Investigating the inflammatory effect of microplastics in cigarette butts on peripheral blood mononuclear cells

Abstract

Cigarette filter microplastics are composed of cellulose acetate that does not undergo biological or photo-degradation. These microplastics are readily dispersed and can be found abundantly in water, soil, and air. These fibers possess high absorption capabilities, allowing them to collect and retain pollutants such as toxic elements. As a result, they are regarded as potential dangers to living organisms. The purpose of this study was to analyze the immune response of human peripheral blood mononuclear cells (PBMCs) when exposed to cigarette filter microfibers, measuring the secretion of the inflammatory cytokines TNFα (tumor necrosis factor-alpha) and IL-6 (interleukin-6). In this study, we examined how used cigarette cellulose acetate microfibers affect the viability of peripheral blood mononuclear cells in an appropriate culture medium at three concentrations: 50, 100, and 200 µg/ml. In addition, this study investigated the release of inflammatory cytokines TNFα and IL6 from PBMCs exposed to 200 µg/ml cigarette filter cellulose acetate. The results showed that increasing the concentration of cellulose acetate fibers of one of the brands in the culture medium has a significant effect on reducing cell viability. The 200 µg/ml in DW is more effective than 50 and 100 µg/ml in reducing cell viability. Peripheral blood mononuclear cells showed an inflammatory immune response when exposed to 200 µg/ml cellulose acetate from cigarette filters. They produced inflammatory cytokines that showed a significant increase compared to the control sample. In general, it can be concluded that cellulose acetate fibers in contact with body cells stimulate them and cause an inflammatory response.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-84784-4.

Introduction

Cigarette filters are an enduring, widespread, and hazardous forms of litter. They represent the most prevalent kind of waste abandoned in nature by consumers^1–3. Approximately 6.5 trillion cigarettes are manufactured and consumed globally on an annual basis⁴. In certain studies, it is projected will rise by 50% by 2025 and reach 9 trillion cigarette butts, resulting in the creation of 1.2 million tons of waste. The most common type of waste found among litter collected from roadsides, parks, and public areas is cigarette filters^5,6.In a citizen-science program (Dive Against Debris), researchers discovered that cigarette butts are the second most frequently encountered type of single-use microplastics on the seabed of the Mediterranean, specifically at depths less than 30 m⁵.When a cigarette is lit, it produces numerous complex, toxic chemical compounds due to the pyrolysis of the tobacco that includes organic species and toxic elements. Contrary to what the general public and consumers believe, cigarette butts are not made of cotton. Furthermore, they do not easily break down under normal light conditions, making them non-biodegradable. The cigarette filters break apart into tiny fragments, scattering throughout the environment and getting diluted in water or soil. Because they are lightweight and small, they can be easily transported by rainfall, floods, runoff, and even wind. In published studies, cigarette filters have been observed in the stomach contents of marine animals such as fish, whales, turtles, and birds that feed on fish and other sea creatures⁷. Many researchers have classified cigarette butts as hazardous waste⁶.

Cigarette filters are composed of roughly 15,000 microfiber strands and cigarette residue, which contains 40 different types of hazardous chemicals². The initial filters used cotton and crepe paper, and subsequently, a range of natural and synthetic substances including finely chopped tobacco, were also assessed.Today, the most commonly used filters are cellulose acetate, which is either enhanced with triacetate or blended with activated carbon⁸.The majority of cigarette filters are made of acetylated cellulose, which allows for frequent replacement⁹.They are created by combining anhydrous acetic acid with cellulose, along with a group of softeners. This process effectively inhibits their decomposition by the cellulase enzyme¹⁰.

Microplastics are often referred to as emerging pollutants, which have garnered the attention and concern of numerous researchers, environmentalists, and politicians in recent years. They exhibit a wide range of dimensions, materials, and applications. Microplastics are small plastic fragments measuring less than 5 mm in size, as well as plastic fibers that are narrower than 5 mm but potentially longer. The natural sources of microplastics are typically categorized into two main types: primary and secondary. The former refers to plastics deliberately manufactured in these dimensions for commercial and industrial purposes, while the latter results from the degradation of larger plastic items due to weathering, impacts, or erosion.

Microplastics can be found in various forms in the environment, including fibers, spheres, and irregularly shaped pieces. These particles can wrap around organisms, causing physical harm through scratching, or they can impact organs and accumulate in tissues, resulting in detrimental effects on living organisms.

