Exploring the role of hepatic lipid droplets in mouse liver toxicity induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin: sequestration, biochemical alterations and gene regulation

Abstract

Dioxins are highly toxic and persistent environmental pollutants. The liver, with its complex enzymatic system, plays a crucial role in their detoxification. Hepatic lipid droplets (LDs) are specialized organelles with diverse biological functions, yet their role in dioxin toxicity remains poorly understood. This study investigates the impact of TCDD, the most potent dioxin congener, on the biochemical properties and functionality of hepatic LDs in exposed mice. Female and male BALB/c mice (12–14 weeks old) received a single oral dose of 15–25 µg/kg bw, designated as TCDD₁₅-F, TCDD₁₅-M, TCDD₂₅-F, and TCDD₂₅-M. TCDD exposure induced a dose-dependent increase in liver weight, suggesting hepatomegaly. The number of hepatic LDs and their protein composition increased, with greater hydroxylation enzymatic activity observed in males. HPLC analysis confirmed that hepatic LDs in both sexes accumulated and stored TCDD, raising concerns about potential long-term effects. Moreover, gene expression analysis revealed significant alterations in hepatotoxicity markers (Ahr, Arnt, Cyp1a1, Gsta1, Gsta3), oxylipin biosynthesis genes (Lox15, Cox2), and LD formation genes (Plin5, Cidec, Ppara, Pparg) in a dose-, time-, and sex-dependent manner. These findings offer insight into how lipophilic pollutants are sequestered and metabolized, aiding strategies to reduce dioxin toxicity and treat related liver disorders.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-21508-2.

Introduction

The term “dioxins” is a broad and non-specific classification that refers to a group of structurally related halogenated aromatic compounds, primarily polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs). Additionally, certain polychlorinated biphenyls (PCBs) and other related compounds exhibit dioxin-like toxicological properties due to their structural and functional similarities^1–4. The most extensively studied and highly toxic congener within this group is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which serves as the prototype congener for assessing the toxicity of other dioxin congeners. The sources of dioxin compounds vary according to their chemical classification, however, under conditions of incomplete combustion, the formation of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/Fs) can occur at significant levels^5,6. Once realized, dioxins persist in the environment due to their intrinsic stability and resistance to physico-chemical factors. On the other hand, their high lipophilicity facilitates their accumulation in biological systems, particularly in lipid-rich tissues of exposed organisms. As these compounds ascend the food chain, their concentrations progressively increase at each trophic level, leading to significant biomagnification in top predators. In both animals and humans, dietary intake represents the predominant route of dioxin exposure^7,8.

Human exposure to dioxins primarily occurs through oral absorption, which is influenced by the nature of the carrier². However, the consumption of lipid-rich foods is recognized as the principal exposure route. Following absorption, dioxins predominantly accumulate in adipose tissues, with notable deposition in the liver^2,9,10.

At molecular level, the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, plays a central role in mediating dioxin toxicity and serves as a key physiological regulator within the body. Beyond its involvement in xenobiotic metabolism, the AhR is crucial for various normal biological functions^11,12. However, dysregulated activation of AhR can lead to a range of toxic effects, driven by complex transcriptional patterns that are specific to the ligand, cell type, and physiological context¹³.

The aryl hydrocarbon receptor (AhR) resides in the cytosol within a protein complex. Upon ligand binding, it translocates to the nucleus, dissociates from chaperones, and dimerizes with ARNT¹⁴. The AhR/ARNT complex binds to dioxin response elements (DREs/XREs), regulating gene expression^15,16. Upon dioxin activation, the AhR signaling pathway regulates numerous genes, with CYP1A1 being the most studied. CYP1A1, a Phase I cytochrome P-450 enzyme, plays a key role in drug metabolism and xenobiotic detoxification^14,17,18. Dioxin-induced AhR activation also upregulates other Phase I/II enzymes, including CYP1A2, CYP1B1, ALDH3, and GSTA1^13,19,20.

The liver is highly susceptible to dioxin-induced toxicity, with hepatotoxic effects strongly influenced by the nature of exposure, whether chronic or acute^21–24. TCDD increases lipid peroxidation, ROS production, and liver damage, while reducing membrane fluidity and GSH levels, and disrupting antioxidant enzyme balance in rat and mouse livers^25–27. In this regards, Peroxygenases constitute a distinct class of cytochromes that mediate selective oxidation reactions without requiring the NAD(P)H cofactor, differing fundamentally from the catalytic mechanism of CYP450 monooxygenases^28–30.

Hepatocytes contain specialized organelles essential for various biological processes. Lipid droplets (LDs) consist of a neutral lipid core encased by a phospholipid monolayer, to which numerous functional proteins are associated. Due to their crucial role in toxicological responses and dynamic protein composition, LDs are believed to exhibit enzymatic activity, facilitating the oxidation of organic toxins and enhancing their excretion through the excretory system^31–34.

Building on these observations, we hypothesized that TCDD exposure disrupts the biochemical composition and functionality of hepatic lipid droplets (LDs) by altering lipid metabolism, oxidative stress responses, and LD-associated enzymatic activity, thereby contributing to hepatotoxicity. Accordingly, this study aimed to investigate the effects of TCDD, the most potent dioxin congener, on hepatic LDs in exposed mice. Specifically, we examine changes in the expression of key genes involved in LD lipid biogenesis, oxidative stress markers, and enzymatic activity. Our findings aim to clarify the role of LDs in TCDD-induced liver toxicity and their involvement in pollutant metabolism and sequestration.

