zebrafish under different treatments (n=10). c. Sperm motility rate in male zebrafish under different treatments (n=10). d. Curvilinear velocity (VCL) of sperm in male zebrafish under different treatments (n=10). e. Average path velocity (VAP) of sperm in male zebrafish under different treatments (n=10). f. Straight-line velocity (VSL) of sperm in male zebrafish under different treatments (n=10). g. Representative images of typical sperm.

In addition to reduced sperm concentration, sperm morphological abnormalities were markedly increased in the TiO₂+PS group, with 90 ± 8.43 % of sperm exhibiting deformities such as enlarged heads or broken tails (Fig. 4b). The malformation rates in the TiO₂ group and PS group were 37 ± 12.18 % and 42 ± 6.40 %, respectively, both significantly higher than in the control group (10 ± 5.12 %).

Following activation, the total motility of sperm in the control group was 82 ± 5.1 %. This was significantly reduced in the TiO₂+PS (40 ± 5.36 %), TiO₂ (55 ± 7.38 %), and PS (50 ± 4.33 %) groups (Fig. 4c. P < 0.05). Furthermore, a significant decrease in key kinetic parameters was observed in the exposure groups compared to the control group. These parameters include curvilinear velocity (VCL), average path velocity (VAP), and straight-line velocity (VSL) (Fig. 4d–f, P < 0.05). Impaired motility markedly compromises the ability of sperm to fuse with oocytes, which is consistent with previous findings [[29], [30], [31]].

Collectively, these data indicate that co-exposure to TiO₂ NPs and nPS exerts a synergistic toxic effect on sperm quality in zebrafish, characterized by decreased concentration, increased malformation rates, and impaired motility.

2.5. Subsequent to exposure to nanoparticle mixtures induced developmental toxicity by the offspring

Previous studies have shown that maternal exposure to TiO₂ nanoparticles (TiO₂ NPs) and nanoplastics (nPS) during pregnancy impairs reproductive capacity, manifested by reduced egg production and fertilization rates, and further induces developmental abnormalities in the offspring [32,33]. To further investigate the potential effects of TiO₂ NPs and nPS on progeny development, in vitro fertilization was performed using sperm collected from exposed male zebrafish, and embryonic development was monitored. The fertilization rate revealed a significantly increased non-hatching rate in the TiO₂ + PS group, with 48 ± 10.15 % of fertilized eggs failing to hatch, compared to 10 ± 4.72 % in the control group. The non-hatching rates for TiO₂ NP and nPS groups were also elevated (20 ± 4.23 % and 30 ± 4.98 %, respectively), though still lower than that of the TiO₂ + PS group (Fig. 5a, P < 0.05).

Fig. 5.

TiO₂ NPs and nPS exposure impaired the development of offspring from male zebrafish. a. Proportion of unhatchability eggs in offspring from control, TiO₂, PS, and TiO₂+PS groups (n=250). b. Survival rate at 3 days post-fertilization (dpf) of offspring from control, TiO₂, PS, and TiO₂+PS groups (n=250). c. Malformation rate of larvae at 3 dpf from control, TiO₂, PS, and TiO₂+PS groups (n=250). d. Representative images of developmental malformations in offspring larvae from CT, TiO₂, PS, and TiO₂+PS groups. CT: untreated male zebrafish; TiO₂: TiO₂ NPs treatment group; PS: nPS treatment group; TiO₂+PS: combined TiO₂ NPs and nPS treatment group. Symbols: # P < 0.05 compared to CT group; $ P < 0.01 compared to CT group; & P < 0.001 compared to CT group; ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.0001.