Inhalation, ingestion, and consumption of seafood are primary and concerning sources through which microplastics enter the human body¹¹. Urban et al.¹² demonstrated that polyethylene micro particles, once inside the body, can be transported to the lymph nodes, liver, and spleen. This translocation indicates their ability to be absorbed and distributed throughout⁷ various parts of the body including the digestive system. Microplastics can exhibit physical, chemical, and microbiological toxicity upon contact with organ surfaces or upon entry into the body. Additionally, they have the potential to accumulate in tissues, potentially leading to harmful effects on human health. Some research conducted in recent years has shown that microplastic particles can potentially affect health, in the form of inflammation, genotoxicity, apoptosis or oxidative stress¹³. In a study conducted by Choi et al.¹⁴, they examined the effects of laboratory-broken pieces of polystyrene microplastics in three different size ranges: 25 − 5, 75 − 25, and 200 − 75 micrometers. They tested these microplastics at three concentrations: 10, 100, and 1000 µg/ml. They used peripheral blood mononuclear cells (PBMCs) and evaluated the amounts of released inflammatory cytokines. Based on the results obtained, they concluded that the size of the polystyrene fragments did not have an impact on cell survival. However, they found a direct relationship between the concentration of microplastics and the secretion of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNFα) cytokines, compared to the control samples¹⁴.

Previous studies have examined the impact of artificially prepared microplastics in the laboratory on cellular immune responses and the secretion of pro-inflammatory cytokines. However, the effect of microfibers, which are the most prevalent type of microplastics found in nature, on human cells has not yet been investigated. Peripheral blood mononuclear cells (PBMCs) possess a strong capability to demonstrate the influence of pollutants on the inflammatory response of cells. They are involved in immune responses and the secretion of inflammatory cytokines. Consequently, PBMCs are frequently utilized in various cell studies and research pertaining to pollutants¹⁵.

The objective of this study is to examine the direct effects of microfibers derived from five popular brands of cigarette filters on PBMCs, as well as their impact on the secretion of pro-inflammatory cytokines TNFα and IL6.

Material and method

This study was conducted at the Shahid Beheshti University of Medical Sciences. All experiments were approved by the Ethics Committee of the Shahid Beheshti University of Medical Sciences (No.IR.SBMU.PHNS.REC.1400.189), and were performed according to their regulations and guidelines. Written informed consent was provided by all the participants.

Measurement of microplastic mass

This research utilized five commonly consumed cigarette brands to maintain ethical principles, they are called by pseudonyms (AB, BK, CG, DW, EM). Ten filters were extracted from each brand. The filters were collected after use in a normal smoking manner. The tobacco residue on the filters and the filter cover paper were separated (without the use of water, so as to not disrupt the filter’s weight). All ten filters were individually weighed, and their average weight was recorded as the weight of a single filter in Table 1. The filters were then placed in a beaker along with 20 ml of distilled water for each filter. The mixture was stirred using a mechanical shaker at a speed of 130 rpm for 48 h. To control and prevent the introduction of microplastics from the air into the sample, the beaker was covered with foil sheets. After this period, hydrogen peroxide was used to eliminate the organic matter from the sample. To achieve this, the sample was placed in a beaker on a hot plate and heated to a temperature of 75 °C. Then, 20 ml of H₂O₂ was added to the beaker while maintaining the sample at 75 °C. After 15 min, an additional 20 ml of H₂O₂ was added to the beaker. Another liter of hydrogen peroxide was subsequently added, with two more additions made at 5-minute intervals. The filters were then pulled through a strainer using a vacuum pump. Subsequently, the filters were dried in an oven at 60 °C for 24 h. The weight of the pre-measured filters was subtracted from the weight of the dried filters, yielding the final weight of the microplastics from the cigarette filters¹⁶. To minimize the potential introduction of other types of microplastics, the dried filters were stored in aluminum packages and utilized during the stages involving capturing images of the microplastic surface using a scanning electron microscope¹⁷, determining the polymer type, and assessing the secretion of inflammatory cytokines from PBMCs.

Table 1.

Mass concentration of microplastic in cigarette filter.

Brands	Description (mg)	Mid weight of a cigarette (g)	Microplastic mass in a filter )g)
AB	Tar 10.5 Nicotine 0.9	0.165 ± 0.05*	0.10
BK	tar 9 Nicotine 0.8	0.166 ± 0.05	0.13
CG	Tar 4 Nicotine 0.4	0.117 ± 0.05	0.11
DW	Tar 10 Nicotine 0.8	0.184 ± 0.05	0.12
EM	Tar 4 Nicotine 0.4	0.182 ± 0.05	0.12

* The ± 0.5 mm represents the standard deviation of the length measurements.

