Nephrotoxic effects of combined exposure to 85 dB(a) noise and 300 ppm toluene in Wistar rats: biochemical and histopathological analysis

Abstract

Toluene, a widely used industrial solvent, and environmental noise, a pervasive physical stressor, are both recognized for their harmful effects on health. This study examined the individual and combined effects of toluene and noise exposure on oxidative stress, renal function, pro-inflammatory response, and kidney histopathology. Twenty-four adult male Wistar rats were randomly divided into four groups: control (C), noise exposure (N; 85 dB SPL white noise, 8–16 kHz), toluene exposure (T; 300 ppm), and combined Noise + Toluene exposure (NT; 85 dB + 300 ppm).

Oxidative stress was assessed by measuring malondialdehyde (MDA) levels and the activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT). Renal function was evaluated using serum urea and creatinine levels, along with serum concentrations of the pro-inflammatory cytokines TNF-α and IL-1β. Histopathological analyses were performed to identify structural alterations in the kidneys.

All exposed groups exhibited significant renal impairment, as indicated by elevated urea and creatinine levels. MDA levels were markedly increased, confirming oxidative stress. Noise exposure induced elevated SOD and CAT activities, whereas toluene exposure led to a reduction in these activities. Combined exposure exacerbated biochemical imbalances, elevated pro-inflammatory cytokine levels, and intensified renal tissue damage, including glomerular atrophy, tubular degeneration, vascular congestion, and leukocyte infiltration in the renal tissue.

These findings demonstrate that toluene and noise are potent nephrotoxic agents whose co-exposure amplifies oxidative and inflammatory injuries. This highlights the critical need to consider combined environmental exposure in toxicological risk assessments.

Graphical Abstract

Graphical Abstract.

Introduction

In today’s industrialized world, exposure to environmental pollutants has become a pressing public health concern, with significant adverse effects documented in various organ systems.¹ Among these pollutants, noise pollution and toluene exposure have emerged as critical areas of study because of their harmful implications for human health. While previous research has explored the renal effects of noise and toluene exposure separately, there remains a significant gap in understanding the potential combined effects of these two pollutants when encountered together.^2–4

The kidneys are highly vascularized organs that play a central role in maintaining systemic homeostasis by regulating the fluid balance, electrolyte concentrations, and clearance of metabolic waste. Beyond their excretory function, they are also critically involved in redox balance and immune modulation in the body. Accumulating evidence suggests that environmental stressors, particularly chemical toxicants and physical agents, compromise renal integrity through mechanisms involving oxidative stress and inflammation. Oxidative stress, characterized by an imbalance between reactive oxygen species (ROS) generation and antioxidant defense systems, is a well-established mediator of renal injury, contributing to tubular damage, glomerular dysfunction, and vascular compromise in the kidney. This redox imbalance often acts in concert with inflammatory processes, triggering the release of proinflammatory cytokines that exacerbate tissue injury.^2,3,5

Noise pollution, which is pervasive in urban and industrial environments, has been extensively linked to an elevated risk of hypertension, cardiovascular disorders, and circadian rhythm disruption. Beyond these systemic manifestations, emerging data suggest that chronic noise exposure induces deleterious effects on renal physiology, potentially through neuroendocrine dysregulation, increased oxidative stress, and vascular endothelial dysfunction. However, the precise molecular and cellular mechanisms underpinning noise-induced nephrotoxicity remain to be elucidated.^6,7

Toluene, a widely used volatile organic solvent prevalent in numerous industrial and household applications, exerts nephrotoxic effects predominantly via biotransformation into reactive metabolites that trigger oxidative damage and inflammatory responses in renal tissue. These metabolites promote lipid peroxidation, mitochondrial dysfunction, and activation of proinflammatory cytokine cascades, thereby compromising glomerular filtration and tubular integrity. Occupational exposure to toluene has been consistently associated with perturbations in renal biomarkers, including elevated serum creatinine and urea levels, as well as histopathological changes such as tubular degeneration and interstitial inflammation.^8,9

While the individual nephrotoxic effects of noise and toluene exposure have been documented, the combined impact of simultaneous exposure to these environmental stressors on renal physiology remains poorly characterized. Given the widespread co-occurrence of noise and toluene in occupational and environmental settings, it is essential to elucidate their interactive effects on renal functions. This understanding is critical for refining risk assessment models and guiding the formulation of targeted mitigation strategies. Co-exposure to noise and toluene is especially prevalent in industrial sectors, including manufacturing, construction, and solvent-utilizing professions,¹⁰ where workers are chronically subjected to multifactorial insults.

This dual exposure scenario poses a heightened threat to renal homeostasis by potentially amplifying oxidative stress, inflammatory responses, and microvascular dysregulation beyond the effects observed with individual exposures alone. Moreover, the systemic repercussions of such combined toxic insults extend beyond the kidneys, implicating critical physiological systems such as the cardiovascular,^7,11 respiratory,¹² and central nervous systems, as well as alterations in neurobehavioral functions.¹³

Accordingly, the present study aimed to investigate the nephrotoxic effects of concurrent exposure to 85 dB(A) white noise and 300 ppm toluene on oxidative stress biomarkers, renal functional parameters, serum concentrations of pro-inflammatory cytokines, and histopathological alterations in the renal tissue of Wistar rats.

Methods

Chemicals

Toluene (C7H8) is a colorless, flammable liquid characterized by a sweet odor. It is widely utilized as a solvent in various industrial processes. The toluene used for inhalation exposure in this study was sourced from “SMSBio, Sud Médical Services, Tunisia.”