Moreover, survival analysis at 3 days post-fertilization (dpf) showed that only 26 ± 6.29 % of larvae from the TiO₂+PS group remained viable, a sharp decline compared to 97 ± 1.58 % survival in the control group (Fig. 5b). Morphological assessment revealed that approximately 63 ± 6.08 % of larvae in the TiO₂+PS group exhibited developmental abnormalities, including yolk sac edema, pericardial edema, and axial curvature. In contrast, the malformation rate in the control group was only 9 ± 3.16 %. Larval deformity rates in the TiO₂ and PS groups were 23 ± 11.99 % and 30 ± 11.65 %, respectively—significantly higher than the control but lower than the TiO₂ + PS group (Fig. 5c, P < 0.05). Representative images of zebrafish embryos at 12 hpf and 3 dpf are shown in Fig. 5d, illustrating the morphological differences between groups.

Regarding TiO₂ NPs, previous studies have shown that prolonged exposure reduces parental egg production and fertilization rates and lowers hatching success. Moreover, TiO₂ NPs exacerbates the reproductive toxicity of other environmental contaminants such as tris(1,3-dichloro-2-propyl) phosphate and bisphenol A. However, the available evidence regarding its effects on offspring development remains limited [[34], [35], [36]]. In our study, zebrafish exposed to nPS exhibited significantly increased rates of unfertilized eggs and malformations, and co-exposure to TiO₂ NPs further aggravated nPS-induced reproductive toxicity, producing more pronounced developmental deformities.

Collectively, these findings demonstrate that TiO₂ NPs and nPS exert varying degrees of toxicity on zebrafish reproductive function and offspring development, with their combined exposure markedly amplifying these effects. We speculate that this enhancement is primarily due to the strong adsorption of nPS onto TiO₂ NPs, forming TiO₂ NPs-nPS complexes that increase the absolute value of the zeta potential and significantly decrease colloidal stability, thereby intensifying ROS generation and genomic damage within zebrafish tissues. Additionally, the ability of TiO₂ NPs to penetrate cellular membranes likely contributes substantially to this process.

2.6. Nanoparticle mixtures impaired reproduction system by PPAR signaling pathway

To explore the molecular mechanisms underlying reproductive impairment caused by TiO₂ NPs and nPS, RNA sequencing was performed on testes collected from zebrafish in the TiO₂+PS and control groups. A total of 32,520 valid transcripts were identified, among which 29 genes were significantly upregulated and 79 were downregulated in the TiO₂+PS group relative to controls (Fig. 6a).

Fig. 6.

TiO₂ NPs and nPS exposure may induce necroptosis in testes of male zebrafish via the PPAR signaling pathway. a. Number of differentially expressed genes in testes tissue between CT and TiO₂+PS groups by RNA-seq analysis. b. GO enrichment analysis of differentially expressed genes in testes tissue between CT and TiO₂+PS groups. c. KEGG pathway enrichment analysis of differentially expressed genes in testes tissue between CT and TiO₂+PS groups. d. Heatmap of RNA-seq results showing gene expression differences between CT and TiO₂+PS testes tissues. e-h. Relative mRNA expression levels of rxr, slc27a6, pparγ and cyp7a1 in testes tissue from CT, TiO₂, PS, and TiO₂+PS groups (n=10). i. SOD levels in testes tissue from CT, TiO₂, PS, and TiO₂+PS groups (n=10). j. MDA levels in testes tissue from CT, TiO₂, PS, and TiO₂+PS groups (n=10). CT: untreated male zebrafish; TiO₂: TiO₂ NPs treatment group; PS: nPS treatment group; TiO₂+PS: combined TiO₂ NPs and nPS treatment group. Symbols: # P < 0.05 compared to CT group; $ P < 0.01 compared to CT group; & P < 0.001 compared to CT group; ∗ P < 0.05; ∗∗ P < 0.01.

GO and KEGG pathway enrichment analyses revealed that the differentially expressed genes were primarily involved in pathways related to metabolism, mineral absorption, and necroptosis (Fig. 6b and c). Notably, the peroxisome proliferator-activated receptor (PPAR) signaling pathway was significantly affected. A heatmap of differentially expressed genes in this pathway is shown in Fig. 6d. In addition, the necroptosis pathway ranked among the top 15 most enriched pathways, consistent with previous findings that the toxic mechanisms of TiO₂ NPs and nPS primarily involve oxidative stress, mitochondrial damage, and endocrine disruption [37,38].