Determining the mass concentration toxic elements

From each selected brand, 0.3 g of smoked filter (of each brand) was weighed and digested in COD vials(16 mm laboratory vial with lid used for liquid analysis). Before use, COD vials were washed with acid and used only after completely dried. For proper digestion, 6 mL of 65% nitric acid and 2 mL of 30% hydrogen peroxide were added. Then the sample was placed in a water bath at 70 °C for 90 min and then heated at 120 °C for 30 min. After digestion, the sample was cooled to room temperature and filtered using 0.45 μm nylon syringe filters to obtain the desired clarity. To minimize errors, a separate syringe head filter was used for each cigarette brand. The types and concentrations of toxic elements in the samples were measured using an inductively coupled plasma spectrometer (ICP-OES Varian, vista-MPX). Control samples were also prepared by the same method. Then the concentrations of toxic elements were calculated in grams¹⁸.

Determination of the lethality of cigarette butts cellulose acetate

Most of the immune cells are PBMCs, which include T, B, and NK lymphocytes and monocytes, and were used in this study¹⁹. MTT test was used to measure cell viability.

Sample preparation

To prepare the samples, the cigarette filters were crushed to a size of 50–500 μm. Subsequently, they were exposed to UV radiation for 30 min to eliminate any contaminants that could potentially impact the results. Following this processing, the filter fibers were directly placed in the suitable cell culture medium and brought into direct contact with the cells.

Cell culture

PBMCs were obtained from blood (random person in the lab) using Ficoll and EDTA. After isolating PBMCs from the blood, a cell culture was conducted. In this step, RPMI culture medium (Bio-IDEA), which is suitable for PBMCs cell culture, along with FBS and trypan blue dye, was utilized to identification of living and dead cells. The PBMCs were suspended in a mixture of 90% RPMI culture medium and 10% FBS, and then transferred to a flask for cell culture. The flask was placed in an incubator at a temperature of 37 °C with 5% CO₂ for 48 h. After this period, 2 ml of trypsin was added the flask and returned to the incubator. Once the cells detached from the flask, the trypsin was neutralized with PBS. The cells adhered to the flask were detached using trypsin and converted into spherical shape. After removing the trypsin, the contents of the flask were centrifuged, and finally, 1 ml of the pellet was re-suspended. A mixture of 20 µl of this suspension and 20 µl of trypan blue dye was prepared, and subsequently, 20 µl this mixture was taken out for cell counting using a Neubauer improved chamber. The remaining cells were seeded in 24-well plates at a density of 5 × 10⁴ cells. After 24 h, cellulose fibers were added to the wells in two steps, with desired concentrations of 50, 100, and 200 µg/ml. To monitor the process, the culture medium was inoculated with 1% Triton X-100. The cells cultured with CA fibers were incubated for 48 h, 100 µl of DMSO was added to each well of the cell culture treated with cigarette microfibers to dissolve the generated formazan, which serves as an indicator of cell viability. The quantity of produced formazan was measured using an ELISA reader (DANA-3200 ELISA READER). After performing the MTT protocols, the light intensity absorbed by the device at the wavelength of 570 nm^14,20,21. The average of three replicates was calculated for each sample, and the percentage of cell viability was determined using Eq. 1 ¹⁴.

1

MTT protocol

MTT steps were performed in the following order.

The selected cells were first cultured and incubated until they adhered and reached a stable state. Then, the test substance was added to the wells and incubated again. After the 48 h, the supernatant was removed and a 25 mM of culture medium containing 0.5 mg/mL of MTT salt solution was added to each well and it was again adjusted in a 5% carbon dioxide incubator at 37 °C for 4 h. The resulting blue-violet formazan crystals were easily recognizable under a microscope. Insoluble crystals were dissolved with 100 µl of DMSO before colorimetry. Then the optical absorbance of the solution was read by an ELISA reader at 570 nm and, a standard curve was formed from the results obtained, which was used to interpret the data.

Analysis of inflammatory cytokines

Inflammatory cytokines are proteins that facilitate communication between cells and the extracellular environment. They are secreted by cells in response to external factors such as physical damage, leading to stimulation and effects on other cells. This response triggers an immune response tailored to combat the introduced pathogen. Consequently, the release of cytokines from the cells demonstrates the immune system’s attempt to combat infection-causing factors or harmful foreign substances. When the immune and inflammatory response persists, resulting in the release of cytokines in the inflamed tissue, it gives rise to auto-inflammatory diseases that ultimately lead to cell death. TNFα is referred to as a pro-inflammatory cytokine since it serves as the initiator of the immune response and represents the initial stage in reacting to the presence of foreign factors within the body²². IL-6 is a unique group of chemokines known as cytokines, which have the ability to stimulate immune cells and activate both the innate and adaptive immune systems by recruiting various types of cells. Therefore, to assess the potential toxicity of cellulose acetate microfibers, cytotoxicity tests and the measurement of pro-inflammatory cytokine release have been conducted²³. IL6 and TNFα are two types of pro-inflammatory cytokines that are released from cells exposed to insults during external stimulation. They affect other cells or the primary cell itself, causing functional changes in the target cells.