Materials and methods

Chemicals and reagents

2,3,7,8-Tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD dissolved in toluene, purity 99%) was purchased from Supelco Inc., USA. A commercial TCDD solution, supplied as a 1-mL ampule of TCDD at 10 µg mL^−l, was placed in a 10-mL capped glass tube and evaporated to dryness under nitrogen flow. For health and environmental safety reasons, residual TCDD was re-dissolved in a minimum volume (100 µL) of dimethyl sulfoxide (DMSO) and diluted to the required concentration in corn oil. Oligonucleotides (Table S1) were supplied by the Primer Synthesizer Unit at the Atomic Energy Commission of Syria (AECS).

Mice and TCDD administration

Twelve-week-old female and male BALB/c mice, with average weight of 20–25 g, were obtained from the Breeding Unit for Inbred Mice of the Department of Molecular Biology and Biotechnology, AECS. All experimental work related to study design and experimental procedure for animals used in this study were performed in accordance with the relevant guidelines and regulations of The ARRIVE guidelines 2.0³⁵. Mice were housed in clean cages, received feed and clean tap water ad libitum and kept under standard 12-h light/dark cycles at 25 ± 2^°Cand 40%–60% humidity³⁰. Two randomly selected groups of female mice and two groups of male mice were used in the experiments, each group consists of twelve animals. Groups I and II of females and males orally received a single 50 µL-dose of corn oil containing 15 µg TCDD/kg bw (body weight), therefore referred to as (TCDD₁₅-F) and (TCDD₁₅-M), respectively. Groups III and IV of females and males orally received a single 50 µL-dose of corn oil containing 25 µg TCDD/kg bw (body weight), therefore referred to as (TCDD₂₅-F) and (TCDD₂₅-M), respectively. The use of this dose is justified by previous studies demonstrating its ability to induce well-characterized toxicological effects in the liver, as well as in both male and female reproductive organs^36–38. While the control groups of females and males orally received a single 50 µL-dose of corn oil alone and are referred to as (CTL-F) and (CTL-M), respectively. Basing on primary results obtained from assaying different concentrations of TCDD for different times of exposure, the dose-time point used in this study is justified by a pronounced hepatotoxicity at these conditions. All mice were maintained on a regular diet. Animals of each group were weighed and euthanized after four consequent time points: 14, 28, 56 and 112 days after treatment, according the literature. Mice were anesthetized with a single intraperitoneal injection of a cocktail of 70 mg/kg Ketamine and 7 mg/kg Xylazine as previously described³⁹. Animals were euthanized using gradual-fill CO₂ inhalation in accordance with AVMA guidelines. Without pre-charging the chamber, animals were placed in the chamber and introduce 100% CO₂ at a flow rate of 50% of the chamber volume per minute, added to the existing air in the chamber. This method complies with the AVMA Guidelines for the Euthanasia of Animals (2020) (https://olaw.nih.gov/policies-laws/avma-guidelines-2020.htm) and was approved by the institutional animal care and use committee (IACUC)⁴⁰. Both blood and ovary samples were collected for further analysis⁴¹. The livers were rapidly removed, rinsed with 0.9% NaCl, weighed, immediately frozen in liquid nitrogen and stored at −20 °C until further use. All experimental protocols used in this study was approved by the AECS Committee of Animal Ethics. All animals’ experiments were carried out in accordance with the guidelines of AECS Committee of Animal Ethics for animal experiments which is adopted from the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments.

Isolation of liver LDs fraction

The isolation of LDs was performed based on their buoyant density by differential centrifugation and using a gradient floating buffer in cooling conditions⁴². An equal quantity (one gram) of liver from each experimental group were pooled (3 g) and vigorously ground in a mortar and pestle in the presence of liquid nitrogen. The liver powder was immediately homogenized with 6 mL of homogenization buffer (HB) (50 mM Tris-HCl, 1 mM EDTA, 1 M sorbitol, pH 7.5). The total homogenate was divided into four fractions of 1.5 mL each, transferred into 2-mL microcentrifuge tube and centrifuged at 5000 ×g for 15 min at 4 °C. The supernatant (about 1 mL) containing LDs was taken into a clean 2-ml tube, overlaid with an equal volume of floating buffer (FB) (50 mM Tris-HCl, 1 mM EDTA, 1 M sorbitol, pH 7.5) and centrifuged at 21,130 ×g for 1 h at 4 °C. After centrifugation, three fractions were obtained, an upper creamy floating layer corresponding to the LDs, an intermediate phase corresponding to cytosolic proteins fraction, and a pellet corresponding the microsomal fraction (M). LD fractions were separately and carefully taken and subjected to a one-step washing with 1 mL of FB, then centrifuged at 21,130 ×g for 1 h at 4 °C. Finally, the respective fractions were re-suspended in 100 µL of suspension buffer (50 mM Tris-HCl pH-7.5, 1 mM EDTA pH-8.0 and 10% glycerol) and stored at 4 °C.

Examination of LDs using fluorescent microscopy

The lipidic core of LDs were stained by Nile Blue dye and examined by a fluorescent microscope. For that, the lipophilic Nile Blue dye was dissolved in dimethyl sulfoxide (DMSO) at concentration of 1 mg mL⁻¹ then diluted 10× and freshly used to stain 1 µL of LDs for 15 min at room temperature. Stained LDs were immediately examined under a fluorescent microscope (Nikon Ti-U microscope supplied with an Olympus FE-4000 camera) using red and green fluorescence filters (excitation, 545 and 480 nm; emission, 620 and 535 nm, respectively) at ×40 magnification. The concentration of proteins associated with LDs was determined by the Bradford method using bovine serum albumin (BSA) as a standard⁴³. The absorbance at 595 nm was measured using a Jenway 6840 spectrophotometer.

Hemocytometer and flow cytometry

LDs were quantified in two ways. Firstly, 10 µL of isolated LDs was diluted and counted using a Marien Field Cell Counting Chamber Neubauer Improved (Germany). The number per milliliter was then determined using a referenced equation. In order to examine LDs using flow cytometry, 18 µL of the extended dye was added to 2 µL of pure LDs 280 µL of PBS (1x) was mixed well, the mixture was incubated in the dark at laboratory temperature for 15 min, and then passed through a flow cytometer (BD FACSCalibur™ Flow Cytometer, Becton, Dickinson and Company, United States) to determine their number and dimensions. The dimensions of LDs were concurrently determined by the fluorometer, and changes in their lipid content were investigated.