To validate the RNA-seq results, several key genes within the PPAR pathway were selected for quantitative PCR analysis. Results demonstrated that rxr and slc27a6 mRNA expression levels were significantly reduced in the TiO₂+PS group compared to controls, whereas pparg and cyp7a1 expression levels were markedly upregulated (P < 0.05. Fig. 6e–h). These findings suggest that dysregulation of the PPAR signaling pathway may contribute to TiO₂ + PS-induced reproductive toxicity in zebrafish.

2.7. Nanoparticle mixtures induce necroptosis of GC-1 spermatogonial cells via PPAR signaling pathway

To further elucidate the effects of TiO₂ NPs and nPS on testicular cells, an in vitro study was conducted using the GC-1 spg cell line. Intracellular ROS levels were measured using the DCFH-DA fluorescent probe (Fig. 7a). The results showed that the TiO₂ + PS group exhibited significantly higher ROS levels than the other three groups.

Fig. 7.

TiO₂ NPs and nPS exposure increase Necroptosis in GC-1 cell line. a. Fluorescence images of ROS staining in GC-1 cells under different treatment conditions (n=3). b. Flow cytometry analysis of necroptosis cell proportions in GC-1 cells under different treatments (n=5). c. AO/EB staining in GC-1 cells under different treatment conditions (n=3). d–j. Western blot analysis and quantification of related proteins in GC-1 cells under different treatments (n=3). K. Immunofluorescence detection of p-RIPK3 in GC-1 cells under different treatment conditions (n=3). CT: untreated group; TiO₂: TiO₂ NPs treatment group; PS: nPS treatment group; TiO₂+PS: combined TiO₂ NPs and nPS treatment group. Symbols: # P < 0.05 compared to CT group;$ P < 0.01 compared to CT group; & P < 0.001 compared to CT group; ns P＞0.05; ∗ P < 0.05; ∗∗ P < 0.01, ∗∗∗ P < 0.0001.

Flow cytometry analysis revealed that exposure to either TiO₂ NPs or nPS alone increased the apoptosis rate of GC-1 spg cells to 10.2 % and 9.33 %, respectively, compared to 3.91 % in the control group. Notably, the TiO₂+PS co-treatment group showed a markedly higher apoptosis rate of 20.6 %, significantly exceeding that of all other groups (Fig. 7b).

Immunofluorescence staining demonstrated that TiO₂ NPs and nPS exposure led to elevated phosphorylation of RIPK1, indicative of activated cell death pathways (Fig. 7c). This observation was confirmed by protein expression, which revealed that treatment with TiO₂ NPs and nPS significantly increased the expression of PPARγ and the apoptosis-associated marker p-p65 in GC-1 spg cells (Fig. 7d–j).

Consistent with the RNA-seq results from zebrafish testis tissue, activation of the PPARγ signaling pathway was confirmed in the GC-1 spg cells following exposure to TiO₂ NPs and nPS. Taken together, these findings suggest that TiO₂ NPs and nPS induce necroptosis in the zebrafish reproductive system via the PPAR signaling pathway.

To further clarify the role of the PPARγ/RIPK3 pathway in the co-exposure to TiO₂ NPs and nPS, we employed GW9662, a PPARγ antagonist, to inhibit the activation of this signaling pathway. After adding GW9662 to the TiO₂ + nPS treatment group, the previously disrupted sex hormone levels showed a significant improvement (Fig. 8a–c). CASA analysis further revealed that sperm quality in male zebrafish was markedly restored, with increased sperm concentration and motility and reduced deformity rates (Fig. 8d–f). The oxidative stress markers SOD and MDA in testicular tissues also returned to levels comparable to the control group (Fig. 8g and h). These findings indicate that GW9662 effectively mitigated TiO₂ NPs–nPS–induced gonadal damage and reduced oxidative stress in male zebrafish.

Fig. 8.

