Experimental animals

Male Wistar rats of three different age groups, i.e. young (4 months), middle-aged (12 months), and old (24 months), were purchased from CSIR-Indian Institute of Toxicology Research, Lucknow, India. Rats were housed in a controlled environment at 22 ± 2 ℃ and relative humidity of 55 ± 15% with a 12-h light/12-h dark cycle, and they had free access to drinking water and nutrient-rich pellets. All animal care and experimental procedures were approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and the Institutional Animal Ethics Committee (IAEC/AU/2019(1)/09), University of Allahabad, India and follow the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications No. 8023, revised 1978). The ARRIVE guidelines checklist for reporting this animal research was used and is provided in the Supplementary file.

Only male rats were used because in female rats, data might be very unpredictable due to hormonal variations associated with the female reproductive cycle¹². After one week of acclimatization, 36 male Wistar rats were divided into six groups (n=6) as follows:

Group 1: Young male rats (aged 4 months, body weight = 115 ± 10 g)

Group 2: Young male control rats (aged 4 months, body weight = 115 ± 10 g)

Group 3: Middle-aged male rats (aged 12 months, body weight = 180 ± 10 g)

Group 4: Middle-aged male control rats (aged 12 months, body weight = 180 ± 10 g)

Group 5: Old male rats (aged 24 months, body weight = 220 ± 10 g)

Group 6: Old male control rats (aged 24 months, body weight = 220 ± 10 g)

Groups 1, 3, and 5 were exposed to passive cigarette smoke for 15 minutes once every day for 30 days and had free access to food and water. Groups 2, 4, and 6 were the respective controls and were not exposed to cigarette smoke at all. The control groups had free access to food and water.

Cigarette smoke generation

An experimental setup consisting of an exposure chamber was constructed to generate cigarette smoke. The exposure chamber was constructed using a plastic material of dimensions 56.4 × 38.5 × 37.1 cm. It had small openings of 371 × 40 mm in both extremities for ventilation. The rats were placed inside the exposure chamber and two cigarettes fixed in a metal holder were lit. A stopwatch was turned on, and the cigarettes were allowed to burn down fully within 15 minutes. The smoke generated inside the chamber was suctioned by a noiseless extractor fan to keep an airflow inside the chamber. A metal grille was placed on top of the cigarette holder to avoid direct contact with the cigarettes and, thus, to prevent the rats from injuring themselves. The inhalation exposure of our study was for different age groups of rats, used as a simulation of environmental tobacco smoke as experienced by non-smokers⁷.

Assessment of biochemical parameters

Blood collection, isolation of erythrocytes, plasma and serum

After 30 days of treatment, the rats were euthanized by standard protocol, and blood samples were collected by cardiac puncture into heparinized syringes.

Anesthetic procedure

A combination of ketamine and xylazine was used to induce general anesthesia. The anesthetic solution was freshly prepared by mixing ketamine (100 mg/mL) and xylazine (20 mg/mL) in a 10:1 ratio (v/v). The anesthetic was administered intraperitoneally (IP) at a dose of 80–100 mg/kg ketamine and 8–10 mg/kg xylazine, based on individual body weight¹³. Injections were performed using a 25-gauge needle in the lower right quadrant of the abdomen to avoid damage to visceral organs.

Following the injection, the animals were monitored continuously. Loss of the righting reflex and absence of response to toe-pinch were used to confirm induction of a surgical plane of anesthesia. Anesthetic depth was rechecked immediately before blood collection¹⁴.

Blood collection via cardiac puncture

Cardiac puncture is a terminal procedure that allows for the collection of large, high-quality blood volumes directly from the heart under deep anesthesia, making it ideal for biochemical and hematological analysis at the endpoint of experiments¹⁵. In contrast, intravenous (IV) blood sampling techniques, such as collection from the tail, jugular, or saphenous veins, are non-terminal and suited for serial sampling in longitudinal studies¹⁶. While cardiac puncture offers better sample purity and volume, IV methods are less invasive and allow for repeated measurements, though they require careful handling to minimize animal stress and ensure welfare¹⁷. Our study required large volumes of blood for various biochemical assays, as mentioned in the following sections. Therefore, cardiac puncture was preferred over intravenous blood sampling.

