Date Published: April 5, 2017
Publisher: Public Library of Science
Author(s): Sarah S. Poulsen, Kristina B. Knudsen, Petra Jackson, Ingrid E. K. Weydahl, Anne T. Saber, Håkan Wallin, Ulla Vogel, Tobias Stoeger.
Pulmonary exposure to multi-walled carbon nanotubes (MWCNTs) has been linked to an increased risk of developing cardiovascular disease in addition to the well-documented physicochemical-dependent adverse lung effects. A proposed mechanism is through a strong and sustained pulmonary secretion of acute phase proteins to the blood. We identified physicochemical determinants of MWCNT-induced systemic acute phase response by analyzing effects of pulmonary exposure to 14 commercial, well-characterized MWCNTs in female C57BL/6J mice pulmonary exposed to 0, 6, 18 or 54 μg MWCNT/mouse. Plasma levels of acute phase response proteins serum amyloid A1/2 (SAA1/2) and SAA3 were determined on day 1, 28 or 92. Expression levels of hepatic Saa1 and pulmonary Saa3 mRNA levels were assessed to determine the origin of the acute phase response proteins. Pulmonary Saa3 mRNA expression levels were greater and lasted longer than hepatic Saa1 mRNA expression. Plasma SAA1/2 and SAA3 protein levels were related to time and physicochemical properties using adjusted, multiple regression analyses. SAA3 and SAA1/2 plasma protein levels were increased after exposure to almost all of the MWCNTs on day 1, whereas limited changes were observed on day 28 and 92. SAA1/2 and SAA3 protein levels did not correlate and only SAA3 protein levels correlated with neutrophil influx. The multiple regression analyses revealed a protective effect of MWCNT length on SAA1/2 protein level on day 1, such that a longer length resulted in lowered SAA1/2 plasma levels. Increased SAA3 protein levels were positively related to dose and content of Mn, Mg and Co on day 1, whereas oxidation and diameter of the MWCNTs were protective on day 28 and 92, respectively. The results of this study reveal very differently controlled pulmonary and hepatic acute phase responses after MWCNT exposure. As the responses were influenced by the physicochemical properties of the MWCNTs, this study provides the first step towards designing MWCNT that induce less SAA.
Multi-walled carbon nanotubes (MWCNTs) exhibit unique electrical, thermic and strengthening properties. But their increased production has also increased the potential risk of human exposure [1;2]. It is well established in rodent models that pulmonary exposure to MWCNTs through inhalation, instillation or aspiration is associated with lung inflammation, genotoxicity, fibrosis and granuloma formation [3–14]. In addition, pulmonary exposure to MWCNTs may increase the risk of developing cardiovascular diseases (CVD) . Indeed, several rodent studies have shown that exposure to MWCNTs and single-walled carbon nanotubes (SWCNTs) induce CVD outcomes such as impaired vasodilation and increased plaque progression [16–19], just as it is well-established that pulmonary exposure to respirable air particulates is linked to increased risk of CVD [20–26]. Also, increased pulmonary expression and increased systemic levels of the acute phase response (APR) protein serum amyloid A (SAA) have been reported after pulmonary exposure to MWCNTs and other engineered nanomaterials (ENMs) [27–33]. Similar to the APR protein C-reactive protein (CRP), elevated plasma levels SAA is a risk factor for CVD in humans [34–37]. SAA (SAA1-4) is a highly conserved family of apolipoproteins associated with high density lipoproteins (HDL). However, species specific differences in the SAA isoforms and their expression exist. In humans, Saa3 is only expressed in mammary gland epithelial cells , whereas Saa1 and Saa2 are expressed both hepatically and extra-hepatically . In mice, Saa3 is expressed in various tissues, including the lung, while Saa1 and Saa2 have previously been considered liver specific .
This study consists of 3 parts with very similar, but not identical, experimental setups. An overview of the parts is presented in S1 Table. Part 2 was first performed, in which the doses 18, 54 and 162 μg MWCNT/mouse were used. We later decided on using lower doses for more relevant measurements, and therefore used doses 6, 18 and 54 μg MWCNT/mouse for part 1 and 3. There is therefore no 6 μg MWCNT/mouse dose for part 2. Similarly, post-exposure days for part 2 were 1, 3 and 28, whereas we later decided to use post-exposure days 1, 28 and 92 in part 1 and 3 to include a more chronic time point. To allow for comparison, the later time point (92 days) for NM-400 and NM-401 exposure was included in part 3. The experimental setups for part 1 and 2 have previously been published [8;9].
The physicochemical properties of MWCNTs are important determinants of their toxic potential. Previous rodent studies have related MWCNT lengths, functionalization levels, and metal impurity content to MWCNT-induced adverse outcomes as inflammation, fibrosis and cancer [8;9;48;49]. Pulmonary exposure to MWCNTs and other ENMs has also been linked to increased risk of developing CVD [16;18;19;31]. We have proposed a mechanism for this increased risk by which pulmonary exposure to MWCNTs induces a strong pulmonary APR . In mice, Saa3 is the most upregulated APR gene in lung after ENM exposure, but Saa1 and Saa2 also have highly upregulated pulmonary expression [9;27;32;61]. Traditionally, circulating acute phase proteins are thought to be of hepatic origin, but we have reported that pulmonary exposure to Printex 90 carbon black nanoparticles induces a strong pulmonary APR, but little to no hepatic APR in mice [27;51]. In contrast, we have also shown that two different MWCNTs induced a hepatic APR in mice of varying potency following pulmonary exposure, even though the experimental protocol used was highly similar to that of the nano-carbon black study . This indicates the involvement of different APR-related mechanisms after MWCNT and nano-carbon black exposure, which could be related to their physicochemical composition. However, very little data is available on the relationship between physicochemical properties of CNT and the APR, including SAA. We therefore assessed the systemic levels of SAA1/2 and SAA3 protein after pulmonary exposure to 14 MWCNTs in female C57BL/6J mice by intratracheal instillation to 3 doses (6, 18 or 54 μg/mouse for NRCWE-040 to NRCWE-049 and NM-402 to NM-403. 18 or 54 μg/mouse for NM-400 to NM-401) and 3 time points (1, 28 and 92 days post-exposure). Using a panel of 14 MWCNT, we were able perform comparisons not possible with the more standard setup of 1 or few MWCNTs seen in the literature.
Pulmonary exposure to MWCNTs induced dose-dependent pulmonary and hepatic acute phase responses. The pulmonary acute phase response was stronger in terms of fold increase and more long lasting than the hepatic acute phase response. Almost all of the 14 studied MWCNTs induced increased plasma levels of SAA3 and SAA1/2 protein on day 1. The OECD standard material NM-401 also induced significantly increased SAA3 levels on day 28 and 92. MWCNT length was identified as protective of increased SAA1/2 levels on day 1, such that a longer length results in lower SAA1/2 levels. Dose and content of Mn, Mg and Co predicted increased SAA3 protein levels on day 1, whereas oxidation and diameter of the MWCNTs were protective on day 28 and 92, respectively. Only SAA3 levels correlated with pulmonary neutrophil influx, and SAA1/2 and SAA3 protein levels did not correlate. The results of this study could provide the initial step towards designing MWCNTs that induce less SAA consequently less risk of inducing CVD following exposure.