Exploring the Dynamic Interplay of Carbon Nanotubes with Apoptosis In-vitro and In-vivo: Insights from Bcl-2 and IAP Families

Abstract

Background:

Carbon nanotubes (CNTs) are carbon-based nanomaterials. They have been widely used in environmental technologies and in biomedical fields.

Objectives:

This study examines the impact of carboxyl-functionalized multi-walled (MWCNTs) and single-walled carbon nanotubes (SWCNTs) on apoptosis in Jurkat cells and Wistar rats.

Materials and Methods:

The methods included functionalizing CNTs, conducting cell culture experiments with Jurkat cells, and performing in-vivo studies on male Wistar rats. Gene expression, particularly the Bcl-2 family, was analyzed using real-time PCR, and statistical analyses assessed group differences.

Results:

Results showed successful CNT functionalization and a slight reduction in cell viability in exposed groups. Gene expression analysis in Jurkat cells treated by MWCNTs and SWCNTs revealed significant upregulation of pro-apoptotic genes (BAD, BAX, BID, BCL2L11) and anti-apoptotic genes (Bcl-2, BCL2L1). Additionally, significant increases in IAP family members, BIRC2 and BIRC3, were observed in these cells. In treated animals, a transient increase in Bcl-2 and BAD expression was observed after 30 days, with no significant changes after 60 days. Unlike Bcl-2 family members, IAP family gene expression (BIRC2, BIRC3) remained unchanged in the treated animal model over the same periods.

Conclusion:

CNT exposure led to significant modulation of apoptotic pathways in Jurkat cells, including alterations in gene expression of some members of the Bcl-2 and IAP family, while the animal models showed transient changes in Bcl-2 family gene expression but no alterations in IAP gene expression. These findings highlight the complex interactions between CNTs and biological systems, underscoring the need for further research on the long-term effects and adaptation mechanisms in organisms exposed to CNTs.

1. Background

Carbon nanotubes (CNTs) are carbon-based nanomaterials known for their remarkable mechanical properties as well as their electrical behavior, optical function, and thermal characteristics. They have been widely used in environmental technologies (e.g., adsorption and filtration) and in biomedical fields, especially for transporting drugs or genes, supporting tissue repair, and enabling biosensor-based detection ( 1 ). CNTs consist of rolled-up graphene sheets and are generally divided into single-walled (SWCNTs) and multi-walled (MWCNTs) configurations according to how many graphene layers constitute the nanotube walls. Despite their wide range of applications, there is growing concern about the possible toxic effects and associated hazards of CNTs on living organisms. CNT Exposure may trigger various biological responses, including oxidative stress, reduced total antioxidant capacity, inflammation, fibrosis, and and cell death through necrotic or apoptotic pathways ( 2 , 3 , 4 ). Zhou et al. (2017) suggest that both SWCNTs and MWCNTs can cause cellular toxicity in both in vitro and in vivo environment through apoptotic and/or necrotic pathways ( 5 ). Kobayashi et al. (2017) have shown that MWCNT exposure predominantly induces inflammation, while SWCNTs induce apoptosis and impair mitochondrial function. Various factors can affect the toxicological properties of functionalized CNTs, particularly the type of functional groups such as carboxyl grafted onto the CNT sidewalls ( 6 ). Apoptosis is mediated through intrinsic (mitochondrial) and the extrinsic (death receptor-mediated) pathways, which are activated by different biomolecular signals ( 7 ). This process is governed by a balance between proteins that promote or inhibit cell death belonging to the Bcl-2 and IAP protein families. The Inhibitors of Apoptosis Proteins (IAPs), such as BIRC2 and BIRC3, are particularly noteworthy for their roles in blocking apoptotic signaling pathways by inhibiting caspase activity. Dysregulation of IAPs has been implicated in cancer progression, making them key targets for understanding cellular responses to CNTs.