Prior to commencing the experiments, the cigarette filters were sterilized using UV light for 30 min to eliminate any interfering factors. A total of 5 × 10⁴ cells were then placed in the wells of 24 plates and allowed to attach to the bottom of each well. Subsequently, the cells, along with the cigarette filter, were placed in an incubator with a CO₂ concentration of 5% and a temperature of 37 °C for 48 h.

After this incubation period, the supernatant was decanted and subjected to testing. To accomplish this, reagents and working standards were prepared according to the instructions provided by the ELISA kit (D6050, DTA00D, R&D systems, USA) and kept the room temperature for 20 min. Following preparation, the absorbance of the complex formed from the samples was measured at 450 nm. The amount of secreted IL6 and TNFα were determined utilizing standard calibration curves^14,20,21. Tables and charts of standard ELISA kits for TNF-alpha and IL6 inflammatory cytokines are given in the supplementary section. (Tables SI-1 and SI-2).

Statistical analysis

Graph-Pad Prism software was used for statistical analysis and generating transparent graphs based on the data. The ANOVA test was employed for normally distributed data, while the Friedman test or Kruskal-Wallis test was utilized for non-normally distributed data. In the graphs, samples with no significant difference were denoted as “ns,” and p-values less than 0.05, 0.01, 0.001, and 0.0001 were represented by *, **, ***, and ****” respectively.

Results

Microplastic mass concentration

As shown in Table 1, the average weight of each AB brand filter is over 60%, BK brand filter is over 75%, CG brand filter is nearly 95%, DW brand filter is over 65%, and EM brand filter is almost 65% microplastic. Based on these findings, it can be concluded that microplastics constitute the largest percentage of the weight of each cigarette butt. Taking into account the global annual cigarette production and the average weight of microplastics found in each cigarette butt (11.6 g), the amount of microplastics entering the environment from cigarette production and consumption (6.5 trillion) was calculated. It is estimated that approximately 0.72 million tons of cellulose acetate microfiber enter the environment from this potential source every year.

SEM results

The morphology of cellulose acetate fibers in cigarette butts was investigated using a scanning electron microscope (SEM-Hitachi SU3500). High-resolution images of the sample surface were captured using a secondary electron detector at three magnifications: 2000x, 400x, and 60x, with a voltage of 15 kV. The recorded images with 60 magnifications are shown in Fig. 1. The obtained images were analyzed using ImageJ to check the diameter and roughness of the fibers that make up cigarette filters. It was observed that the diameters of fibers in different brands were not the same. Even in specific filter, there were fibers with different diameters.

Fig. 1.

Images taken by scanning electron microscope (SEM) with 60× magnification of used cigarette filters.

Figure 1 provides images of cellulose acetate fibers utilized in various cigarette filter brands. The images demonstrate a noticeable intertwining of the fibers, contributing to the complexity of the filter’s structure. A comparison between images captured from used and unused filters, magnified at 400x, shows the impact of heat-induced burning on certain filters. This effect is evident through the destruction of fiber segments or the presence of foreign objects on them. Conversely, some filters exhibit no alteration in their cellulose acetate fibers (Figure SI-1).

Using images with a resolution of 2000 in ImageJ, the average diameter of the fibers was measured (Table 2). By measuring the diameter in different parts and in many fibers visible in SEM images, the average diameter of the fibers was calculated. Table 2 also includes the approximate length of each cellulose acetate filament compared to the length of the filters, showing the compression of the filaments to increase cohesion and maximize filter use.

Table 2.

The dimensions of the strand and the cigarette filter.

Brands	Average cigarette filter length (mm)	The average of length of a cellulose acetate strand (mm)	The average of diameter of a cellulose acetate strand (µm)
AB	20	14 ± 0.5*	28.95
BK	27	15 ± 0.5	27.83
CG	26.5	13 ± 0.5	41.67
DW	25	15 ± 0.5	27.56
EM	27	13 ± 0.5	30.95

* The ± 0.05 g represents the standard deviation of the weight measurements.