Cardiac puncture was performed as a terminal procedure under deep anesthesia. The rat was placed in dorsal recumbency, and the xiphoid process was used as a landmark. A 21–23 gauge, 1-inch needle attached to a 5 mL syringe was inserted at a 30–45° angle just left of the sternum into the thoracic cavity. The needle was directed toward the heart while negative pressure was applied until blood was aspirated. A maximum volume of 6–10 mL of blood was collected, depending on the animal’s weight¹⁶. Blood was transferred to heparin or serum-separator tubes as required.

The red blood cells and plasma were separated by centrifugation of the collected blood at 800g at 4℃ for 10 min, and subsequent removal of the upper buffy coat and 15% of packed red blood cells (PRBCs). Post-separation, the RBCs were washed thrice with ice-cold phosphate buffer saline (PBS) (10 mM; pH 7.4) and used for the assessment of oxidative stress parameters such as reduced glutathione (GSH), malondialdehyde (MDA), and plasma membrane redox system (PMRS). Plasma was frozen and stored at -80°C for analysis of biochemical parameters such as the ferric-reducing ability of plasma (FRAP), advanced oxidation protein products (AOPPs), and protein carbonyls (PCOs).

Following collection, euthanasia was confirmed via cervical dislocation and observation of the absence of respiratory movement and cardiac activity for at least 5 minutes¹⁸.

Lipid peroxidation assay in erythrocytes

By quantifying the amount of malondialdehyde (MDA) in erythrocytes, lipid peroxidation products were identified using the technique of Esterbauer and Cheeseman¹⁹. Three mL of PBS with 0.5 mM glucose (pH 7.4) was used to suspend 0.2 mL of PRBCs. One mL of 10% trichloroacetic acid (TCA) and two mL of 0.67% thiobarbituric acid (TBA) were also added to the 0.2 mL of suspended PRBCs, which were then heated for 20 min at 90–100℃ before being cooled. After five minutes of centrifuging the mixture at 1000g, the absorbance of the supernatant was measured at 532 nm. The extinction coefficient (e ¼ 153000 M^-1 cm^-1) was used to get the MDA concentration (in nmol/mL PRBCs).

Estimation of plasma protein carbonyl content

The Levine²⁰ method was used to assess the carbonyl content of plasma proteins. Two tubes containing plasma samples from each group were designated as a test and a blank control. The test sample received 4.0 mL of a reagent cocktail consisting of 10 mM 2,4-dinitrophenylhydrazine (DNPH) produced in 2 M HCl, while the blank control received 4.0 mL of 2 M HCl alone. After vortexing, the contents were incubated at 37℃ for one hour in the dark. To promote protein reactions, the tubes were shaken sporadically every 10 min. After adding 20% TCA (w/v) to each tube, the mixture was allowed to sit in ice for 10 min.

The protein pellets that accumulated at the tube’s bottom were then separated by centrifuging the tubes for 20 min at 850g. The supernatant was disposed of with care. Protein pellets were extensively cleaned with an ethanol-ethyl acetate (1:1, v/v) solution to eliminate any remaining DNPH and lipid remains. The protein pellets were then incubated for 10 min at 37℃ after being dissolved in 6 M guanidine hydrochloride. By measuring the supernatant’s spectra at 370 nm and comparing each sample to the control, the carbonyl content was calculated. The Lowry et al.²¹ technique was used to calculate the plasma total protein content. An absorption coefficient of 22000 M^-1 cm^-1 was used to determine the carbonyl concentration, and the results were reported in nmol/mg protein.

Estimation of AOPPs (advanced oxidation protein products) in plasma

With a small modification, the Witko-Sarsat et al.²² method was used to measure the amount of AOPPs in plasma. After diluting the initial plasma five times, 10 mL of 1.16 M potassium iodide were added to 2 mL of plasma. Twenty mL of glacial acetic acid were then added to the solution after two minutes. At 340 nm, the reaction mixture’s absorbance was measured instantaneously. A volume of 2.0 mL of chloramine-T solution (0–100 mmol/L) was used as a calibrator, while 2 mL of PBS served as a blank. Mmol of chloramine-T equivalents per liter of plasma (mmol/L) was used to express the amount of AOPPs present.

Determination of erythrocytes plasma membrane redox system (PMRS) activity

As previously reported by Rizvi et al.¹¹, the reduction of ferricyanide was used to evaluate the PMRS activity in erythrocytes. To reach a final volume of 2 mL, PRBCs (0.2 mL) were suspended in PBS that contained 5 mM glucose and 1 mM newly made potassium ferricyanide. After 30 min of incubation at 37℃, the suspensions were centrifuged for 10 minutes at 800g. Using 4, 7-diphenyl-1, 10-phenanthroline disulfonic acid disodium salt, the ferro cyanide content of the collected supernatant was determined. The absorbance was measured at 535 nm (e ¼ 20500 M^-1 cm^-1). The results are reported in µmol ferrocyanide/mL PRBC/30 min.