2. Objectives

Although many studies have examined the cytotoxic and apoptotic effects of CNTs ( 8 , 9 , 10 ), the molecular mechanisms remain inconsistent across reports. Some studies attribute CNT-induced apoptosis to oxidative stress, mitochondrial dysfunction, or caspase activation ( 11 , 12 , 13 ), whereas others describe minimal or context-dependent effects influenced by CNT type, dose, and surface chemistry ( 14 , 15 ). These contradictory findings underscore the need for integrated analyses of apoptotic regulators across different biological models.

However, limited information is available on how carboxylated SWCNTs versus MWCNTs coordinately regulate both pro-apoptotic (Bcl-2 family) and anti-apoptotic (IAP family) genes across cellular and whole-animal systems, especially over extended exposure periods. Few studies have combined physicochemical characterization with multi-timepoint gene-expression profiling to elucidate how functionalized CNTs influence apoptotic and survival pathways ( 9 , 10 , 11 , 12 ).

The present study addresses these gaps by evaluating the expression of Bcl-2 and IAP family genes in parallel in vitro (Jurkat cells) and in vivo (rat PBMCs) models following exposure to carboxylated CNTs. By integrating CNT characterization (FTIR, TEM, and zeta potential) with qRT-PCR at 30- and 60-day intervals, this study provides a time-dependent and comparative assessment of apoptotic and survival responses under CNT exposure.

3. Materials and Methods

3.1. CNS Preparation and Functionalization

The carbon nanotubes were prepared at the Oil Industry Research Institute in the Department of Biotechnology and Environment. Functionalization of the CNTs involved dispersing them in a concentrated mixture of sulfuric acid and nitric acid (3:1) and sonicating the mixture in an ultrasonication bath at 40 ºC for 4 hours. The CNTs were then filtered through a PTFE membrane filter with a 0.2-mm diameter and rinsed with deionized water. The accuracy of CNT functionalization was verified using FTIR spectroscopy. The carboxylated CNTs were subsequently dispersed in phosphate-buffered saline (PBS, pH 7.4) and autoclaved for further cellular assays. After functionalization, these carboxylated multiwall carbon nanotubes have 2-7 wt% COOH as determined by titration, typically exhibited a high zeta potential, ranging from -30 to -70 mV depending on pH ( 16 ).

3.2. Cell Culture and MTT Assay

Jurkat cells, human T lymphocyte cells in suspension form (suitable for apoptosis studies), were chosen for apoptosis measurement. They were obtained from the Iranian National Cell Bank. The cells were cultured in RPMI-1640 medium. RPMI-1640 medium was supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 2mM L-glutamine. FBS is a common supplement in cell culture media that provides nutrients, growth factors, and hormones to support cell growth and proliferation. L-glutamine is an amino acid that is essential for cell growth and metabolism. The cells were incubated at 37 °C in a humidified incubator with 5% CO₂. After the Jurkat cells reached 70% confluency, they were washed with PBS solution and seeded into flat-bottom 24-well plates at a density of 2×10⁵ cells.mL-1.well^-1. The cells were then exposed to SWCNT or MWCNT preparations, which were dispersed in PBS at a concentration of 100 mg.mL^-1 for 72 hours. The MTT assay was used to determine cell viability by measuring mitochondrial function. Briefly, cells were treated with MTT solution at a final concentration of 1.0 mg.mL^-1 and then incubated for an additional 2 hours. The resulting formazan was solubilized in acidified isopropanol, and the absorbance of the organic phase was measured at 570nm using a BioTek ELX800 microplate reader ( 17 ).