Determination of protein concentration

The Bradford Assay (Bio-Rad) was used to determine the concentration of proteins bound to LDs⁴³. The extinction coefficient at a wavelength of 595 nm was measured using quartz cuvettes (Hellma Analytics, USA) and a spectrophotometer (JENWAY 6850 UV/Vis. Spectrophotometer).

Enzymatic activity

The potential fatty acid oxygenation activities of LD-associated proteins were assayed using a rapid test based on the measurement of aniline hydroxylation as described before^44,45. This was performed by incubating an increasing number of LDs containing about 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8 and 25.6 µg of total proteins with 1 M of aniline in a final volume of 1 mL of 0.1 M potassium phosphate buffer, pH 7.4. The enzymatic activity was initiated, at room temperature, by adding 20 mM of cumene hydroperoxide as an oxygen donor. The accumulation of hydroxy aniline was spectrophotometrically measured at 310 nm.

HPLC analysis of TCDD

For the solubilization of LDs and analysis of TCDD content, 100 µL of LDs were mixed with 1 mL of acetone/hexane (1:1, v/v) in 2 mL-tube and mixed intermittently on a vortex shaker for approximately 20 min. The samples were then centrifuged at 15,100 rpm for 20 min at 4 °C. The extraction was repeated three times and the supernatant (organic phase) was collected and passed through a 0.22 μm aseptic filter (MS Nylon Syringe Filter, USA) into a clean opaque vial. The entire organic phase was evaporated to complete dryness under a gentle stream of nitrogen gas and 400 µL of acetonitrile solution was added to the vial for subsequent analysis by HPLC (HPLC, Jasco, Japan) as described in Sicupira et al., 2019⁴⁶ with some modifications. The analysis was performed using a C18 column (ZORBAX Eclipse XDB C, 150 × 4.6 mm, 5 μm) of the type described in the ZORBAX Eclipse XDB C18 paper (2019). The mobile phase was acetonitrile, the temperature was 30 °C, the wavelength was 232 nm, the flow rate was 0.5 mL/min and the syringe volume was 20 µL. An internal standard that was spiked into the samples at a constant concentration prior to extraction and analysis. The peak areas of TCDD were then normalized to the corresponding internal standard signal to subtract all technical variations.

Genes, primers and transcripts analysis

Three sets of genes were selected to evaluate the hepatic transcriptional changes as a function of TCDD exposure. Set-I refers to key genes involved in the reception and detoxification pathway of TCDD. The selected key genes were regrouped in three sets; Set-I comprises key genes known as potential mediators in the TCDD-hepatotoxicity pathway, e.g., aryl hydrocarbon receptor (Ahr), aryl hydrocarbon receptor nuclear translocator (Arnt), cytochrome P450 1A1 (Cyp1a1), glutathione S-transferase α−1 (Gsta1) and glutathione S-transferase α−3 (Gsta3). Set-II comprises genes with key roles in lipid metabolism and oxylipins biosynthesis, e.g., the cyclooxygenase (Cox2) and lipoxygenase-15 (Lox15). Set-III contains key genes encoding lipid droplets-associated proteins, e.g., Perilipin 5 (Plin5), Cell death inducing DFFA-like effector c (Cidec), peroxisome proliferator-activated receptor α (Ppara) and peroxisome proliferator-activated receptor gamma (Pparg). Gene expression was normalized by using a set of reference genes, including actin-beta (Actb), ribosomal protein L13a (RPL13a), hypoxanthine phosphoribosyl transferase 1 (Hprt1) and succinate dehydrogenase complex flavoprotein subunit A (SdhA). Table S1 summarizes genes name abbreviation, NCBI-accession number, forward and reverse oligonucleotides and expected amplicon size. Changes in relative transcriptional abundance of three sets of genes in response to TCDD exposure were analyzed by reverse-transcription quantitative PCR (RT-qPCR) as previously described (Hammoudeh et al., 2020).

For total RNA extraction, 30 mg of liver from each animal group were ground in the presence of liquid nitrogen and the total RNA was extracted using RNeasy kit according to the manufacturer’s instructions (Qiagen, Germany). DNA traces were removed by treating the samples for 1 h at 37 °C with 2 U of RNase-free RQI DNase (Promega, USA). RNAs were diluted to 200 ng µL⁻¹using RNase-free water and stored at − 80 °C. An aliquots of 1 µg total RNA were used for first-strand cDNA synthesis using M-MLV RT (Invitrogen), for more details please refer to Hanano et al.⁴⁷,.

Real-time PCR was performed in 96-well plates using an AriaMx Real-time PCR System from Agillent technologies, USA. In brief, 25 µL reaction mixtures contained 0.5 µM of each specific oligonucleotide primer for the target and reference genes, 12.5 µL of SYBR Green PCR mix (Bio-Rad, USA) and 100 ng cDNA. RT-qPCR conditions were as described before⁴⁵. Each point was replicated in triplicate and the average of CT was taken for calculation of the relative quantification RQ = 2^(−ΔΔCT).

Statistics

All data are expressed as means ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism software, version 10. Comparisons between control and treatments were evaluated by ANOVA two-way analysis followed by Dunnett’s or Tukey’s tests. Each measured value was compared based on two main factors—dose and animal gender. Differences between treatment groups were considered significant when p < 0.05 or p < 0.001.