Polymer assay

Spectroscopy and verification of synthetic polymers in the cigarette filter microfibers was performed using Fourier Transform Infrared Spectroscopy (FTIR) using ATR (Thermo Nicolet) with a diamond crystal. A 0.5 mm sample was extracted and placed on the crystal surface of the device. FTIR-ATR spectra were recorded in the mid-infrared range of 4000 to 500 cm^− 1 (Figure SI-2). Analysis of the obtained data was done first using OMNIC 9 programs, and then further analysis was done using the Open Specy program. In OMNIC, the transmitted intensity was first converted into absorbed intensity. Using pre-identified and registered spectra, the Hummel library evaluated the percentage similarity of microplastics in the sample to registered microplastics and thus, identified the type of polymer. The Sigma Aldrich table helped to determine the type of polymer based on the peaks observed in the recorded waves and the absorption wavelength of the functional groups. The peaks identified in the FTIR-ATR spectra of the analyzed samples showed significant similarities, which indicated the presence of the same functional groups in their polymer structure. When comparing these spectra with those in the OMNIC program library, a 77.69% match was observed with cellulose acetate butyrate polymer. Since the match percentage is less than 80% the Open Specy program was also used in the second step to identify the polymers²⁴. Figure 2 shows the cigarette filter spectra. Examining the spectra and comparing them with the spectra in the Open Specy library showed that the AB, BK, CG, and DW samples had a 97% match, while the EM sample showed a 98% match with the pure cellulose acetate spectrum.

Fig. 2.

Identification of the polymer of cigarette filters by ATR spectrum. The red color of the cellulose acetate waves recorded in the Open Specy library, the white color of the ATR waves of the checked cigarette filters.

Determining the mass concentration toxic elements

The measured concentrations of toxic elements in cigarette filters were reported in parts per billion (ppb) in the acidified digested sample of cigarette filter (Table SI-3). These values were converted to elemental concentrations per gram of cigarettes (Eq. 2). Calibration graph were constructed for the assayed elements and the method was evaluated by calculating the limit of detection (LOD) and limit of quantitation (LOQ) values, which are given in Table SI-4.

2

where A: The concentration of toxic elements in the digested sample (mg/L); B: Final volume of the digested solution²⁵.

B, Ba, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Sn, Zn are elements that were observed in the examined cigarette filters. Among the 13 elements detected in the cigarette butts of 5 filters, Zn showed the highest concentration in the BK sample, with a concentration of 40.34 µg/g. In the AB brand, Ni had the highest concentration with a concentration of 10.9 µg/g, followed by Sn and Zn with concentrations of 5.27 and 3.57 µg/g, respectively. Co had the lowest amount in this brand, with a concentration of 0.14 µg/g. In the BK sample, Zn and Cr had the highest and lowest concentrations, respectively, with values of 40.34 and 0.07 µg/g.

The highest and lowest concentrations in the examined samples were observed in CG, which was Fe with a concentration of 16.4 µg/g and Co with a concentration of 0.13 µg/g. In DW brand, Zn had the highest concentration with 5.74 µg/g and Ba had the lowest concentration with 0.13 µg/g. In the EM brand, the highest value was recorded for Sn at 22.25 µg/g, while Co had the lowest concentration at 0.13 µg/g. Co and Cd, showed values below 10 ppb in all samples. Overall, the observed elemental concentrations were as follows: AB − 35.3 µg/g, BK − 58.3 µg/g, CG − 37.4 µg/g, DW − 20.6 µg/g, and EM − 42.44 µg/g. Figure 3 shows the comparison of elemental concentrations in the studied brands with the control sample. There are substantial differences in the elemental concentration among the studied cigarette brands and the control sample.

Fig. 3.

The average concentration of heavy metals presents in each gram of cigarette filter of the examined brands compared to the control.

Determination of the lethality of cigarette butts cellulose acetate

Figure 4 shows the survival percentage of PBMCs cells after 48 h of exposure to cellulose acetate fibers. According to the percentages obtained from Eq. 2, the highest percentage of viable cells was observed at a concentration of 50 µg/ml of the BK sample and lowest value belongs to EM, At the concentration of 200 µg/ml BK showed the highest and EM the lowest percentage of survival. PBMCs showed a high survival in the concentration of 50 µg/ml cellulose acetate of all brands. But comparing EM with the control sample, has shown a significant effect on toxicity and cell death with an average survival percentage of 58.34 of PBMCs in 50 µg/ml of cellulose acetate (p < 0.05). Treatment with EM sample showed the most cytotoxic effect on cells in three concentrations, which led to a significant decrease in the viability of PBMCs. DW at 200 µg/ml, unlike 50 and 100 µg/ml of cellulose acetate, had a significant effect on reducing cell viability. (p < 0.5). Figure 4 Comparison of survival percentage between cells treated with cellulose acetate from different brands and the control sample. As shown in the figure, the toxicity of EM and DW samples increased significantly with increasing concentration (p < 0.01).

Fig. 4.

Viability percentage of PBMCs after 48 h exposed to cellulose acetate present in cigarette filter. (A) Cell viability in 50 µg/ml of cigarette filter cellulose acetate. (B) Cell viability in 100 µg/ml of cigarette filter cellulose acetate. (C) Cell viability in 200 µg/ml of cigarette filter cellulose acetate.

