In vivo toxicity evaluation of boron nitride nanosheets in Bombyx mori silkworm model

Highlights

• In vivo toxicity of BN NSs is evaluated in silkworm model.

• BN NSs cause no obvious damage to the growth, silk properties and tissues of silkworm.

• The expressions of genes about some specific functions and pathways greatly changed.

Abstract

Boron nitride nanosheets (BN NSs), a novel material with a structure similar to graphene, have attracted much attention due to their extraordinary properties. A deep in vivo study of the toxicity of BN NSs is indispensable, which can help to understand their potential risk and provide useful information for their safe application. However, so far as we know, the systematic in vivo toxicity evaluation of BN NSs hasn’t been reported. In this study, silkworm (Bombyx mori) was used as a model to investigate the toxicity of BN NSs, by continuously feeding silkworm larvae with BN NSs at various mass concentrations (1%, 2%, 3%, 4%). The toxicity was evaluated from the levels of animal entirety (mortality, silkworm growth, cocoons and silk properties), tissues (pathological examination) and genes (transcriptomic profiling). The results show that the exposure to BN NSs causes no obvious adverse effects on the growth, silk properties or tissues of silkworm, but the expressions of genes in midgut concerned with some specific functions and pathways are significantly changed, indicating that BN NSs may have potential danger to lead to dysfunction. This study has performed in vivo toxicity evaluation of BN NSs and provided useful safety information for the application of BN NSs.

Introduction

Boron nitride nanosheets (BN NSs) are a kind of graphene analogs with a two-dimensional morphology and sheet-like structure, which possess higher thermal and chemical stability than carbon materials (Chen et al., 2019; Guiney et al., 2018; Mateti et al., 2018). As a promising catalyst support, BN NSs have drawn more and more attention of researchers in recent years, because they can prevent supported catalyst from being sintered on hot spots (Lin et al., 2002; Wang et al., 2011). It was reported that graphene-based nanomaterials could generate intracellular reactive oxygen species and thus provoke dramatic toxicity (Bianco, 2013; Seabra et al., 2014). Thus, it is necessary to evaluate the toxicity of BN NSs which have a similar shape with graphene and graphene oxide. However, the corresponding study is rather scarce. Liu et al. (2017) studied the cytotoxicity of BN NSs by exposing HepG2 cells to BN NSs for 24 h, the results demonstrated that low exposure concentrations (0.2–2 μg mL⁻¹) of BN NSs were cytotoxic, which can act as a chemosensitizer and restrain transmembrane transporter activity. Zhang et al. (2019) evaluated the toxicity of BN NSs to bacterial membranes (used Escherichia coli as an example), revealing that BN NSs could cause damage to bacterial outer and inner membrane. However, so far, the in vivo toxicity analysis of BN NSs in animal models has not been reported, which is rather important since the interactions between nanomaterials (NMs) and animal systems are very complicated. NMs uptake by animals may cause novel metabolism patterns, immune response, clearance and biodistribution, which can provide more useful information for evaluating the likely hazards of NMs to mankind (Mangalampalli et al., 2017). Some researchers have analyzed the toxicity of BN nanotubes (BN NTs) and nanospheres (BN NSPs) by performing animal experiments, for example, Kodali et al. (2017) explored the toxicity of BN NT mixtures using mice model (40 μg per mouse) and found that they could induce inflammation due to NLRP3 inflammasome activation, Wang et al. (2017) used Caenorhabditis elegans model to analyze the toxicity of BN NSPs and revealed that the latter could promote oxidative stress levels by increasing reactive oxygen species production (higher than 100 μg mL⁻¹). However, it was reported that the toxicity of NMs was strongly dependent on their shapes (Abramenko et al., 2018; Li et al., 2015; Yuan et al., 2018), the toxicity or biological effects of BN NSs can not be predicted directly from the conclusions of other BN NMs. Hence, it is necessary to study the in vivo toxicity of BN NSs by using a suitable animal model.