Evaluation Of FRAP (total antioxidant capacity of plasma)

With minor adjustments, the Benzie and Strain²³ method was used to assess plasma’s capacity to reduce ferric acid; 300 mM acetate buffer (pH 3.6), 20 mM ferric chloride, and 10 mM 2,4,6-tripyridyl-s-triazine (made in 40 mM hydrochloric acid) were combined in a 10:1:1 ratio to create the FRAP reagent. A volume of 3 mL of FRAP reagent were combined with 100 μL of plasma, and the mixture was thoroughly mixed. Using a UV-VIS spectrophotometer (Evolution 201, Thermo Fisher), the absorbance was measured at 593 nm every 30 s for 4 min. Using the standard calibration curve, the plasma’s FRAP values (mmol Fe (II) per L) were determined.

Evaluation of reduced glutathione and total plasma thiol

The procedure outlined by Garg et al.²⁴ was used to measure the amount of reduced glutathione in erythrocytes, and the published method²⁵ was used to estimate the plasma-SH groups. Based on the -SH group’s capacity to reduce 5, 50-dithiobis, 2-nitrobenzoic acid (DTNB) and produce a yellow-colored anionic product, the optical density of which was measured at 412 nm, both evaluations were made. The standard plot was used to determine the GSH concentration, which is given as mg/mL PRBCs. The unit of measurement for the -SH group concentration is nmol/mL of plasma.

Sialic acid (SA) level

The Spyridaki and Siskos²⁶ approach was used to calculate the SA content of the erythrocyte membrane. The amount of membrane SA is reported as mg/mg membrane protein, and the quantity of sialic acid was determined by creating a calibration graph using standard solutions of SA.

Protein carbonyl (PCO) level

Erythrocyte membrane PCO was measured according to the procedure of Levine et al.²⁰. Absorbance was measured at 370 nm and the amount of PCO was calculated using an extinction coefficient of 22000 M^-1 cm^-1. The concentration of PCO is expressed as mmol/mg membrane protein.

Statistical analysis

The statistical analysis was performed using Graph-Pad PRISM version 5.01 software. The values reported are expressed as mean ± standard deviation (SD). The statistical analysis was carried out by one-way analysis of variance (ANOVA) followed by a post hoc Bonferroni test. A p<0.05 was considered statistically significant.

Effect of cigarette smoke generation on the antioxidant levels in young, middle-aged and old rats

Effect of cigarette smoke on FRAP level

With respect to control rats, a significant decrease (p<0.05) is observed in the FRAP level in the groups inhaling smoke (YC=662.18, YS=527.53, MC=553.84, MS=396.10, OC=372.74, OS=277.59) (Figure 1). This decrease can be attributed to the increase in oxidative stress and, therefore, a gradual decline in the endogenous antioxidant levels.

Fig 1

Effect of cigarette smoke on the antioxidant parameters in RBCs and plasma of rats: a) The ferric reducing ability of plasma (FRAP) value gives the antioxidant capacity measured in the plasma of rats (μmol Fe/L plasma); b) The plasma membrane redox system (PMRS) measures the overall redox balance in erythrocytes of rats exposed to cigarette smoke (μmol ferrocyanide/mL PRBC); c) The effect of cigarette smoke on young, middle-aged, and old rats in terms of DPPH % inhibition; d) The intracellular GSH level (mg/mL) in RBC

Effect of cigarette smoke on PMRS activity

Our observed results show a significant increase (p<0.05) in PMRS levels in the treated rats in comparison to the control group (YC=0.989159892, YS=1.513279133, MC=2.210840108, MS=3.035772358, OC=3.462872629, OS=4.141463415) (Figure 1). This suggests an activation of PMRS in response to a disturbed redox state caused due to cigarette smoke.

Effect of cigarette smoke on plasma DPPH radical scavenging activity

The percentage inhibition in terms of DPPH radical scavenging was found to be significantly decreased (p<0.05) in each of the three groups, i.e. the young group, middle-age group, and old-age group when compared to their respective control groups (YC=31.57%, YS=24.04%, MC=26.81%, MS=19.33%, OC=16.32%, OS=10.84%) (Figure 1). However, on comparing the three groups, the percentage inhibition was recorded highest in the young group (24.04%), and intermediate in the middle-age group (19.33%). Interestingly, it was lowest in the old-age group (10.84%) suggesting an age-dependent loss of antioxidant activity.