3.3. In-vivo Studies

Twenty-four male Wistar rats obtained from the Razi Vaccine and Serum Research Institute in Tehran, Iran and housed in an animal facility within Institute of Biochemistry and Biophysics at Tehran University under controlled environment. The conditions included a steady temperature of 22 ± 2 °C and a consistent 12 h light cycle from 08:00 to 20:00. Three weeks before commencing the experiments, we randomly assigned the rats to three equally sized groups (n = 8 rats per group). The first group was subjected to treatment with MWCNT, the second group received SWCNT treatment, while the third group functioned as a control and received a PBS treatment. These injections were administered intraperitoneally. Throughout the duration of the study, the rats resided in the aforementioned animal facility and were provided unrestricted access to both food and water. All protocols described here were approved by the Animal Care and Use Committee of Islamic Azad University (Ethical permission number: IR.IAU.SRB.REC.1395.49). Intraperitoneal injection (i.p.) of ketamine (80 mg.kg^-1) and xylazine (10 mg.kg^-1) was used for euthanasia in rats. At the 30-day and 60-day marks, we gathered 2 mL of whole blood from each rat. The last step entailed employing Ficoll density gradient centrifugation to separate peripheral mononuclear cells (PBMCs) and extract total RNA.

3.4. RNA Extraction and cDNA Synthesis

Total RNA isolation from Jurkat cells and rat PBMCs was performed using QIAzol Lysis Reagent. The quality of the extracted RNA was assessed by measuring its absorbance at 260/280 nm and by conducting electrophoresis on agarose gels with ethidium bromide. Subsequently, complementary DNA (cDNA) was synthesized using the QuantiTect Reverse Transcription Kit from Qiagen, located in Hilden, Germany. The RT-qPCR assays were performed using a commercial RT2 profilerTM PCR Array Human Apoptosis kit, (PAHS-D-012, SABiosciences a Qiagen company, USA), which includes pre-validated primers with optimized amplification efficiency and specificity. B-actin was used as the reference gene. Real-time PCR was conducted using SYBR Green Master Mix, along with appropriate primers (2 pg.mL), and 200 ng of cDNA. The real-time PCR was carried out on a BIO-RAD CFX96 system from Bio-Rad, USA. The PCR amplification program consisted of an initial cycle at 95 °C for 15 minutes, followed by 40 cycles of 95 °C, 60 °C, and 72 °C for 30 seconds each. Real-time PCR reactions were performed in triplicate. The quality of the resulting graphs, melting curves, and quantitative data analysis were assessed using CFX Manager software (version 1.1.308.111) from Bio-Rad, USA.

3.5. Statistical Analysis

The statistical analyses were conducted in triplicate, and the outcomes were presented as the mean with the standard deviation. Eeach comparison involved two independent groups and independent t-tests were used to evaluate the differences in gene expression levels. Since experimental design did not include simultaneous multi-factor or multi-dose interactions, t-testing was considered statistically appropriate and sufficient for the pairwise comparisons performed. Therefore, group differences were assessed using the Student’s t-test in SPSS 21.0 software. Statistical significance was determined when the probability (P) values were less than 0.05.

4. Results

4.1. CNT Functionalization

The optical absorption of CNTs, both in their crude and carboxylated forms, after preparation was measured using FTIR spectroscopy. As shown in Figure 1, the results demonstrate the accuracy of the synthesis and carboxylation of SWCNTs and MWCNTs nanotubes ( 11 , 12 ).

Figure 1.

The FTIR spectra of untreated CNT A) and the carboxylated CNT B).

The presence of functional groups on both SWCNTs and MWCNTs was confirmed by FTIR spectroscopy. Analysis of the FTIR spectra for acid-treated carbon nanotubes revealed distinct peaks centered at approximately 1637 and 1280 cm^-1, which were attributed to the stretching vibrations of carboxyl groups (C=O and C–O), indicating the successful introduction of these functional groups onto the nanotube surfaces. Furthermore, the FTIR spectra exhibited an additional band at around 1033 cm^-1, corresponding to the bending vibrations of hydroxyl groups (O–H). A broader band observed at approximately 3400 cm^-1 was also linked to the stretching vibration of hydroxyl groups (O–H). The presence of these characteristic peaks confirmed the successful functionalization of both SWCNTs and MWCNTs with carboxyl and hydroxyl groups.

The structural properties of carboxylated SWCNTs and MWCNTs were investigated using transmission electron microscopy (TEM). TEM images revealed that the nanotubes had lengths ranging from 1 to 10 μm, with diameters of approximately 10 nm for SWCNTs and 20 nm for MWCNTs.

