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. 2018 Apr 23;7(5):809–816. doi: 10.1039/c8tx00032h

Effects of exposure of adult mice to multi-walled carbon nanotubes on the liver lipid metabolism of their offspring†

Hong-yu Zhang a,‡, Ru-long Chen a,‡, Yang Shao a, Hua-lin Wang a, Zhi-guo Liu a,✉
PMCID: PMC6115901  PMID: 30310658

graphic file with name c8tx00032h-ga.jpg Objective: To explore the toxicity of multi-walled carbon nanotubes (MWCNTs) on the liver lipid metabolism of offspring mice and the possible mechanisms involved.

Abstract

Objective: To explore the toxicity of multi-walled carbon nanotubes (MWCNTs) on the liver lipid metabolism of offspring mice and the possible mechanisms involved. Method: Virgin female (16–18 g) and male (18–20 g) C57BL/6 mice were randomly divided into two groups: Control group and Test group. After anesthesia with chloral hydrate, the mice were administered 50 μL saline or dust solution by intratracheal instillation (Control group: 50 μL saline; Test group: 15 mg kg–1 MWCNTs). Mice were injected with these doses once a week for 13 weeks. Then, male and female mice in the same group were allowed to mate to produce offspring. The pups were fed with normal diet until the end of the experiment (12 weeks old). The offspring mice were sacrificed by decapitation to detect the blood biochemistry and the expression of genes and proteins. Results: Compared with the Control group, MWCNTs significantly reduced the weight of offspring mice (male and female) and led to histopathological changes in the liver tissues. The expression of liver fat synthesis gene significantly increased (P < 0.05 or P < 0.01). The expression of genes and proteins involved in the inflammatory reactions appeared to be abnormal (P < 0.05 or P < 0.01). Conclusion: Exposure of adult mice to MWCNTs can affect the expression of fatty acid synthesis genes in the liver tissues of offspring mice, leading to disruption of liver function and accumulation of lipid droplets in the hepatocytes. The imbalance between M1 and M2 liver macrophage phenotypes may be one of the underlying mechanisms of action of MWCNTs leading to disordered fatty acid synthesis in offspring mice.

Background

Dust is one of the major factors causing air pollution that endangers public health. Many studies have demonstrated that dust pollution can cause a variety of cardiovascular diseases,1–6 respiratory diseases,7 obesity8–10 and even mental illness.11,12 This is because the small size of the particulate matter can absorb more toxic and harmful ingredients and reach deeper parts of the lungs. Ultrafine particles can even enter into the blood circulation through the blood-brain barrier, causing more serious damage to the body.13

Carbon nanotubes have a wide range of applications in plastics,14 battery electrodes,15 and water purification systems.16 Carbon nanotubes can penetrate the cell membrane, gastrointestinal mucosal barrier, blood–brain barrier, skin barrier, and blood–air barrier in the lung.17 The liver is the largest and most enriched organ, in which the carbon nanotubes can accumulate.18 Exposure of adult mice to carbon nanotubes can alter the levels of malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH) in the mouse liver.19,20 Carbon nanotubes can also affect the proliferation and differentiation of stem cells. Carbon nanotubes inhibit adipogenic differentiation through the bone morphogenetic proteins (BMPs) signaling pathway.21 Many scholars believe that the activation of the nuclear factor-κB (NF-κB) signaling pathway is the main mechanism of action of carbon nanotubes.22

Moreover, some scholars have paid attention to the toxic effects of nanomaterials on reproductive organs. There is evidence that nanomaterials can cause cytotoxicity in the murine Leydig cells23 and mouse spermatogenic cells in vitro.24 Nanomaterials can cause some damage to the mouse testicular tissue in vivo.25

Apart from causing toxicity in male reproductive organs, nanoparticles can also affect the cell cycle of human ovarian granulosa cells and cause apoptosis.26 Respiratory exposure to carbon nanoparticles during middle and late gestation may produce allergic or inflammatory effects in male offspring, and may provide initial information on the potential developmental immunotoxicity of nanoparticles.27 Respiratory exposure to metallic nanoparticles during pregnancy can also induce stereotyped impairment of lung development with a lasting effect in adult mice.28 Therefore, exposure to carbon nanotubes in adulthood can alter the development of reproductive system, and this effect on developmental programming will be permanent for future generations. Even if lack of relevant environmental stress, this genetic change can also be passed through the sperm or egg to the next generation, and lead to the emergence of the same metabolic phenotype, the abnormal formation of various organs.