The necroptosis induced by TiO₂ NPs and nPS exposure was suppressed by GW9662.a. Measurement of 17β-Estradiol concentration in zebrafish gonadal tissue from CT, TiO₂+PS, TiO₂+PS+DMSO, TiO₂+PS+GW9662 groups (n=10). b. Measurement of Testosterone concentration in zebrafish gonadal tissue (n=10). c. Measurement of 11β-hydroxyandrostenedione concentration in zebrafish gonadal tissue (n=10). d. Sperm concentration in male zebrafish from CT, TiO₂+PS, TiO₂+PS+DMSO, TiO₂+PS+GW9662 groups (n=10). e. Sperm motility in male zebrafish under different treatments (n=10). f. Sperm abnormality rate in male zebrafish under different treatments (n=10). g. SOD levels in testes tissue from CT, TiO₂+PS, TiO₂+PS+DMSO, TiO₂+PS+GW9662 groups (n=10). h. MDA levels in testes tissue from CT, TiO₂+PS, TiO₂+PS+DMSO, TiO₂+PS+GW9662 groups (n=10). i. AO/EB staining in GC-1 cells under different treatment conditions (n=3). j and o. Flow cytometry analysis of necroptosis cell proportions in GC-1 cells under different treatments (n=5). k-n. Western blot analysis and quantification of related proteins in GC-1 cells under different treatments (n=3).

In GC-1 spg cells, AO/EB staining demonstrated that GW9662 significantly decreased the proportion of necroptotic cells (Fig. 8i). Flow cytometry analysis further confirmed that cell death was markedly lower in the TiO₂ + nPS + GW9662 group compared with the TiO₂ + nPS group (Fig. 8j–o). Consistent with these observations, Western blot analysis showed that the levels of necroptosis-related protein markers p-MLKL and p-RIPK3 were significantly reduced in the TiO₂ + nPS + GW9662 group (Fig. 8k–n).

Overall, GW9662 markedly reduced oxidative stress in zebrafish gonadal tissues and rescued sperm quality by inhibiting the activation of the PPARγ pathway, which aligns with the findings from our previous RNA-sequencing analysis.

A pivotal discovery of this study is that co-exposure to TiO2 NPs and nPS instigates considerable synergistic toxicity in the male zebrafish reproductive system, with severity far surpassing that of single exposure. This “1 + 1>2″ effect is not merely a simple additive toxicity, but rather, it unveils unique interactions between the two nanopollutants. The present study proposes that the distinctiveness of this synergistic effect manifests primarily at two levels:

Firstly, distinctive physicochemical interactions act as the point of departure for the manifestation of synergistic effects. As demonstrated by our characterization results, TiO₂ NPs and nPS rapidly form stable hybrid aggregates in water. The distinguishing characteristic of this interaction is its high adsorption rate and low desorption rate, which leads to the formation of complexes that possess physicochemical properties that are distinct from those of their individual components [39,40]. Secondly, the cascade amplification from physical adsorption to biological effects constitutes the core mechanism of synergistic effects. The experimental data, collected both in vitro and in vivo, indicate a significant activation of the PPARγ/RIPK3-mediated necrotic apoptosis. It is hypothesised that synergistic effects may intensify cellular stress through multiple pathways [41,42].

3. Conclusion

In the male zebrafish model, exposure to TiO2 NPs and nPS resulted in varying degrees of reproductive system damage, with significantly enhanced toxicity observed under co-exposure conditions. The deposition of TiO₂ NPs–nPS complexes in the testes has been shown to induce necroptosis through the PPAR pathway, resulting in impaired reproductive capacity and developmental abnormalities in offspring. The present study discloses the deleterious effects of combined TiO₂ NPs and nPS contamination on the reproductive health of aquatic organisms, thus giving rise to concerns regarding the potential risks to human health.

4. Experimental section

Ethical statement

All experimental procedures involving animals were reviewed and approved by the Ethics Committee of Xiangya Hospital, Central South University (Approval No.:2024091060).