Additionally, zeta potential measurements were performed using a Zeta Sizer to determine the surface charge of the carboxylated nanotubes. The results showed that the carboxylated SWCNTs had a zeta potential of approximately -38 mV, while the carboxylated MWCNTs had a zeta potential of approximately -40 mV.

4.2. Cell Viability

The MTT assay showed that exposure of Jurkat cells to carboxylated SWCNTs and MWCNTs did not significantly reduce cell viability compared with untreated controls (Fig. 2). The calculated cell viability percentages, compared to the control group, were 88% for MWCNT and 95% for SWCNT (Fig. 2) ( 11 , 12 ).

Figure 2.

Cell viability of Jurkat cells treated by SWCNT and MWCNT. Data are presented as mean ± SD from three independent experiments, and statistical analysis using Student’s t-test confirmed the absence of significant cytotoxicity (p > 0.05).

4.3. Gene Expression of Bcl-2 Family in SWCNTs-COOH and MWCNTs-COOH -Treated Jurkat Cells

The investigation of gene expression patterns within the Bcl-2 gene family in the Jurkat cell line revealed a wide spectrum of responses following treatment with both MWCNTs and SWCNTs. Figure 3 shows the expression levels of Bcl-2 family genes following exposure to carboxylated MWCNTs and SWCNTs.

Figure 3.

The expression level of BCL2 family genes following the effect of carboxylated MWCNT and SWCNT. Data are presented as mean ± SD of three independent experiments; p < 0.05 was considered significant. * Statistically different from the control group.

As shown in Figure 3, several genes, including BAD, BAX, Bcl-2, BCL2L1, BID, and BCL2L11, exhibited increased expression in Jurkat cells when subjected to SWCNTs and MWCNTs treatment in comparison to the non-treated cells. Additionally, the BAG1 gene showed elevated expression upon exposure to MWCNTs, whereas the BAG4 gene demonstrated increased expression in the presence of SWCNTs. Conversely, the remaining genes (BAG3, BAK1, BCL2L1, BCL2L10, BCL2L12, BCLAF1, BIK, BNIP1, BNIP2, BNIP3, BNIP3L, HAK, and MCL1) did not display significant changes when comparing treated cells to control cells.

4.4. Bcl-2 Gene Expression in CNT-Treated Rat PBMCs

Table 1 presents a comprehensive analysis of the mean alteration in Bcl-2 family gene expression across the three studied groups following 30- and 60-day periods in the animal study. After 30 days of treatment, the results show an increase in Bcl-2 and BAD gene expression in both treatment groups compared to the control group, with statistical significance (p = 0.038 and 0.003, respectively).

Table 1.

The mean value of all gene expression in the three studied groups in the animal phase after 30 and 60 days.

	Group	Mean +_SD (30 days)	P-value (30 days)	Mean +_SD (60 days)	P-value (60 days)
BCL2	PBS	1.00±0.21	0.038	1.10±0.35	0.268
	MWCNT	2.27±0.63		1.37±0.22
	SWCNT	2.30±0.37		1.22±0.41
BCL2L1	PBS	1.09±0.21	0.208	1.07±0.12	0.371
	MWCNT	12.02±8.77		2.02±1.33
	SWCNT	20.94±11.85		5.22±1.41
BAD	PBS	1.0±0.21	0.003	1.00±0.54	0.569
	MWCNT	5.33±1.33		1.36±0.23
	SWCNT	6.16±1.7		2.26±0.7
BAX	PBS	1.00±0.34	0.247	1.00±0.45	0.276
	MWCNT	2.99±1.26		1.32±0.06
	SWCNT	2.10±0.65		1.54±0.51
BID	PBS	1.00±0.25	0.301	1.00±0.23	0.512
	MWCNT	1.31±0.37		1.37±0.64
	SWCNT	1.98±0.64		1.20±0.50
BIRC2	PBS	1.0±0.37	0.115	1.22±0.17	0.547
	MWCNT	0.18±0.098		1.34±0.41
	SWCNT	0.52±0.14		1.62±0.11
BIRC3	PBS	1.0±0.50	0.110	1.21±0.29	0.371
	MWCNT	0.14±0.053		1.44±0.03
	SWCNT	0.18±0.082		1.02±0.26