In addition, in this study, we found that the respiratory toxicity of nanomaterials has different susceptibilities to different periods.29 Therefore, it is important to pay attention to the toxic effects of nanomaterials on offspring mice at different physiological stages. The studies on in vivo toxicity by carbon nanotubes now focus on the aspects of respiratory and pulmonary systems,30,31 skin,32 and cardiovascular system.33 However, studies on the biological effects of carbon nanoparticles on the liver are limited; in particular, their effects on the formation of liver tissues or even intergenerational transmission have not yet been reported. Therefore, it is necessary to study the toxicity of carbon nanoparticles (MWCNTs) on the liver lipid metabolism in offspring mice. Hence, we established a mice model and exposed adult mice to MWCNTs. We then examined whether these genetic changes can be passed on to the next generation and determined the possible underlying mechanisms.

Materials and methods

Maintenance and treatment of mice

Mice were treated humanely with minimum suffering during experiments, and all the procedures were approved by the Ethics Committee of Wuhan Polytechnology University (approval number 20160046).

Virgin female (16–18 g, 5 weeks old) and male (18–20 g, 6 weeks old) C57BL/6 mice were bought from the Hubei Research Center of Laboratory Animals. The experimental conditions were as follows: room temperature, 22–25 °C; relative humidity, 50 ± 10%; and a 12 h light/dark cycle. Food and water were provided ad libitum. MWCNTs were purchased from the Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences. The physicochemical characteristics of MWCNTs are as follows: purity, 99.9%; outer diameter, 8–15 nm; length, 10–50 μm; and special surface area, <90 m2 g–1. According to the results reported by Zhang et al., 10 and 60 mg kg–1 MWCNTs can induce gene expression changes in the liver of mice, particularly in the TNF-α and NF-κB signaling pathways.34 Herein, we mainly studied the toxic effects of perinatal exposure to MWCNTs in offspring mice using MWCNTs at a set the dose of 15 mg kg–1. The MWCNTs were dissolved in 0.9% saline, broken by cell ultrasound for 15 min and preserved in the dark at 4 °C. After adaptive feeding for 1 week, 20 male and 20 female mice were randomly chosen and divided into two groups: Control group and Test group. The mice were anesthetized using chloral hydrate and administered with 50 μL saline or dust solution by intratracheal instillation (Control group, 50 μL saline and Test group, 15 mg kg–1 MWCNTs). Mice were administered with these injections once a week for 13 weeks. Then, the male and female mice in the same group were allowed to mate to produce the next generation of mice, considered as F1 generation. After spontaneous delivery, the pups were adjusted to 6 (3 male and 3 female pups) per dam. When the pups weaned, a male and a female offspring were randomly selected from each litter (10 mice per group) and fed with normal diet until the end of the experiment (F1 mice 12 weeks old). The F1 generation mice were sacrificed by decapitation, and trunk blood and liver tissue were collected and stored at –80 °C. The body weight was measured every week from the start to the end of the study.

Examination of liver function

The concentrations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum were measured by colorimetric assay (Jiancheng Biological Institute, Nanjing, China). The detection procedures were completed using the Beckman DU800 automatic biochemical analyzer; a parallel reference was set for all the parameters to ensure the true validity of data.

Histology of the liver

Hematoxylin and Eosin (H&E) staining was carried out to observe the pathological changes in the liver. A small amount of liver tissue was cut into pieces ranging from 2 to 5 mm2. The liver sample was fixed in 4% paraformaldehyde for 24 hours. Then, we performed routine paraffin embedding and made slices of 4 μm thickness. The sections were deparaffinized, hydrated, stained with H&E, dehydrated, cleared and finally mounted in neutral resin. An optical microscope was used to observe the pathological and morphological changes.