4.1. Characterization of nanoparticles

TiO₂ NPs and polystyrene nPS were purchased from Wanjing New Materials Co., Ltd. (Hangzhou, China), with an average diameter of 30–50 nm and a purity of ≥99.9 %. The morphology and particle size of TiO₂ NPs and nPS were characterized using scanning electron microscopy (SEM; Sigma 300, Zeiss, Germany).

4.2. Zebrafish maintenance and experimental design

Adult male zebrafish (Danio rerio) used in this study were obtained from the Institute of Hydrobiology, Chinese Academy of Sciences. A total of 400 healthy 3-month-old males were randomly divided into four groups (n = 100 per group) and maintained under standard aquaculture conditions (28 ± 0.5 °C, 14 h light/10 h dark photoperiod). The TiO₂ group was exposed to a suspension containing 50 μg/L titanium dioxide nanoparticles (TiO₂ NPs), while the PS group received 100 μg/L polystyrene nanoplastics (nPS). The combined TiO₂+PS group was treated with both TiO₂ NPs and nPS at concentrations of 50 μg/L and 100 μg/L, respectively. All groups were fed daily with commercial feed at approximately 2 % of their body weight.

4.3. In vitro fertilization and sperm analysis

Following the exposure period, male zebrafish were used for artificial fertilization. Sperm was collected by gently pressing the abdomen and immediately diluted in pre-chilled Hank's Balanced Salt Solution (HBSS, pH 7.4; Thermo Fisher Scientific, USA). Sperm quality and motility were analyzed within 2 min using a computer-assisted sperm analysis system (CASA, Hamilton Thorne HTM-IVOS II). A 10 μL aliquot of diluted sperm suspension (1:10 in HBSS) was mixed with 100 eggs (sperm-to-egg ratio of 1:10), and 1 mL of system water (28 °C) was added to activate fertilization. After 30 s, the fertilization process was terminated by washing the eggs three times with fresh embryo medium (0.3 % Instant Ocean).

4.4. Embryo incubation and developmental assessment

Fertilized eggs (n = 200 per group) were transferred to 6-well plates (10 embryos/mL per well) and incubated at 28.5 °C under a 14 h light/10 h dark photoperiod. Hatching rate was recorded at 48 h post-fertilization (hpf), while survival and malformation rates were assessed at 72 hpf using a stereomicroscope.

4.5. Zebrafish pigmentation analysis

Adult zebrafish were anesthetized in 0.02 % MS-222 (Sigma) until opercular movement ceased (approximately 3 min). Fish were then placed laterally on a transparent Petri dish lined with moist sponge. High-resolution lateral images were taken using a Nikon digital camera (Japan) under fixed LED cold lighting and against a white background, ensuring full extension and visibility of the caudal and anal fins. Fish were immediately transferred to fresh water for recovery. Pigmentation analysis was performed using ImageJ software (v1.53).

4.6. Steroid quantification by liquid chromatography-tandem mass spectrometry (LC-MS/MS)

Following treatment, zebrafish from each group were euthanized humanely and transferred into silanized tubes, then snap-frozen on dry ice. Gonads were rapidly dissected and frozen in liquid nitrogen, followed by fine grinding and freeze-drying using a Mikro Dismembrator S (Sartorius, Göttingen, Germany). Approximately 50 mg of the dried tissue was transferred into a 2-mL microcentrifuge tube and resuspended in 900 μL of ultrapure Milli-Q water (Millipore, Burlington, MA) and 100 μL of Milli-Q water containing deuterated internal standards. A stainless-steel bead was added to each tube, and samples were homogenized at 3.3 m/s for 1.5 min using an Omni Bead Ruptor 24 (Kennesaw, GA). The homogenates were then centrifuged at 8000 g for 5 min. Supernatants were collected into glass tubes and extracted twice with methyl tert-butyl ether (MTBE, 1:3 v/v). The remaining pellet was resuspended in 1 mL Milli-Q water, re-homogenized, and extracted using the same procedure. All MTBE fractions from each sample were combined and evaporated to dryness under a nitrogen stream at 45 °C. The residues were reconstituted in 1 mL methanol and passed through Phree columns (Phenomenex, Torrance, CA) to remove residual phospholipids. The columns were washed with an additional 1 mL methanol, and the resulting 2 mL eluate was evaporated to dryness and reconstituted in 150 μL of 50 % methanol for analysis. Steroid hormones were separated and quantified using an Acquity UPLC system (Waters, Milford, CT) coupled to a Xevo TQ-S triple quadrupole mass spectrometer, as previously described [21].