However, no significant changes were observed in BCL2L1, BAX, and BID gene expression in the treatment groups relative to the control. Analysis after 60 days, as shown in Table 1, indicated that the expression of Bcl-2, BCL2L1, BAD, BAX, and BID genes in both treatment groups did not differ from the control group.

4.5. Gene Expression of IAP Family in SWCNTs-COOH and MWCNTs-COOH -Treated Jurkat Cells

Analysis of gene expression in the studied cell line for the IAP family showed that some genes, such as BIRC2 and BIRC3, were upregulated in response to both types of carbon nanotubes. In contrast, other genes in this family showed little to no change in expression (Fig. 4).

Figure 4.

The expression level of IAP family genes following the effect of carboxylated MWCNT and SWCNT in Jurkat cells. Data are presented as mean ± SD of three independent experiments; p < 0.05 was considered significant. * Statistically different from the control group.

4.6. IAP Gene Expression in CNT-Treated Rat PBMCs

After 30 days, Table 1 shows that the expression levels of the BIRC2 gene in both treatment groups remained unchanged compared to the control group (p = 0.115), indicating no statistical significance. The MWCNT group had a mean expression of 0.18 ± 0.098, and the SWCNT group had a mean of 0.52 ± 0.14, reflecting a non-significant decrease.

Similarly, the expression levels of the BIRC3 gene in both treatment groups did not differ from the control group (p = 0.110). The MWCNT group recorded a mean of 0.14 ± 0.053, and the SWCNT group had a mean of 0.18 ± 0.082, showing a non-significant decrease.

After 60 days, Table 1 confirms that the expression levels of both BIRC2 and BIRC3 genes in the treatment groups remained unchanged compared to the control group.

5. Discussion

The toxicity of CNTs is a significant concern due to their potential harm to human health and the environment. CNTs have notable effects on cells due to their ability to penetrate cell membranes ( 2 , 6 ). They induce oxidative stress, inflammation, and genotoxicity within cells, disrupting cellular signaling and potentially causing cell death ( 18 , 19 ).

Although numerous studies have explored the cytotoxic and apoptotic effects of carbon nanotubes (CNTs), the molecular mechanisms underlying CNT-induced apoptosis remain controversial. Some reports indicate that CNTs trigger apoptosis through oxidative stress, mitochondrial dysfunction, and caspase activation, whereas others describe minimal or even protective cellular responses depending on CNT type, surface charge, and degree of functionalization ( 8 ). These findings indicate the context-dependent nature of CNT-induced cell death and the need for a more integrated understanding of how pro- and anti-apoptotic regulators respond under varying biological conditions.

Functionalized CNTs have been extensively investigated in Jurkat cells, revealing that surface chemistry plays a pivotal role in determining cellular responses, including viability, proliferation, and oxidative balance ( 9 , 10 ) . Further studies have examined apoptotic signaling pathways, such as caspase-2 and TNF family gene induction following CNT exposure ( 11 , 12 , 13 ). However, Zhou et al. (2017) reported that COOH functionalization reduced CNT cytotoxicity by promoting aggregation ( 5 ), whereas Ursini et al. (2016) observed increased inflammatory and toxic responses in BEAS-2B cells, indicating that surface modification can lead to divergent biological outcomes ( 14 ). Additionally, CNT-mediated anti-caspase-3 siRNA delivery has been shown to mitigate neuronal apoptosis and neurodegeneration further illustrating the complex and sometimes contradictory nature of CNT–cell interaction ( 15 ).