Quantitative real-time PCR (RT-PCR)

The RT-PCR method was used to detect the expression of fatty acid synthesis genes and inflammatory genes in F1 generation mice. Primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd [shown in ESI Table 1:† interleukin-1 (IL-1) β, interleukin-6 (IL-6), cluster of differentiation 11C (CD 11C), arginase-1 (ARG1), cluster of differentiation 206 (CD 206), and cluster of differentiation 86 (CD 86)]. RNA was extracted from the liver tissue using the PureLink® Pro 96 total RNA Purification Kit (Thermo Fisher, America). The concentration and purity of the samples were determined using an ultramicro spectrophotometer. The RNA samples were reverse-transcribed into cDNA using a high-capacity reverse cDNA transcription kit (Applied Biosystems, Foster City, CA) following the manufacturer's instructions. The gene expression was measured using 2× SYBR Green PCR Master Mix (Applied Biosystems) in the 7500 Real-Time PCR System (Applied Biosystems). Data were presented as 2–ΔΔCt. Reaction conditions: 50 °C, 2 min; 5 °C, 10 min; 95 °C, 30 s; 60 °C, 30 s; 40 cycles.

Western blot analysis

The liver tissues were washed 2–3 times with cold PBS, cut into small pieces and placed in the homogenizers. A 10-fold volume of RIPA lysis buffer and some protease inhibitor were also added into the homogenizers. The homogenates were transferred to 1.5 mL centrifuge tubes after being thoroughly homogenized on ice. They were kept in a rotating ice bath for at least 30 min and centrifuged at 12 000g for 10 min; then, the supernatant was collected for analysis. SDS-PAGE electrophoresis was performed on 40–80 μg of total protein. Specific experimental methods were performed as described previously.35 The film was scanned and archived. Alpha software was used to analyze the optical density of the target band. ARG1 (66129-1-lg), CD206 (18704-1-AP), IL-1β and IL-6 were bought from Proteintech Group, Inc. β-Actin (sc-47778; 1 : 500) and goat anti-mouse IgG-HRP (sc-2005; 1 : 5000) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

Statistical analysis

All data were expressed as means ± SEM (n = 8). The data were analyzed using the SPSS 13.0 software (SPSS Inc., Chicago, IL, USA). Comparison between groups was made using two-sample T-tests. The values of p < 0.05 were considered to be statistically significant.

Results

MWCNTs exposure of adult mice changed the body weights and the liver organ coefficient

Coefficients in F1 mice

MWCNTs exposure did not change the gestation days, litter sizes, sex ratios and birth weights of F1 offspring (see Table 1). After 12 weeks, the body weights of F1 male and female mice in the Control group were (29.8 ± 1.6) g and (27.5 ± 0.3) g, respectively. The body weights of Test group mice ((22.3 ± 0.4) g) were significantly lower than those of the Control group mice ((25.1 ± 1.1) g) (P < 0.01; see Fig. 1A). Compared with the Control group mice, the organ coefficient of liver of male mice in the Test group increased, and was statistically significant (P < 0.05; see Fig. 1B).

Table 1. The gestation days, litter size, sex ratio and birth weight of F0 generation offspring.
  Gestation days (day) Litter size (mm) Male–female sex ratio (%) Birth weight (g)
Control group 20.13 ± 0.51 9.52 ± 2.13 54.90 ± 5.73 1.52 ± 0.14
Test group 20.34 ± 0.28 9.11 ± 2.27 55.81 ± 2.18 1.63 ± 0.21
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Fig. 1. Effect of MWCNTs exposure on (A) body weights and (B) liver organ coefficients of F1 offspring (12 weeks old). n = 8 mice per group. *P < 0.05 vs. control; **P < 0.01 vs. control.

Fig. 1

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Effect of MWCNTs exposure of adult mice on liver enzyme activity in offspring serum

Compared to the Control group, the concentrations of ALT and AST in the serum of Test group male mice significantly increased, and the value was statistically significant (P < 0.05; see Fig. 2). However, MWCNTs had no significant effect on the two enzymes of female offspring mice (P > 0.05; see Fig. 2).

Fig. 2. Effect of MWCNT exposure on the Biochemical indexes of offspring mice (12 weeks old). n = 8 mice per group. *P < 0.05 vs. control.

Fig. 2

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Pathological observation of livers of offspring mice

Mice were sacrificed, decapitated and the abdominal cavity was removed. The histological changes in the liver tissues were observed by naked eye. The liver tissues of the Control group showed bright red, sharp edges as well as smooth surfaces, and no abnormal changes were found. The liver tissues of the Test group (male and female) were dark red with a blunt edge and stick to the surrounding tissues. H&E staining of the liver showed hepatic sinus dilation, unclear lobular structure, irregular arrangement of liver cells, significant inflammatory cell infiltration, and some lipid droplets. Even in female MWCNTs-exposed mice, the hepatocyte cytoplasm was loose and highly vacuolated (see Fig. 3).