Results and discussion

Effect of TCDD on body and liver weight

The body weight and liver weight of male and female mice in both the experimental and control groups were measured. Body weight was recorded at the start of the experiment, prior to TCDD administration, and again at the time of sacrifice. At the beginning of the experiment, the mean body weight of female and male mice in the control TCDD₁₅ group was 19.13 ± 0.88 g and 23.25 ± 1.14 g, respectively. Over time, female mice in the TCDD₁₅ group exhibited an increase in mean body weight, while male mice showed a decrease, reaching 20.6 ± 1.6 g and 21.67 ± 2.11 g, respectively (Fig. 1A). The mean body weight of both female and male mice in the TCDD₂₅ group fluctuated over time. While the mean weight of TCDD₂₅-exposed females decreased up to day 56, a significant increase was observed by day 112. A similar trend was noted in TCDD₂₅-exposed males (Fig. 1B). These findings suggest that TCDD exposure induces gender-, dose-, and time-dependent variations in body weight, highlighting differential physiological responses to dioxin toxicity. At the time of sacrifice, liver weights were recorded for both TCDD-treated and control mice, with females averaging 1.26 ± 0.12 g and males 1.47 ± 0.06 g. In the TCDD₁₅ group, the average liver weight significantly increased in females to 1.66 ± 0.18 g, while in males, it showed a non-significant increase to 1.53 ± 0.25 g. In the TCDD₂₅ group, liver weights were significantly elevated in both sexes, reaching 1.43 ± 0.05 g in females and 1.72 ± 0.23 g in males (Fig. 1C). These results suggest a dose-dependent effect of TCDD on liver weight, potentially indicating hepatomegaly as a response to dioxin exposure.

Fig. 1.

Variations in body weight, liver weight, and liver-to-body weight ratio in TCDD-exposed females and males. (A) The mean weight of animals of experimental group receiving a dose of 15 µg/Kg/B.W (TCDD₁₅-F and TCDD₁₅-M) was compared with the mean weight of the control group (CTL-F and CTL-M, respectively) at four time points (14, 28, 56 and 112 days). (B) The mean weight of animals of experimental group receiving a dose of 25 µg/Kg/B.W (TCDD₂₅-F and TCDD₂₅-M) was compared with the mean weight of the control group (CTL-F and CTL-M, respectively) at four time points (14, 28, 56 and 112 days). (C) The mean liver weight of females (F) and males (M) receiving TCDD₁₅ and TCDD₂₅ was compared with that of the control group (CTL). (D) Represents the ratio of liver weight to body weight at four experimental time points (14, 28, 56 and 112 days) for female groups (TCDD₁₅-F and TCDD₂₅-F) and male groups (TCDD₁₅-M and TCDD₂₅-M) compared to their respective controls (CTL). Two-way ANOVA followed by Dunnett’s test or Tukey’s test was used to analyze the data using GraphPad Prism 10. A p-value of less than 0.05 was considered statistically significant (*), while a p-value of less than 0.01 was considered highly statistically significant (**), and so on. A p-value ≥ 0.05 was considered not statistically significant (ns).

Subsequently, the liver-to-body weight ratio was calculated and analyzed statistically (Fig. 1D), raising significant differences between both TCDD-treated groups (TCDD₁₅ and TCDD₂₅) and their respective controls. In females, the liver-to-body weight ratio was significantly increased in both TCDD₁₅ and TCDD₂₅ groups on days 14, 56, and 112, with no significant differences detected on day 28. In males, significant increases were observed on days 28 and 56 in the TCDD₁₅ group, and on days 14 and 112 in the TCDD₂₅ group. These findings indicate a time- and sex-dependent impact of TCDD on liver weight relative to body mass.

To discuss on these data, it is well recognized that dioxins cause irreversible damage in both humans and animals, with the liver being particularly susceptible to TCDD-induced toxicity, leading to various hepatotoxic effects^23,24. The severity of TCDD-induced hepatotoxicity is largely dose-dependent. In this study, female and male mice were acutely exposed to a single oral dose of 15 and 25 µg/kg, which is comparable to the dose used for rats (22 µg/kg) but significantly lower than the doses reported for mice (146 µg/kg) and guinea pigs (1 µg/kg)^48–51. While the effects of TCDD on body weight remain inconclusive, the impact on liver weight and liver-to-body weight ratio was evident. Under our experimental conditions, TCDD exposure resulted in significant alterations in body and liver weight. In this context, it was reported that the chronic exposure to dioxin promotes hepatomegaly and liver fibrosis development in mouse^50,52. Moreover, these findings align with previous studies reporting similar effects, whether using a higher single-dose concentration or lower concentrations administered over multiple exposures^53–55. Similar effects have been reported, particularly when a fasting strategy was implemented following oral TCDD administration for 72 h⁵⁶ or even up to 168 h⁵⁷. Additionally, consecutive administration of doses approaching the LD₅₀ for up to 30 days resulted in comparable outcomes⁵⁸. Furthermore, TCDD has been shown to induce wasting at higher exposure levels⁵⁹, whereas lower doses have been associated with weight gain⁶⁰.

TCDD exposure impacts physico-chemical properties of hepatic LDs

Hepatic lipid droplets (LDs) were isolated from the livers of both control and TCDD-exposed mice (TCDD₁₅ and TCDD₂₅) for both sexes at four time points (14, 28, 56, and 112 days). The integrity and purity of the LDs were assessed using Nile blue staining and fluorescence microscopy. The droplets exhibited spherical shapes with nonuniform sizes and red fluorescence (Fig. 2A). LDs from all experimental groups were examined, with Fig. 2B displaying the LDs from TCDD₁₅-F, TCDD₂₅-F, TCDD₁₅-M, and TCDD₂₅-M at the designated time points. Overall, the micrographs revealed no significant differences in LD morphology or size across groups. Flow cytometry analysis determined the mean LD dimensions to be 180 ± 5 nm in females and 160 ± 5 nm in males, with no observed variations between control and TCDD-exposed animals.