Analysis of inflammatory cytokines

ELISA results show that TNFα secretion from cells treated with EM cigarette microfibers was significantly different from the control group (p < 0.01) and the amount of TNFα release from cells in the treatment with AB, BK and DW samples, there was no significant difference from the control group, which are marked with ns in the graph. In the control sample, an average of 40.17 pg/ml of the cytokine TNFα was released in three repetitions of the test. However, when treated with cellulose acetate microfibers from the cigarette filter under the same conditions, the average values of TNFα released were 41.6 pg/ml (AB), 61.94 pg/ml (BK), 116.7 pg/ml (CG), 41.94 pg/ml (DW), and 132.1 pg/ml (EM). The EM and CG samples had the highest toxicity they increased the release rate of TNFα from PBMCs by up to 4-fold and 2.9x.

The IL-6 concentrations indicated that the release of either cytokine from samples AB, BK and DW did not differ significantly from the control sample. There was no significant effect of fiber concentration on IL-6. However, the CG and EM samples displayed an increase in IL-6 compared to the control group. The control sample released an average of 40.9 pg/ml of IL-6 in three tests over a period of 48 h. Under the same conditions, the cells in contact with cellulose acetate released averages of 46.0 pg/ml (AB), 71.3 pg/ml (BK), 92.6 pg/ml (CG), 45.5 pg/ml (DW), and 102.9 pg/ml (EM) of IL-6. The EM sample exhibited a three-fold increase in IL-6 cytokine secretion. Consequently, when compared to the other investigated samples, the EM sample induced greater damage and irritation to human cells.

Figure 5 shows that the EM brand had a greater effect on the release of the cytokine TNFα than IL-6.

Fig. 5.

Inflammatory response of PBMCs after 48 h of exposure to 200 µg/ml cellulose acetate in cigarette filter compared to control. (A) inflammatory cytokine IL-6 (pg/ml), (B) pro-inflammatory cytokines TNFα (pg/ml).

Discussion

In recent years, production of solid waste has increased significantly, and despite the utilization of various waste management techniques, the quantity of waste deposited in the environment continues to escalate. This problem is especially prevalent in certain coastal cities worldwide, resulting in a substantial influx of waste into animal habitats. Such a scenario poses a grave threat to the well-being and biodiversity of animals, plants, and humans²⁶. Microplastics are one of the largest categories of waste, and their quantity is rising due to the extensive utilization of plastics and microplastics in everyday life. Their prevalence and distribution are determined by both environmental and human activities, indicating areas with higher accumulation of plastic and microplastic debris²⁷.

Among the litter gathered from streets, parks, and beaches, cigarette filters are the most frequently encountered form of debris^1,28. The number of cigarette filters found on beaches has increased in recent years because the general cigarette smoker believes that cigarette filters are made of cotton and that photo-degradation or biodegradation by microorganisms is possible. Cigarette filters are microplastics that do not biodegrade due to acetylation, and the photo-degradation of these microplastics is only possible under special laboratory conditions. The prevalence of cigarette filters among all types of discarded waste has led to the failure of waste management systems in collecting and disposing of this waste. As a result, cigarette butts constitute the majority of waste collected from sidewalks, parks, and beaches.

The purpose of cigarette filters is to prevent substances and particles produced by smoking from entering the body¹⁰. If these filters have high efficiency and absorb the most chemicals produced, they become a potential source of pollutants such as benzene, PAHs, and toxic elements¹. The results of this study indicate that the toxic elements observed in these five brands range from 0.07 to 40.34 µg/g. Several studies have been conducted to investigate the quantity of toxic elements in cigarettes. The results of the research conducted by Abdullahnejad et al.²⁹ on the amount of toxic elements in tobacco of 9 different cigarettes brands showed that iron with the amount of 473.5 ± 78.3 µg/g is the highest and chromium with 1.33 ± 0.03 µg/g is the lowest concentration element found in tobacco leaves. A comparison of these results with the outcomes of studies examining the presence of toxic elements in the filters of 4 commonly used cigarettes demonstrates that a small amount of toxic elements present in tobacco is absorbed by cellulose acetate fibers as a consequence of cigarette combustion. In a 2017 study carried out on the northern shores of the Persian Gulf, Dobaradaran et al.³⁰ investigated the levels of toxic elements in cigarette filters that were left on the beach. Comparing the findings of this research with the study done by Dobradaran et al. showed that iron, manganese and cadmium have the most differences between fresh cigarette butts that have not been dumped in the environment and those collected from the environment. This comparison shows the effectiveness of cellulose acetate microfibers in cigarette butts in removing and transporting toxic elements. Depending on the surrounding water or soil environment, these microfibers can absorb or release toxic species like other microplastics. As a result, this study can support and confirm the observations and conclusions of Holmes et al.³¹ on the transport of toxic elements by microplastics.