Despite these valuable contributions, significant knowledge gaps persist. Previous research has largely focused on isolated apoptotic markers or single experimental models, with limited insight into how carboxylated single-walled (SWCNT-COOH) versus multi-walled (MWCNT-COOH) CNTs coordinately regulate pro-apoptotic (Bcl-2 family) and anti-apoptotic (IAP family) genes across both cellular and whole-animal models. Moreover, most studies have assessed only single time points and have not integrated surface characterization with temporally resolved gene-expression profiling in vivo ( 9 , 10 , 11 , 12 ).

To address these gaps, the novelty of the present study lies in its parallel examination of the Bcl-2 and IAP gene families across in vitro (Jurkat cells) and in vivo (rat PBMCs) systems exposed to carboxylated CNTs. By combining detailed physicochemical characterization (FTIR, TEM, and zeta potential) with qRT-PCR analysis over 30- and 60-day intervals, this study provides a comparative and time-dependent understanding of apoptosis and survival regulation under CNT exposure, linking gene-level responses to oxidative stress and mitochondrial perturbation. This dual-system, multi-timepoint approach advances previous single-gene or single-model investigations by offering mechanistic insights into the dynamic balance between pro-apoptotic and anti-apoptotic signaling in response to CNTs.

The results reveal a complex interplay of molecular responses and provide valuable insights into the potential effects of CNT exposure on apoptosis regulation and gene expression. Recent studies ( 20 , 21 , 22 ) have highlighted that oxidative stress and mitochondrial signaling play central roles in CNT-induced apoptosis, with surface carboxylation affecting cellular redox balance and apoptotic pathways. Our present study extends these findings by simultaneously examining multiple Bcl-2 and IAP family members under both in-vitro and in-vivo conditions, thus providing a broader mechanistic context for understanding how CNT functionalization influences cell death regulation.

The BAD gene exhibited a significant increase in expression in Jurkat cells when subjected to SWCNT and MWCNT treatments. BAD is a key regulator of apoptosis, and its increased expression indicates a robust pro-apoptotic response in Jurkat cells ( 23 , 24 ). The BAG1 gene displayed increased expression upon exposure of Jurkat cells to MWCNTs. BAG1 is a member of the BAG family of proteins known to interact with chaperone proteins and participate in various cellular processes, including apoptosis regulation ( 25 , 26 ). Conversely, the BAG4 gene demonstrated increased expression in the presence of SWCNTs. The upregulation of BAG4 in response to SWCNT exposure highlights a differential effect of SWCNT compared to MWCNT. This finding underscores the importance of considering the type of carbon nanotube in toxicity assessments. Several other genes, including BAX, Bcl-2, BCL2L1, BID, and BCL2L11, also exhibited heightened expression levels in Jurkat cells when exposed to both SWCNT and MWCNT treatments that further emphasizes the potential activation of apoptotic pathways in response to CNT exposure ( 27 , 28 ). The simultaneous upregulation of both pro-apoptotic (BAD, BAX, BID, and BCL2L11) and anti-apoptotic (Bcl-2 and BCL2L1) genes underscores the complexity of the cellular response. The balance between these factors may determine whether the cells ultimately undergo apoptosis or survive in response to CNT exposure.

In the animal study, a significant upregulation of Bcl-2 gene expression was observed in both SWCNT and MWCNT treatment groups after 30 days. This finding suggests that CNT exposure may initially suppress apoptosis in the animal model, potentially promoting cell survival. However, the lack of significant changes in gene expression after 60 days indicates that this effect may not be sustained. Previous studies have reported conflicting results regarding the impact of CNT exposure on Bcl-2 expression, some showing upregulation, while others have reported downregulation or no significant changes. These inconsistencies further highlight the complexity of CNT-animal interactions, which can vary depending on factors such as CNT type, concentration, duration of exposure, and the specific animal model used.

The significant increase in the expression of the BAD gene in response to both MWCNT and SWCNT in the animal study after 30 days may suggest that apoptosis was triggered as a protective mechanism against potential cellular damage in response to CNTs. Similar to Bcl-2, the lack of sustained changes in BAD expression after 60 days raises questions about the long-term effects of CNT exposure on apoptosis regulation in the animal model.