Fig. 2.

Fluorescence microscopy images of hepatic lipid droplets (LDs). The images show LDs under fluorescence microscopy after staining with Nile Blue dye. The number of LDs within the female (TCDD₁₅-F and TCDD₂₅-F) and male (TCDD₁₅-M and TCDD₂₅-M), as well as the control (CTL-F and CTL-M) groups at four time points (14, 28, 56 and 112 days) at 40x magnification. Negative control show no autofluorescence in the absence of LDs. Scale bars represent 10 μm.

In the control group, the hepatic LD count, determined using a hemocytometer, was approximately 6.8 × 10⁸ LD/mL in females and 3.1 × 10⁸ LD/mL in males (Fig. 3A, B). In the TCDD₁₅ group, LD counts significantly increased, reaching 9.1 × 10⁸ LD/mL in females and 9.3 × 10⁸ LD/mL in males. A similar trend was observed in the TCDD₂₅ group, with LD counts rising to 7.1 × 10⁸ LD/mL in females and 6.8 × 10⁸ LD/mL in males. These findings indicate that TCDD exposure markedly enhances hepatic LDs accumulation, with the most pronounced increase observed in male mice of the TCDD₁₅ group, independent of time points.

Fig. 3.

Number of hepatic lipid droplets (LDs) in the experimental groups. (A) The female groups (TCDD₁₅-F and TCDD₂₅-F) were compared to the CTL-F. (B) The male groups (TCDD₁₅-M and TCDD₂₅-M) were compared to the CTL-M at four time points (14, 28, 56 and 112 days). Furthermore, the differences in concentration of LDs-associated proteins in TCDD-treated groups in comparison to their controls are illustrated in (C) for (TCDD₁₅-F and TCDD₂₅-F) groups and (D) for (TCDD₁₅-M and TCDD₂₅-M) groups, respectively. Two-way ANOVA followed by Tukey’s test was used to analyze the data using GraphPad Prism 10. A p-value of less than 0.05 was considered statistically significant (*), while a p-value of less than 0.01 was considered highly statistically significant (**), and so on. A p-value ≥ 0.05 was considered statistically insignificant (ns).

To assess the functional characteristics of hepatic LDs, the protein concentration of LD fractions was measured. As shown in Fig. 3C and D, the mean concentration of LD-associated proteins in the control groups was 1.15 mg/mL in females and 1.3 mg/mL in males. In the TCDD₁₅ group, the mean concentration was 1.16 mg/mL in females and 1.85 mg/mL in males. In the TCDD₂₅ group, protein concentrations increased to 2.75 mg/mL in females and 3.15 mg/mL in males. These findings suggest that TCDD exposure induces differential alterations in LD-associated protein concentrations in a dose- and time-dependent manner, with distinct effects in males and females.

Numerous studies have demonstrated that LD number and size are dynamic and can be influenced by various factors, including diet, lipotoxicity, and metabolic status. These alterations may reflect changes in lipid metabolism and the capacity of cells to generate new LDs^61,62. Interestingly, TCDD-induced effects on LD size and number are not species-specific, as similar responses have been observed in plants and fungi exposed to TCDD. This suggests a fundamental role for LDs in the cellular sequestration of such toxicants^45,63–65. Although the precise mechanism underlying LD growth and size expansion remains incompletely understood, the CIDE (cell death-inducing DFF45-like effector) protein family has been identified as a key contributor to this process. Notably, CideC (also known as FSP27) plays a crucial role in mediating LD fusion and facilitating lipid transfer from smaller to larger LDs, particularly in mammalian adipocytes^66–69. Similarly, peroxisome proliferator-activated receptors (PPARs) have been identified as key regulators of LD formation and development by modulating perilipin gene expression and triglyceride storage. Among them, PPARα (Ppara) and PPARγ (Pparg) play crucial signaling roles in various tissues, particularly in the liver and kidney, where they influence lipid metabolism and storage dynamics^70–72.

TCDD exposure enhances the hydroxylation activity of the hepatic LD fraction

The functionality of LDs is closely linked to their associated proteins, particularly those with enzymatic activity. To assess this, the hydroxylation activity of LD fractions from both experimental and control groups was measured using the aniline as a prototype substrate. As shown in Fig. 4A, hydroxylation activity increased significantly in hepatic LDs fractioned from male mice on day 14 in both TCDD₁₅ and TCDD₂₅ groups compared to controls, followed by a decline at later time points. Notably, a highly significant increase was observed in LDs from TCDD₁₅ males (p-value ≤ 0.01), while a moderate increase was detected in TCDD₁₅ females (p-value ≤ 0.05) (Fig. AB). However, no significant changes were found in TCDD₂₅ males and females compared to their respective controls. These findings suggest that TCDD exposure induces a marked increase in hepatic LDs hydroxylation activity, with a more pronounced effect in males.

Fig. 4.

Hydroxylation activity of hepatic lipid droplet (LD) associated proteins. (A) The hydroxylation activity of LDs proteins in both male and female groups at 14, 28, 56 and 112 days after exposure compared to their respective controls (CTL). (B) Represents comparison the averaged values across the four time points of hydroxylation activity in both TCDD₁₅-F and TCDD₁₅-M as well as in TCDD₂₅-F and TCDD₂₅-M compared to CTL averaged values across the four time points. Two-way ANOVA followed by Dunnett’s test was used to analyze the data using GraphPad Prism 10. A p-value of less than 0.05 was considered statistically significant (*), while a p-value of less than 0.01 was considered highly statistically significant (**), and so on. A p-value ≥ 0.05 was considered not statistically significant (ns).