Domestic silkworm (Bombyx mori), as a representative of lepidoptera, is an ideal invertebrate animal model with a short growth cycle. Compared with mammal models such as rabbit, rat and mouse, silkworm will not arouse disputes in ethical levels but have similar pharmacokinetics and median lethal doses with mammals (Abdelli et al., 2018; Hamamoto et al., 2004; Panthee et al., 2017). Compared with non-mammal models such as zebra fish, Drosophila and Salamandra laurenti, silkworm will not cause biosafety issues since it has already lost the ability to survive outside, moreover, the size of silkworm is more suitable for handling (Kaito et al., 2002; Ma et al., 2019b). Besides, silkworm has many other virtues as an animal model, for instance, silkworm is highly sensitive to toxicants and can monitor any harmful substance at extremely low amount (about one-hundred-thousandth of human lethal amount) (Ma et al., 2019b; Qin et al., 2010), most materials which are harmful to human beings can be detected by silkworm model (National Institute of Genetics, 1978.; Qin et al., 2010), it also has a distinct genetic background and rich genetic resources with abundant morphological mutation and genetic death systems (Meng et al., 2017). Owing to the outstanding advantages as an animal model, silkworm has gained much attention in many fields, including but limited to human disease investigation (Hanaoka et al., 2008; Meng et al., 2009; Mukherjee et al., 2015), classical genetics (Song et al., 2016; Tan et al., 2013), environmental monitoring (Ikeya et al., 1996; Shi et al., 1998; Tian et al., 2010) and medicinal studies (Hamamoto et al., 2009; Orihara et al., 2008). For example, researchers have found that some genes related to human genetic diseases have high sequence similarities to Bombyx mori silkworm genes, according to which models of human genetic diseases (such as Phenylketonuria, Parkinson’s disease, Hermansky-Pudlak syndrome and so on) can be established (Chen et al., 2014; Chen et al., 2016; Fujii et al., 2012; Tabunoki et al., 2013). In recent years, silkworm has also attracted numerous attention in nanotoxicity study, the toxicity and biological effects of many NMs were evaluated by silkworm model, such as graphene quantum dots (GQDs) (Ma et al., 2019a), BSA capped gold nanoclusters (BSA-Au NCs) (Ma et al., 2019b), CdTe quantum dots (CdTe QDs) (Li et al., 2017; Liu et al., 2014; Yan et al., 2016) and so on (Cheng et al., 2017; Ni et al., 2015; Xing et al., 2016). In previous reports, our group studied the safety limit and biological effects of graphene QDs in silkworm by intravascular injection (Ma et al., 2019a), we also explored the toxicity of BSA-Au NCs in silkworm by acute injection (Ma et al., 2019b). Liu and coworkers realized the toxicity evaluation of CdTe QDs by investigating their effects on silkworm hematopoiesis (Liu et al., 2014). Cheng et al. directly fed silkworm with Cu and Ag nanoparticles (NPs) and examined the biological effects of the two NPs, through characterizing the growth status and silk properties of silkworm (Cheng et al., 2017).

In this study, the in vivo toxicity of BN NSs was evaluated by silkworm model, via feeding silkworms with BN NSs at different concentrations (mass fraction: 1%, 2%, 3% and 4%) during the whole 5th instar until cocooning. The growth status, silk properties, cell morphology and gene expression level of silkworms were characterized. The biodistribution and clearance of BN NSs were also studied. The results indicate that BN NSs can be easily cleared and cause no obvious hazards to the growth, silk properties and tissues of silkworms. However, there is a significant change of the expression levels of genes related to some specific functions (such as metabolism, catalytic ability, immunity, xenobiotics biodegradation and digestion), suggesting that BN NSs may have potential danger to silkworm. To the best of our knowledge, it is the first study systemically investigating the in vivo toxicity of BN NSs, which will shed light on the safety evaluation of BN NSs for their potential applications.