Fig. 3. H&E staining of liver tissue (×400).

Fig. 3

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MWCNT exposure of adult mice can alter the synthesis of fatty acids in the livers of offspring mice

To further confirm whether exposure of adult mice to MWCNTs can affect the synthesis of liver fatty acids in offspring mice, we examined some genes in the liver tissue, such as sterol regulatory element-binding protein 1 (Srebp-1), lipoprotein lipase (Lpl), peroxisome proliferator-activated receptor (Ppar)-α, acetyl CoA carboxylase (Acaca)-α and insulin induced gene 2 (Insig-2). Compared with the corresponding Control group, Srebp-1 and Insig-2 gene expression increased, but the expression of the other three genes decreased (P < 0.05 or P < 0.01; see Fig. 4A and B).

Fig. 4. Effect of MWCNT exposure of adult mice on the fatty acid synthesis genes in liver tissues of offspring mice. A: Male; B: Female. n = 8 mice per group. *P < 0.05 vs. control; **P < 0.01 vs. control.

Fig. 4

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MWCNTs exposure of adult mice can alter the balance of M1/M2 macrophage phenotype in the livers of offspring mice

The expression of marker genes of M1 and M2 macrophages in liver tissues were measured by RT-PCR. Compared with the Control group, the expression of M1 marker genes (IL-1β, IL-6 and CD11c) in the Test group significantly increased, the expression of M2 marker genes (CD206 and ARG1) decreased, and the expression of CD86 increased (P < 0.05 or P < 0.01; see Fig. 5A–D).

Fig. 5. Results of PCR analysis indicating alteration in the balance between macrophages M1 and M2 in the liver of offspring mice caused by MWCNTs exposure of adult mice. A and C: Male; B and D: Female. n = 8 mice per group. **P < 0.01 vs. control.

Fig. 5

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The expression of M1 macrophage marker protein IL-6 by western blot analysis was consistent with that obtained by PCR analysis (P < 0.05). The expression of IL-1β protein in Test group female mice decreased (P < 0.05), which was contradictory to the result by PCR; the expression of male IL-1β did not change. The expression of M2 macrophage marker proteins ARG1 and CD206 in female mice was consistent with the results produced by PCR, i.e., all expressions decreased (P < 0.05); however, the expression of ARG1 and CD206 protein in male mice increased (P < 0.05; see Fig. 6A and B) (P < 0.05).

Fig. 6. Results of western blot analysis indicating alteration in the balance between macrophages M1 and M2 in the liver of offspring mice caused by MWCNTs exposure of adult mice. A: M1 macrophage; B: M2 macrophage. n = 8 mice per group. **P < 0.01 vs. control.

Fig. 6

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Discussion

In recent years, the rates of non-alcoholic fatty liver disease have been increasing and have become a type of developed areas’ and affluent societies’ disease. Obesity, diabetes, long-term use of hormones, improper diets and lifestyle, and environmental pollution are the causes of these pathogenic conditions. However, the specific mechanism still lacks understanding.

Our experiment focused on the effect of MWCNTs exposure of adult mice on the fatty acid synthesis in the liver of offspring mice. The results indicated that MWCNTs exposure significantly reduced the weight of offspring mice (male and female) and the indices of male liver organs increased except for those of female organs. MWCNTs exposure could lead to histopathological changes in liver tissues, and parts of the mice liver tissues showed fat accumulation. We then examined the increasing expression of the liver fat synthesis genes. In order to investigate the causes of abnormal liver fatty acid synthesis in offspring mice by MWCNTs, we detected the inflammatory factors and found that their expression also appeared to increase abnormally.