Peroxygenases (POX) are LD-associated enzymes capable of hydroxylating a wide range of substrates. While a direct role of POX activity against TCDD has not been demonstrated, its activity has been reported against other lipophilic xenobiotics, such as aflatoxins, suggesting that LD-associated proteins may play a role in metabolizing AFB₁, leading to the formation of its more polar metabolite, AFB₁−8,9-epoxide³⁰. Indeed, POX activity may influence the response to TCDD indirectly through the hydroxylation of mono- and polyunsaturated fatty acids, which generate signaling molecules. These, in turn, activate the lipoxygenase pathway, potentially contributing to the enhanced response observed in males—a gender-specific effect of TCDD exposure^37,38. The enrichment of LDs with such enzymatic activity could be attributed to the activation of key enzymes involved in the metabolism of lipophilic toxicants. Notably, certain CYP450 isoforms have been detected in hepatic LDs, with their abundance increasing under diet-induced hepatic steatosis^73–75.

TCDD exposure may potentially alter the lipid composition of hepatic lipid LDs

Hepatic LDs isolated from mice in the experimental groups were analyzed using flow cytometry after Nile Blue staining to assess variations in lipid composition. The results revealed shifts in LD core lipid characteristics, reflected by one or more fluorescence intensity peaks. Notably, only LDs from TCDD-exposed females exhibited alterations, with a delayed modification detected in TCDD₁₅-F at day 56 (Fig. 5A). A similar shift was observed earlier in TCDD₂₅-F at day 14. No changes were detected in LDs from other experimental groups. These findings were further corroborated by a colored plot, where distinct fluorescence intensities indicated different lipid types (Fig. 5B). These data suggest that TCDD alters the neutral lipid composition of hepatic LDs in females in a dose- and time-dependent manner. While the high dose induces an early and pronounced alteration, the low dose leads to a delayed response.

Fig. 5.

Characterization of hepatic lipid droplets (LDs) using flow cytometry. (A) The number of Nile Blue dye-stained LDs is represented by peaks of varying height and extent, according to the number of LDs present in female (TCDD₁₅-F and TCDD₂₅-F) and male groups (TCDD₁₅-M and TCDD₂₅-M), in comparison to their respective controls (CTL). The resulting peaks from the sample analysis indicate either a single population of LDs, represented by a single peak, or two populations of LDs, represented by two consecutive peaks of varying height, suggesting a distinction in fluorescence intensity, which is indicative of a discrepancy in lipid content. (B) Colored plot represents a distinct fluorescence intensity, thereby indicating a different lipid type. The plots illustrate the densities observed in the experimental groups, as previously designated. The absence of pink coloration was observed in the central region of two female groups (TCDD₁₅-F, day 56) and (TCDD₂₅-F, day 14), in comparison to the other groups and the control.

This finding aligns with the observations of Angrish et al., who reported that TCDD exposure alters the lipid composition of LD core, leading to a reduction in free saturated fatty acids and an increase in unsaturated fatty acids within hepatic TAG⁵⁷. Similarly, our results can be supported by previous findings indicating that TCDD exposure increases free fatty acid and TAG levels while decreasing cholesterol levels in rat serum⁷⁶. The TCDD-induced alterations in LD lipid composition may be attributed to the activation of key genes involved in fatty acid metabolism, such as LOXs and COXs, which are known modulators of inflammatory responses and carcinogenesis, suggesting the use of LDs lipids as biomarkers to assess cell pathology and toxicology under various conditions^77,78. Furthermore, previous studies have shown that acute exposure to lipophilic toxins, including AFB₁, significantly alters both liver and serum lipid content in rats, affecting the expression of genes responsible for lipid synthesis and lipoprotein metabolism^79,80. Notably, it has been suggested that TCDD-induced modifications in LD lipid composition play a critical role in determining their capacity to sequester lipophilic toxins^81–83.

The sequestration of TCDD into hepatic LDs

The sequestration of TCDD within hepatic LDs was analyzed using an HPLC system. A standard curve was generated using graded concentrations of TCDD (0.313, 0.625, 1.25, 2.5, and 5 ng/mL), and its linearity was confirmed (Fig. 6A and B). Blank and spiked samples, along with experimental group samples, were prepared following the same protocol and compared with the standard TCDD (Fig. 6C). Based on TCDD-spiked LD samples, the extraction efficiency was calculated to be approximately 83.8%, ensuring reliable quantification of TCDD concentrations in experimental samples, which were expressed as pg/g of liver fresh weight. UV-HPLC spectra confirmed the presence of TCDD in LDs isolated from both female and male mice in both experimental groups TCDD₁₅ and TCDD₂₅ across all time points. The retention time (4.8 min) matched that of the standard TCDD (STD), confirming its identity (Fig. 6D and E). Quantitative analysis revealed that TCDD concentrations in hepatic LDs ranged from 122 to 1558 pg/g FW in females and from 3.4 to 758.9 pg/g FW in males (Fig. 6F). A comparison of mean concentrations between females and males using the Mann-Whitney test indicated a statistically significant accumulation of TCDD in both sexes, with a notably higher concentration observed in females (p < 0.05) (Fig. 6G).

Fig. 6.

Detection and quantification of TCDD using HPLC. (A) depicts the linear series of the standard TCDD, while B illustrates the specific peaks for increasing concentrations (0.313, 0.625, 1.25, 2.5, 5 ng/ml) of TCDD, utilizing a C18 column, wavelength 232 nm and flow rate 0.5 mL/min. (C) shows the specific peaks for standard TCDD (STD), Spike and Blank. (D and E) depict the samples from female and male groups, respectively, that were administered with TCDD₁₅ and TCDD₂₅ at four time points (14, 28, 56 and 112 days) in comparison to the STD, respectively. (F) TCDD concentrations in female and male LDs. (G) the mean concentrations were compared between females and males, and the results of the Mann-Whitney test indicated significant differences at p < 0.05 (*).