BAX is another pro-apoptotic protein involved in the regulation of cell death ( 29 , 30 ). In the animal study, the expression of the BAX and BID genes did not show significant alterations after 30 days of CNT exposure. BID, another key regulator of apoptosis, is involved in the cross-talk between the intrinsic and extrinsic apoptotic pathways ( 31 , 32 ). This suggests that the initial response of the animal model to CNTs may not involve changes in BAX and BID expression. However, it is important to consider that the absence of significant changes does not necessarily indicate the lack of biological effects. Similar results were observed for BCL2L1 expression in the animal study. BCL2L1 encodes a protein similar to Bcl-2 and plays a role in preventing apoptosis by inhibiting the release of cytochrome c from mitochondria ( 27 , 33 ).

In Jurkat cells, significant upregulation of BIRC2 and BIRC3 (members of the IAP family) was observed following exposure to both SWCNTs and MWCNTs. This finding underscores the involvement of IAP family members in cellular responses to CNT-induced stress. IAPs are known to inhibit caspase activation and regulate NF-κB signaling pathways, which play crucial roles in cell survival and apoptosis ( 34 , 35 , 36 ). Their upregulation may represent a cellular mechanism to counteract pro-apoptotic signals induced by CNT exposure, as evidenced by the concurrent upregulation of pro-apoptotic genes such as BAD and BAX.

In contrast, the unchanged expression of IAP family members in the animal models after 30 and 60 days of CNT exposure may reflect adaptive mechanisms or compensatory processes that mitigate the initial cellular stress observed in Jurkat cells. The diminished alteration in Bcl-2 and IAP gene expression observed after 60 days of exposure may reflect a physiological adaptation rather than a lack of exposure effect. Prolonged CNT contact can stimulate compensatory cellular mechanisms such as antioxidant responses and activation of DNA repair and survival pathways, which may normalize apoptotic gene expression toward baseline levels. This adaptive recovery has also been noted in other long-term nanomaterial exposure studies, where initial molecular disturbances gradually decline as homeostasis is re-established ( 8 ).

The divergence between in vitro and in vivo IAP gene expression likely arises from differences in CNT uptake, biodistribution, and clearance. In cultured Jurkat cells, CNTs are directly in contact with cells at a defined concentration, leading to robust cellular stress and IAP regulation. In contrast, in animals, systemic administration subjects CNTs to dilution, protein corona formation, and organ-level sequestration, which can reduce the effective cellular dose reaching. Moreover, clearance mechanisms and tissue barriers further limit sustained cellular exposure, which may explain why IAP upregulation was observed only in vitro while Bcl-2 family responses were more transient in vivo ( 8 , 37 , 38 , 39 ).

In fact, the lack of sustained changes in Bcl-2 family gene expression after 60 days suggests that the influence of CNT exposure on apoptosis regulation may be transient or less pronounced in this animal model. Indeed, the results indicate that exposure to CNTs, both MWCNTs and SWCNTs, initially leads to the upregulation of specific genes involved in apoptosis regulation. However, these effects may not be maintained over a longer exposure period. The intricate balance between pro- and anti-apoptotic factors is crucial, as it may determine whether cells ultimately undergo apoptosis or survive in response to CNTs exposure.

6. Conclusion

These findings highlight the complex regulation of apoptosis in response to CNTs exposure and reveal differences in IAP gene expression between in vitro and in vivo models. While cellular studies provide detailed mechanistic insights, animal studies offer a broader systemic perspective, emphasizing the importance of context in nanotoxicology. Future studies should focus on elucidating the molecular pathways linking CNTs exposure the regulation of Bcl-2 and IAP family members, including interactions with TRAIL and caspases. In addition, dose–response studies and validation at the protein level are recommended to better understand temporal dynamics and resolve discrepancies between in vitro and in vivo observations. While this study focused on gene expression changes, further evaluation of histopathological alterations and biochemical toxicity markers is warranted to better correlate molecular responses with tissue-level effects.