Multiple studies have confirmed that children and marine creatures consumed by humans ingest cigarette filters³². Studying the cytotoxicity and inflammatory response of Micro/Nano plastics has recently become a major research topic. Most of these studies have been conducted on the subject of investigating the effect of polystyrene Micro/Nano plastics because it is widely available³³. In studies conducted in recent years, MPs have been identified in various human samples, including feces, placenta, liver, lungs, sputum, and body wash effluent³⁴. Recently, in a study in Italy, out of 10 men who were investigated, microplastics in the dimensions of 2–6 micrometers from 1 to 5 in irregular and fragmented shapes were observed in the semen of 6 of them. These microplastics are mainly polyethylene and polystyrene, but other types of polymers are also seen among them³⁵.

Numerous studies on various species have demonstrated that ingested microplastics accumulate in the intestines of diverse organisms³⁶. A review of studies indicates the observation and identification of microfibers in lung tissue, confirming their inhalation-based entry and transfer³⁷.

It has been observed in animal studies that microplastics are capable of passing through the intestinal wall, depending on their size³⁶.

In 2019, Cox and colleagues³⁸ investigated the intake of microplastics through inhalation and ingestion in American children and adults. The findings of this study indicate that individuals receive between 97 and 170 microplastic particles per day from these two pathways, and this quantity varies based on geographical location and dietary habits. Seafood is recognized as the primary source of microplastic transfer to humans. The research conducted by Schwabel³⁹ has confirmed the presence of microplastics ranging in size from 50 to 500 micrometers, at a count of 20 particles per 10 g of human feces. These findings underscore the importance of exploring the toxic effects of microplastics on bodily cells.

In the present study, we examined the toxic impact of cellulose acetate fibers derived from cigarette butts on PBMCs. Similar work used ready-made plastic pellets to investigate toxicity^14,20,39,40.

PBMCs exhibit sensitivity to pathogenic infections and the introduction of foreign substances.

Consequently, many scientists in the field of immunology including autoimmune disorders, infectious diseases, hematological malignancies, vaccine development, and transplant immunology, frequently utilize these cells. PBMCs are highly valuable for drug development and enhancement as they offer an accurate representation of an individual’s immune response. Thus, these cells have been utilized in this study to investigate the impact of cellulose acetate toxicity on the immune system.

In this study, cellulose acetate microfibers found in cigarette butts were used. These microplastics were collected immediately after use and before release into the environment. The amount of toxic elements and the mass of microplastics were measured and the toxicity evaluated through in vitro assessment with PBMCs. The microfibers in the filters of five different brands of cigarettes were prepared in three concentrations of 50, 100 and 200 mg/ml. The results showed that the presence of cellulose acetate microfibers in cigarette filters has a pathogenic effect leading to cell death through direct contact. In addition, as the concentration of fibers increased, the fractional cell survival decreased substantially. Huang et al.⁴¹ observed that at a concentration of 200 mg/ml, the EM and DW sample yielded the lowest percentage of viable cells. However, polystyrene particles at a concentration of 500 µg/ml did not show toxicity to human. In the present study, cigarette filter cellulose acetate microfibers at a concentration of 50 µg/mL from EM, as well as all five brands at concentrations of 100 and 200 µg/mL, showed lethal effects on PBMCs.

In addition, the release of the inflammatory cytokines IL-6 and TNFα by PBMCs at a concentration of 200 mg/ml was investigated. The secretion of pro-inflammatory cytokines from cells treated with cellulose acetate indicated that this group of microplastics was recognized by PBMCs as an external adverse factor. Thus, immune cells were stimulated and an immune inflammatory response was triggered. The increased release of inflammatory cytokines compared to the control sample led to the activation of the host’s defense system and an increase in its overall performance. Huang et al.⁴¹ concluded that polypropylene particles with sizes smaller than 20 μm and concentrations of 100 and 1000 µg/mL show a significant toxic effect on PBMCs. Given that the concentrations of heavy metals found in the cigarette filters analyzed are below 50 µg/g, and referencing studies by Rikans and Yamano⁴², Das and Büchner⁴³, and Pal et al.⁴⁴, heavy metals like lead, nickel, and cadmium can cause toxicity and cell lethality at levels exceeding 1 mg/L. Therefore, the toxicity and lethality observed in PBMCs in this study may be attributed to the presence of microplastics within the cigarette filters. However, the release of cytokines was not significant for particle sizes in the range of 25–200 μm¹⁴. In the present study, cigarette cellulose acetate microfibers with approximate diameters of 31.5 μm and lengths between 50 and 500 μm, had a significant effect on the secretion of inflammatory cytokines IL-6 and TNFα. Prolonged cytokine release sends a self-destructive signal to cells, ultimately leading to cell death⁴⁵.