Consistent with our findings, previous studies have demonstrated that LDs can trap and accumulate lipophilic polycyclic aromatic hydrocarbons, such as polychlorinated biphenyls (PCBs), thereby reducing their toxicity to other cellular organelles^84–86. Moreover, a similar process has been observed in plants, as shown in a study by Hanano et al., which reported the ability of date palm LDs to sequester TCDD in an aqueous medium^87,88.

TCDD triggers transcriptional alterations in key genes associated with hepatotoxicity

The gene expression analysis revealed sex-, dose-, and time-dependent variations in response to TCDD exposure. The first group of analyzed genes included AhR, ARNT, and key detoxification enzymes from phase I (Cyp1a1) and phase II (Gsta1, Gsta3) pathways. Significant differences in expression levels were observed across experimental groups. The highest increase in Ahr expression occurred in males (TCDD₁₅-M, day 14; TCDD₂₅-M, day 28) and females (TCDD₁₅-F and TCDD₂₅-F, days 56 and 112) (Fig. 7A-D). Arnt expression followed a similar pattern in some but not all cases, with notable increases in TCDD₁₅-M, TCDD₂₅-M (days 14 and 28), and TCDD₁₅-F, TCDD₂₅-F (day 56). Cyp1a1, a well-established marker of TCDD exposure, showed a pronounced upregulation, particularly in female groups, while male groups exhibited increased expression at earlier time points (TCDD₁₅-M, TCDD₂₅-M, days 14 and 28). Similarly, Gsta1 and Gsta3 expression was elevated in TCDD₁₅-F at all time points and in TCDD₂₅-F at most time points. In males, Gsta1 and Gsta3 were significantly upregulated in TCDD₁₅-M (day 14) and moderately increased in TCDD₂₅-M (days 28 and 56) (Fig. 7A-D). Overall, these results underscore the critical role of AhR signaling and detoxification pathways in TCDD-induced liver toxicity.

Fig. 7.

Relative expression (QR) analysis of key genes associated to TCDD-hepatotoxicity. The figure depicts the RQ-based relative differences in gene expression among all experimental groups, which have been classified into three categories according to their function. The genes of interest are Ahr, Arnt, Cyp1a1, Gsta1, and Gsta3. (A) and (B) depict the expression of the genes of interest in female and male administered TCDD₁₅ (TCDD₁₅-F and TCDD₁₅-M) at (14, 28, 56 and 112 days). (C) and (D) show the expression of the same genes in female and male administered TCDD₂₅ (TCDD₂₅-F and TCDD₂₅-M) during the same periods. The y-axis represents log₁₀ RQ values. A two-way ANOVA was conducted to analyze the data, followed by Dunnett’s test using GraphPad Prism 10. The p-value less than 0.05 considered statistically significant (*), less than 0.01 considered highly statistically significant (**), and so on. A p-value ≥ 0.05 was considered to be statistically insignificant (ns).

TCDD toxicity is primarily mediated through the activation of the AhR pathway^89–91, where AhR dimerizes with ARNT and binds to dioxin response elements (DREs), inducing the expression of target genes^92–94. Consistent with previous studies, our findings confirm that TCDD exposure activates Ahr, Arnt, and Cyp1a1 expression, with notable sex-dependent differences^95–99. Moreover, prior research has highlighted differential Ahr expression between males and females, influencing sensitivity to TCDD¹⁰⁰. Also, the observed increase in Cyp1a1 and Gstgene expression aligns with reports of elevated transcription levels within 24 to 48 h post-exposure in TCDD-dosed rats¹⁰¹. Unlike studies suggesting transient gene activation, our results indicate that the effects of TCDD persist long after exposure. The TCDD-induced Ahr expression remained elevated at 112 days, and Gsta1 and Gsta3 levels were upregulated up to 56 days, particularly in females. This prolonged response is likely linked to the AhR/ARNT pathway’s role in activating GST, a key enzyme in detoxification processes¹⁰². Interestingly, our findings mirror those of Wu et al., who reported that Gstm6 expression in TCDD-exposed mice was not necessarily correlated with Ahr and Arnt levels. Similarly, Gsta1 and Gsta3 expression varied between sexes and exposure groups (TCDD₁₅ vs. TCDD₂₅)¹⁰³. The persistent upregulation of Gst genes suggests a role in the resistance of BALB/c mice to TCDD-induced toxicity. This aligns with studies on aflatoxin B₁, where selective Gsta gene expression was linked to hepatocarcinogenesis resistance, highlighting the protective role of Gst in detoxifying toxic metabolites¹⁰⁴.

TCDD modulates the expression of key genes involved in Oxylipin biosynthesis

TCDD exposure influences the biosynthesis of oxylipins, including leukotrienes and prostaglandins, which are key inflammatory mediators. Variations in gene expression levels between study groups are depicted in Fig. 8A-D. Notably, Lox15 and Cox2 expression increased in both TCDD₁₅-F and TCDD₁₅-M on day 56. In contrast, a lower induction of Lox15 was observed in TCDD₂₅-F and TCDD₂₅-M between days 14 and 56. Similarly, Cox2 expression was elevated in TCDD₂₅-F and TCDD₂₅-M at days 14 and 28. These findings indicate that TCDD modulates the expression of key genes involved in oxylipin biosynthesis, suggesting a potential role for leukotrienes and prostaglandins in the liver’s response to TCDD exposure.

Fig. 8.

Relative expression (QR) analysis of key genes involved in oxylipin biosynthesis. The figure depicts the RQ-based relative differences in gene expression among all experimental groups, which have been classified into three categories according to their function. The genes of interest are Lox15, Cox2. (A) and (B) depict the expression of the genes of interest in female and male administered TCDD₁₅ (TCDD₁₅-F and TCDD₁₅-M) at (14, 28, 56 and 112 days), while graphs (C) and (D) show the expression of the same genes in female and male administered TCDD₂₅ (TCDD₂₅-F and TCDD₂₅-M) during the same periods. The y-axis represents log10 RQ values. A two-way ANOVA was conducted to analyze the data, followed by Dunnett’s test using GraphPad Prism 10. The p-value less than 0.05 considered statistically significant (*), less than 0.01 considered highly statistically significant (**), and so on. A p-value ≥ 0.05 was considered to be statistically insignificant (ns).