4.7. Determination of oxidative stress parameters

Testes were collected from each group of male zebrafish and homogenized in 0.01 mol/L PBS using a homogenizer. Nine milliliters of PBS were added per gram of tissue. The homogenates were centrifuged at 3000 g for 20 min at 4 °C. The supernatants were collected, diluted with PBS, and stored at −80 °C until analysis. Measurements were performed using ELISA kits purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd. (Shanghai, China). Total protein concentrations of zebrafish samples were quantified using a BCA assay kit (Wuhan Boster Biological Technology Co., Ltd., China) for normalization.

4.8. Tissue processing and histological analysis

Following treatment, zebrafish were euthanized by spinal transection under anesthesia with 0.02 % MS-222 (Sigma-Aldrich, E10521). Whole fish were fixed in 4 % paraformaldehyde (PFA, pH 7.4; Sigma-Aldrich, 158127) at 4 °C for 24 h. Fixed tissues were dehydrated through a graded ethanol series (70 %, 80 %, 95 %, 100 %), cleared with xylene (Sigma-Aldrich, 214736), and embedded in paraffin using a HistoCore Arcadia H embedding system (Leica Biosystems). Serial sagittal sections (4 μm thick) were prepared using a rotary microtome (Leica RM2235) and mounted onto poly-L-lysine–coated slides (Thermo Fisher Scientific, J2800AMNZ).

4.9. Hematoxylin and eosin (H&E) staining

Paraffin-embedded sections were deparaffinized in xylene, rehydrated through a graded ethanol series (100 %, 95 %, 70 %), and rinsed with distilled water. Sections were stained with Harris hematoxylin (Sigma-Aldrich, HHS32) for 5 min, differentiated with 1 % acid ethanol (70 % ethanol containing 1 % HCl), and blued in Scott's tap water substitute (0.5 % NaHCO₃ and 2 % MgSO₄). Cytoplasmic staining was performed with 0.5 % aqueous eosin Y solution (Sigma-Aldrich, 318906) for 1 min. After dehydration and clearing, slides were mounted using neutral balsam mounting medium (Sigma-Aldrich, 03989) and examined under a light microscope.

4.10. Transmission electron microscopy (TEM)

Tissue samples were collected and fixed in electron microscopy fixative solution (Servicebio, Wuhan, China), then post-fixed in 1 % osmium tetroxide for 1–2 h. Dehydration was performed through a graded ethanol series (30 %, 50 %, 70 %, 80 %, 90 %, 95 %). After acetone treatment, samples were embedded in resin. Ultrathin sections (70–90 nm) were prepared using an ultramicrotome and stained with lead citrate and uranyl acetate (saturated in 50 % ethanol) for 5–10 min each. Sections were examined using a Hitachi H-7650 transmission electron microscope (Thermo Fisher Scientific, Waltham, MA).

4.11. Quantitative PCR (qPCR)

Following anesthesia with 0.02 % MS-222 (Sigma-Aldrich), treated male zebrafish were sacrificed and testes were rapidly dissected under RNase-free conditions. Total RNA was extracted using TRIzol® reagent (Invitrogen, Cat. No. 15596026) according to the manufacturer's instructions. Briefly, 30 mg of testicular tissue was homogenized in 1 mL TRIzol, followed by phase separation with 200 μL chloroform and RNA precipitation with isopropanol. The RNA pellet was washed with 75 % ethanol and dissolved in 30 μL RNase-free water. RNA concentration and quality were assessed prior to reverse transcription using the PrimeScript™ RT Reagent Kit (Takara, Cat. No. RR037A). Quantitative PCR was performed with SYBR Green chemistry to amplify target genes including rxr, slc27a6, pparγ, cyp7a1, and gapdh. Gene expression levels were analyzed using the comparative CT method (2^–ΔΔCT). Primer sequences are listed in Supplementary Tables S1–2.