Effect of cigarette smoke on GSH level

GSH is a strong determinant of the redox state and the intracellular GSH level is significantly reduced (p<0.05) in the smoke-treated groups amongst all age groups compared to their respective control groups (YC=0.0217875, YS=0.0173055, MC=0.015189, MS=0.009462, OC=0.00996, OS=0.0056025) (Figure 1). The decrease in GSH levels denotes a reduction in antioxidant levels and increased oxidative stress.

Effect of cigarette smoke on plasma protein oxidation level

Protein carbonyl (PCO) content is significantly increased (p<0.05) in all the smoke-treated groups of different age groups when compared to their respective controls (Figure 2). Advanced oxidation protein products (AOPP) show levels of protein oxidation products which have significantly increased (p<0.05) in the treated groups compared to control rats (YC=26731.89, YS=36235.97, MC=35353.47, MS=44951.55, OC=47646.04, OS=56132.37) (Figure 2). The increase in oxidized protein products amongst all groups suggests an equally negative impact of cigarette smoke on health due to a rise in oxidative stress.

Fig 2

Effect of cigarette smoke on the anti-oxidant parameters in RBCs and plasma of rats: a) Protein carbonyl content in the plasma of control rats and cigarette smoke-treated rats (mmol/mg protein); b) Advanced oxidation protein products (AOPP) level in plasma of young, middle-aged, and old rats treated with cigarette smoke (μmol/L plasma)

Effect of cigarette smoke on plasma paraoxonase-1 aryl esterase (PON-1)

Our results showed a significant decrease (p<0.05) in the plasma paraoxonase-1 activity in all the cigarette smoke-treated groups in comparison to their respective control groups (YC=0.097484964, YS=0.078130126, MC=0.060843813, MS=0.056843448, OC=0.05562238, OS=0.04080554) (Figure 3). Further, the old age treated group showed the lowest PON-1 activity when compared to young age and middle age groups.

Fig 3

Effect of cigarette smoke on the anti-oxidant parameters in RBCs and plasma of rats: a) Lipid peroxidation content (nmol/mL RBC) is measured in terms of MDA levels. The effect of cigarette smoke on young, middle-aged, and old rats in terms of: b) sialic acid content (mM) in plasma; c) plasma paraoxonase-1 activity (U/mL)

Effect of cigarette smoke on plasma sialic acid content

Sialic acid content was found to be significantly increased (p<0.05) in all the cigarette smoke-treated groups of different age groups when compared to their respective controls (YC=1246242.77, YS=1538535.64, MC=1663391.13, MS=2126011.56, OC=2282466.28, OS=2700192.67) (Figure 3). However, the highest sialic acid content was found in the old age group and the lowest in the young age group, while the middle age group showed intermediate sialic acid content.

Effect of cigarette smoke on malondialdehyde content in erythrocytes

MDA is a content of lipid peroxidation and our results show significant increase (p<0.05) in MDA content in the treated rats with respect to control group (YC=9679.95, YS=12317.79, MC=13946.10, MS=16913.51, OC=17606.13, OS=19989.28) (Figure 3). The result suggests an increase in malondialdehyde content due to increased oxidative stress.

Limitations

This study provides important insights into the redox imbalance induced by passive cigarette smoke exposure in male Wistar rats; however, several limitations should be considered while interpreting the findings. First, the sample size was relatively small (n=36), which, while sufficient for preliminary analysis, may limit the statistical power to detect subtle biochemical changes or subgroup variations. Secondly, the exposure model involved controlled passive smoke exposure in a laboratory setting, which may not precisely mimic the chronic, variable exposure encountered in real-world human scenarios. Additionally, the duration of exposure was limited to 30 days, and the long-term effects of sustained redox imbalance could not be evaluated. Finally, while multiple oxidative stress markers were assessed, the study did not include functional or histopathological endpoints (e.g. tissue damage, behavioral outcomes), which could have provided a more comprehensive understanding of physiological consequences. Despite these limitations, the study establishes a foundational link between passive cigarette smoking and oxidative stress in vivo. Future studies with larger, more diverse animal cohorts, longer exposure durations, and broader endpoints are warranted to validate and expand upon these findings.