Comparing the effects of survival and cytokine release among the examined brands showed that the EM brand produced the greatest decrease in fractional cell survival. In addition, both EM and CG brands had the greatest effect on inflammatory cytokines releases due to their toxicity and stimulatory properties. This effect may be attributed to the larger mean diameter of the cellulose acetate fibers in these two brands compared to the others. Comparing the toxic effect at low concentrations and the identification of cigarette cellulose acetate as a pathogen by thee immune cells, shows the potential negative impacts of this microplastic on health.

Thus, the management of this type of waste requires attention. Educating people and raising awareness about the dangers of discarding cigarette butts, formulating effective prevention laws and enforcing laws, creating appropriate infrastructure for the proper disposal of cigarette butts, and considering studies and planning focused on recycling and combining functional compounds in cigarette filters from There are measures that can help reduce the incidence of these lesions following efforts to curb smoking. For example, Arroyo et al.⁴⁶ conducted a study in which they successfully recycled cigarette filters obtained from the environment. They used electrospinning and acid hydrolysis processes to convert cellulose acetate found in cigarette butts into thin films of cellulose acetate nanofibers (TFCAN). These layers are widely used in thermal insulation, protective clothing and filtration⁴⁶. In a separate study, Abu Danso et al.⁴⁷ showed that cellulose acetate in cigarette filters can be extracted and used to effectively absorb diclofenac from water. cellulose acetate is a valuable and widely used material that is produced in considerable quantities annually apart from its use as a cigarette filter. Recycling these materials from discarded cigarette filters along with effective waste management, helps to protect the environment, the well-being of living organisms, and preserve natural and economic resources⁴⁸. However, educating consumers rather than seeking biodegradable filters is the most effective approach to reducing cigarette waste⁴⁹.

Conclusions

In this study, we investigated the quantities of toxic elements that remain from tobacco after smoking in cellulose acetate cigarette filters, the average mass of the microplastics that form in the cigarette filter, the average diameter of the microplastic filaments in the cigarette butt, and the impact of the microplastics in the cigarette butt on PBMCs. The results of this study indicate that the toxic elements found in various brands are consistent, and their concentrations are high. The average masses of microplastics produced by different filter brands were comparable and constitutes the majority of the total mass of cigarette butts. The average diameter of microplastic particles in cigarette filters across different brands was consistently observed within the range of 27 to 42 micrometers. In 2021, Stock et al.⁴⁹ observed in a study that polyethylene particles with a size of 10 micrometers were not absorbed by cells, and their maximum absorption was in the range of 5 micrometers, and polyvinyl chloride particles were not absorbed by cells in the range of 10 to 100 micrometers, they concluded that the reduction of cell viability in exposure to PVC (136.5) and PE (90.1) was due to the accumulation of these particles outside the cell. According to the results of the observations of Stock and his colleagues and the results obtained in this study, it can be concluded that CG and EM brands showed a greater effect in reducing the survival rate of PBMCs due to the larger diameter of cellulose acetate fiber particles. and increased the release of more inflammatory cytokines. TNFα and the inflammatory chemokine IL-6 were elevated, stimulating an immune system response⁵⁰. The continuation of this process and the release of additional inflammatory cytokines from the cell led to cell death⁵¹. Consequently, these findings indicate that microplastics in cigarette filters possess the capability to trigger the immune system and were identified by it as an external factor that poses a threat. Therefore, the management of this hazardous waste is imperative. It is recommended that future studies evaluate the immune response induced by cell exposure to microplastics in filters collected from parks and beaches to obtain a better survey of real-world potential exposures.

Electronic supplementary material

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Author contributions

Monire Soltani: Conceptualization; methodology; formal analysis; investigation; writing - Original Draft; Visualization, Abbas Shahsavani: Conceptualization; methodology; resources; Visualization; Supervision; project administration, funding acquisition, Philip K. Hopke: Conceptualization; review & editing, Nadali Alavi Bakhtiarvand: Conceptualization; methodology; investigation; Writing- Reviewing and Editing, Mehrnoosh Abtahi: Conceptualization; methodology; investigation; Writing- Reviewing and Editing, Masoumeh Rahmatinia: Writing—review & Editing, Majid Kermani: Formal analysis; Writing—review & Editing.

Funding

This study was funded by Shahid Beheshti University of Medical Sciences (grant number:32345).

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.