4.12. RNA-seq and bioinformatics analysis

Total RNA was extracted from zebrafish testis samples using TRIzol reagent (Invitrogen). RNA purity (A260/A280 ratio between 1.8 and 2.2) and integrity (RIN ≥8.0) were assessed using a Nanodrop spectrophotometer and Agilent 2100 Bioanalyzer, respectively. Sequencing libraries were prepared using the NEBNext® Ultra™ II RNA Library Prep Kit for Illumina, and library quality was verified with the Agilent 2100 Bioanalyzer. Paired-end 150 bp sequencing was performed on an Illumina NovaSeq 6000 platform.

Raw reads were quality-checked using FastQC, and clean reads were aligned to the zebrafish reference genome (GRCz11) using Hisat2. Gene expression was quantified using featureCounts, and differentially expressed genes (DEGs) were identified using DESeq2 (v1.34.0) with a cutoff of |log₂FoldChange| ≥ 1 and adjusted p-value (padj) < 0.05. Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the clusterProfiler package (v4.0), with a significance threshold of p.adjust <0.05.

4.13. Cell culture and flow cytometry analysis

GC-1 spermatogonial cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10 % fetal bovine serum (FBS; Gibco) under standard conditions (37 °C, 5 % CO₂). Cells were treated with TiO₂ nanoparticles (TiO₂ NPs) and nanopolystyrene particles (nPS) for 24 h. After treatment, both adherent and floating cells were collected, washed with PBS, and resuspended in Annexin V binding buffer (BD Biosciences). Then, 5 μL Annexin V-FITC and 5 μL propidium iodide (PI, 20 μg/mL) were added, followed by incubation in the dark for 15 min.

Apoptosis was evaluated using a BD FACSCanto II flow cytometer (BD Biosciences), and 10,000 events were recorded per sample. Data were analyzed using FlowJo v10.8 software. Apoptotic cells were defined as Annexin V⁺/PI⁻ (early apoptosis) and Annexin V⁺/PI⁺ (late apoptosis/necrosis). Untreated cells and cells treated with staurosporine (1 μM for 4 h) served as negative and positive controls, respectively.

4.14. Reactive oxygen species (ROS) detection

Cells were seeded in 24-well plates and subjected to the indicated treatments. Intracellular ROS levels were detected using a ROS assay kit (Beyotime, Guangzhou, China). According to the manufacturer's instructions, DCFH-DA was diluted 1:1000 in serum-free medium to a final concentration of 10 μM. After removing the culture medium, 100 μL of diluted DCFH-DA was added to each well and incubated at 37 °C for 20 min. Cells were then washed three times with PBS and observed under a fluorescence microscope for imaging.

4.15. Statistical analysis

All data were analyzed using SPSS 25.0 software (IBM, Chicago, IL, USA). Data are presented as mean ± standard deviation (SD). Differences between two or more groups were analyzed using one-way ANOVA or Student's t-test. Levene's test was used to assess homogeneity of variance. Dunnett's T3 test was applied when variances were unequal; otherwise, the least significant difference (LSD) test was used. A p-value <0.05 was considered statistically significant. Graphs were generated using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA).

CRediT authorship contribution statement

Gang Xiang: Writing – original draft, Software, Methodology, Investigation, Data curation, Conceptualization. Yi Yin: Methodology, Data curation. Ang Deng: Methodology, Investigation. Tao Li: Visualization, Software. Jie Dai: Visualization, Methodology. Sihan He: Validation, Software, Investigation. Guanteng Yang: Supervision, Funding acquisition. Yunjia Wang: Writing – review & editing, Supervision, Software, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Data availability

Data will be made available on request.