The liver is an important organ for fatty acid synthesis. Synthetic fatty acids will combine with apolipoprotein, cholesterol, etc., to produce very low-density lipoprotein (VLDL). VLDL enters the blood and is transported to other tissues for storage or use. Once the liver cells are damaged with cell arrangement disorder by the xenobiotics, ALT and AST in the cytoplasm of liver cells will be directly released into the blood, leading to an increase in the levels of both these indicators. Fatty acids in the hepatocyte cannot enter into the blood and lead to the accumulation of fat droplets. These typical abnormalities in the liver are consistent with our experimental results. Therefore, exposure to MWCNTs in adulthood can affect the normal function of liver tissues, leading to improper lipid metabolism of liver cells, accumulation of fatty acids in hepatocytes, and further development into nonalcoholic fatty liver in offspring mice. This is consistent with the results of Jackson P's research.36 The MWCNTs affect the liver fatty acid synthesis in offspring mice in two ways: on the one hand, MWCNTs affect the development of male or female germ cells and also alter some genes involved in the liver lipid synthesis, leading to abnormalities in the fatty acid synthesis in the liver of offspring mice; on the other hand, the MWCNTs accumulate in the parent mice, enter the fetus through the placental barrier or breast milk,37 and affect the normal development of the fetal liver. Our experiment showed that MWCNTs can alter the expression of fatty acid synthesis genes, such as Srebp-1, Lpl, Ppar-α, Acaca-α and Insig-2, in the liver of offspring mice. Srebp-1, which is an important nuclear transcription factor, regulates the expression of enzymes for the synthesis of endogenous cholesterol, fatty acids, triglycerides and phospholipids to maintain blood lipid dynamic balance. Studies have shown that Srebp-1 and its target gene network abnormalities can cause the formation of non-alcoholic fatty liver tissues.38,39 The overexpression of Srebp-1 increases the mRNA expression level of Acaca-α. Acaca-α is involved in the first step of fatty acid synthesis,40 promotes the process of steatosis, results in fat accumulation in the liver and leads to the occurrence of non-alcoholic fatty liver tissues. Lpl hydrolyzes triglycerides and secretes glycerol and free fatty acids; the latter can be taken up by hepatocytes and participate in the body's energy metabolism.41 Ppar-α promotes VLDL and high-density lipoprotein metabolism and fatty acid oxidation. Insig-2 significantly inhibits the key enzymes involved in liver fat synthesis, so that Acaca-α, fatty acid synthase and Stearyl-CoA desaturase-1 are significantly reduced, which significantly decreases the liver weight.42 MWCNTs exposure of adult mice can elevate the expression levels of fatty acid synthesis genes in the liver of offspring mice and promote the synthesis of liver fatty acids. Liver cells accumulate more fatty acids and thus lead to liver cell steatosis, affecting the normal function of liver cells.

Inflammation is usually considered to be a major cause of the toxicity of nanomaterials.43,44 Oxidized multiwalled carbon nanotubes were injected into the subcutaneous tissue of mice, which can activate and attract the surrounding macrophages (M1 and M2 macrophages) towards the affected tissues.45 Macrophages have three major functions in inflammation: phagocytosis, antigen presentation, and production of various cytokines and growth factors.46 M1 macrophages promote anti-inflammatory immune responses by activating the adaptive immune system, while M2 macrophages down-regulate anti-inflammatory immune responses. Both of them play key roles in the initiation, maintenance, and selectivity of inflammation. Our experimental results indicate that exposure of adult mice to MWCNTs could activate M1 macrophages and inhibit M2 macrophages in the liver tissue of offspring mice, such that macrophage representative protein and gene expressions increase or decrease. MWCNTs can induce the differentiation of macrophages into M1 type and inhibit the M2 type conversion of macrophages. This is consistent with the results reported by other groups.47–49 Maternal and paternal mice were subjected to long-time low degree inflammation, which can lead to the alterations in the uterine environment, affect germ cells and also influence the offspring development.46,50–52

In summary, exposure of adult mice to MWCNTs can affect the expression of fatty acid synthesis genes in the liver tissue of offspring mice, leading to destruction of liver function and accumulation of lipid droplets in hepatocytes. The expressions of genes and proteins of M1 macrophages and M2 macrophages in liver tissue were increased or decreased. The imbalance between M1 and M2 macrophages may be one of the mechanisms of action of MWCNTs, leading to fatty acid synthesis disorder in offspring mice.

Conflicts of interest

There are no conflicts of interest to declare.

Supplementary Material

Click here for additional data file. (72.9KB, pdf)

Acknowledgments

This study was supported by the National Natural Science Foundation of China (81370246), and the Fundamental Research Funds for the Wuhan Polytechnic University (2016J07), Hubei science and technology plan project (2017CFB479) and the Project of scientific research project of Hubei Provincial Department of Education (B2017079).

Footnotes

†Electronic supplementary information (ESI) available. See DOI: 10.1039/c8tx00032h

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