Lipid droplets (LDs) serve as a major reservoir of arachidonic acid (AA), a key substrate for COX, LOX, and CYP450 enzymes, which are known to localize within LDs^105,106. In this context, studies have reported that TCDD enhances the activity of arachidonic acid-metabolizing enzymes, including COX, LOX, and CYP450, potentially influencing inflammatory and metabolic pathways^107,108.

TCDD induces the transcription of key genes involved in LD-associated proteins

The expression of key genes involved in LD biogenesis, including Plin5, CideC, Ppara, and Pparg, exhibited distinct variations across experimental groups (Fig. 9A-D). Notably, a significant upregulation of these genes was observed at specific time points in female groups, particularly in TCDD₁₅-F on day 56 and TCDD₂₅-F on day 14. However, at other time points, their expression levels showed a marked decline. In male groups, gene expression displayed a different pattern, with increased levels detected at various time points. The most pronounced upregulation occurred in TCDD₁₅-M between days 14 and 56, extending up to 112 days in TCDD₂₅-M (Fig. 9A-D).

Fig. 9.

Relative expression analysis of key genes associated with LD-related proteins. The figure depicts the RQ-based relative differences in gene expression among all experimental groups, which have been classified into three categories according to their function. The genes of interest are Plin5, Cidec, Ppara, Pparg. (A) and (B) depict the expression of the genes of interest in female and male administered TCDD₁₅ (TCDD₁₅-F and TCDD₁₅-M) at (14, 28, 56 and 112 days). (C) and (D) show the expression of the same genes in female and male administered TCDD₂₅ (TCDD₂₅-F and TCDD₂₅-M) during the same periods. The y-axis represents log₁₀ RQ values. A two-way ANOVA was conducted to analyze the data, followed by Dunnett’s test using GraphPad Prism 10. The p-value less than 0.05 considered statistically significant (*), less than 0.01 considered highly statistically significant (**), and so on. A p-value ≥ 0.05 was considered to be statistically insignificant (ns).

Perilipins constitute the fundamental components of the LDs architecture^109,110. Plin5 is predominantly expressed in tissues characterized by high oxidative activity, such as the heart, skeletal muscle and liver¹¹¹. Lin et al., demonstrated that Plin5 plays a crucial role in the remodeling of LDs in hepatic stellate cells (HSCs). It promotes increased fat content by stimulating the expression of pro-lipogenic genes and suppressing pro-lipolytic gene expression. Additionally, Plin5 inhibits HSC activation, further contributing to the regulation of lipid metabolism in the liver¹¹².

This finding aligns with our data, which show a significant increase in Plin5 gene expression in the experimental groups. This increase corresponds with a rise in the number of LDs and a significant upregulation of CideC, Ppara, and Pparg gene expression. Moreover, our results are consistent with previous studies indicating that the expression of Ppara and Pparg is regulated based on the substances to which an organism is exposed. For instance, Ppara is activated by benzofuran, galactosamine, and TCDD, but suppressed by hepatotoxins such as acetaminophen, lipopolysaccharide, silica nanoparticles, and trovafloxacin¹¹³.

Sex-specific differences in Pparg expression have also been found in the follicular helper T-cell (TFH) response in male mice¹¹⁴. Our results also indicate that TCDD induces the expression of CideC, a member of the cell death activator protein family. This finding is significant considering CideC’s role in mediating LD growth, LD-LD communication, and lipid exchange, particularly in adipocytes. The increased gene expression of Plin5, CideC, Ppara, and Ppargcorrelates with the elevated number of LDs in the experimental groups. This increase in LDs may also be influenced by additional mechanisms, such as the AhR-CD36 pathway, which enhances fatty acid uptake and lipid accumulation in hepatocytes, further contributing to LD formation¹¹⁵.

Conclusions

The current study underscores the profound effects of TCDD exposure on the biochemistry and functionality of hepatic lipid droplets (LDs) in both female and male BALB/c mice. The findings reveal that hepatic LDs play a pivotal role in sequestering and storing TCDD, a highly toxic and persistent dioxin congener, raising concerns about its long-term impact on liver health. TCDD exposure led to significant changes in LD abundance, lipid composition, and associated protein content, accompanied by substantial alterations in the expression of key genes involved in dioxin detoxification, oxylipin biosynthesis, and LD formation. These molecular and biochemical changes occurred in a dose-, time-, and sex-dependent manner.

The observed disruptions suggest that TCDD exposure may impair lipid metabolism and contribute to liver dysfunction, with a potential link to hepatocellular carcinoma, particularly in females. The elevated expression of genes linked to liver carcinogenesis highlights the need for further research into the long-term risks associated with chronic dioxin exposure. Moreover, the study emphasizes the crucial role of hepatic LDs in the body’s defense against lipophilic toxins like TCDD, shedding light on their function in toxin sequestration and metabolism. These insights could guide future strategies for mitigating dioxin-induced toxicity and developing therapeutic interventions to counteract liver damage and other disorders associated with environmental pollutant exposure.

Supplementary information

Below is the link to the electronic supplementary material.

Author contributions

N.H. performed the experiments and wrote the first draft of the manuscript. C.S. co-supervised the work. A.H. supervised the work and wrote the final version of the manuscript. All authors reviewed the manuscript.

Founding

The authors state that no founding was received for this work.

Data availability

Data supporting this study are available in the manuscript and supplementary data.

Declarations

Competing interests

The authors declare no competing interests.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.