The following analytical-grade reagents were obtained from Sigma-Aldrich (Germany): 2-thiobarbituric acid (TBA), 2,6-di-tert-butyl-4-hydroxytoluene (BHT), trichloroacetic acid (TCA), hydrogen peroxide (H₂O₂), 2-methoxyphenol (guaiacol), bovine liver catalase, and epinephrine (4-(1-hydroxy-2-methylamino-ethylbenzene-1,2-diol).

In vivo experimental protocol

Twenty-four male Wistar rats obtained by the Pasteur Institute of Tunis were divided into four groups of six animals each. The control group (C) was also transferred to a separate chamber located in a noise-free environment, with background noise levels maintained below 40 ± 5 dB (A) and free from toluene exposure (clean air). This ensured consistent environmental conditions comparable to the other groups.

Exposure system

Following the established protocol described in our recent study by Ben Attia et al.,¹² rats were subjected to daily exposure to noise and/or inhaled Toluene for six weeks (5 days/week) from 8:00 am to 2:00 pm. This exposure was conducted within specially designed Plexiglas chambers, incorporating a generation system, an exposure system, and a monitoring system, as detailed by Hinners et al.¹⁴

The acoustic exposure system was established using the Audacity 2.3.2 audio software, where the noise level was consistently set at 85 dB (A) to deliver an octave-band noise (8–16 kHz) through a sound speaker. This speaker was uniformly positioned to ensure that all rats were subjected to the same intensity of noise. The noise level was meticulously monitored with an integration sound level meter with Class 1 accuracy, Type 2,238 Bruel and Kjaer, ensuring precise exposure as per the established protocol.¹²

For the toluene exposure system, a concentration of 300 ± 10 ppm was maintained for six hours within the exposure chamber. This was achieved by injecting liquid toluene (SMSBio, Sud médical services, Tunisia) using an isocratic pump into a mixing vessel connected to the chamber. Air circulation was facilitated by a fan at the inlet, and an adjustable airflow was ensured by a centrifugal fan at the outlet. The toluene concentration was continuously monitored and analyzed using gas chromatography–mass spectrometry (Agilent Technologies 6,890 N Network GC System). The chamber also featured several circular openings to allow researchers to monitor and sample the toluene levels, with the temperature kept stable at 23 ± 1 °C, monitored via a digital thermometer.

To explore the potential interactions between noise and toluene, a co-exposure experiment was conducted, following a protocol similar to the individual exposure experiments for both noise and toluene. In this setup, the simultaneous generation of noise and toluene allowed for the examination of their combined effects on the subjects, providing valuable insights into the interaction between these two environmental stressors. Figure 1 presents an illustrative schematic of the experimental exposure chamber design.

Fig. 1.

Schematic diagram of the chamber used for rat exposure to noise and toluene.

The exposure chamber, constructed from plexiglass, has an internal volume of 0.43 m³. Six rats were introduced into the chamber daily, where they were subjected to a 6-h exposure protocol from 8:00 AM to 2:00 PM.

Sampling and analysis of toluene

To verify and maintain the target atmospheric concentration of toluene (300 ppm) within the exposure chamber, preliminary calibration trials were conducted. Quantitative air sampling was conducted in accordance with the NIOSH Method 1,501 (NIOSH, 2003), a validated and widely adopted protocol for determining aromatic hydrocarbons in workplace environments. Active sampling was performed using a calibrated personal air sampling pump (Casella CEL, Apex 182180B-K5), which drew ambient air through activated charcoal sorbent tubes specifically designed for volatile organic compound (VOC) capture.

Following collection, each charcoal section was extracted with 1 mL of carbon disulfide (CS₂), and the tubes were gently agitated for 30 s to ensure optimal desorption of toluene from the adsorbent matrix. The resulting extracts were subsequently analyzed using gas chromatography coupled with mass spectrometry (GC–MS; Agilent Technologies 6,890 N Network GC System), enabling the sensitive and specific quantification of toluene concentrations. This methodological approach ensured the reliability and traceability of exposure levels throughout the experimental protocol.

Animals and treatments

24 Male Wistar rats, aged between 2 and 3 mo (180-200 g), were maintained in animal housing a week before the start of the experiments to ensure an adaptation period. Animals were placed in number of two per cage to avoid stress factors and received a commercial pellet diet (Industrial Society of Food, Sfax, Tunisia) and water ad libitum. Rats were kept under closely controlled environmental conditions (12 h light/dark cycle, room temperature 23 ± 1 °C). All experimental procedures followed the guidelines outlined in the Care and Use of Animals and were approved by the Ethics Committee of the National School of Veterinary Medicine of Sidi Thabet (Réf: 162020/FMT) according to the International Council for Laboratory Animal Science (ICLAS) recommendations.

Experimental design

A cohort of twenty-four male Wistar rats was partitioned into four groups, each comprising six rats. These rats were exposed simultaneously to noise and Toluene for six hours per day, five days per week, for six consecutive weeks. Before the exposure period, the rats were housed in cages in a noise-free and toluene-free environment. During the exposure period, rats in the noise and/or toluene exposure groups (N_{(85 dB)}, T_{(300 ppm)}, NT_{(85dB + 300 ppm)}) were relocated daily to rooms containing the exposure model for their daily exposure regimen. The control group (Group C) was also transferred to separate chambers with background noise levels maintained below 40 ± 5 dB (A) and free from toluene exposure (clean air), ensuring consistent environmental conditions comparable to the other groups.

The groups were as follows:

Group C: Control rats housed under standard laboratory conditions.

Group N _{(85 dB)}: Rats exposed to noise at 85 dB (A).

Group T _{(300 ppm)}: Rats exposed to toluene at 300 ppm.

Group NT _{(85dB + 300 ppm)}: Rats co-exposed to noise (85 dB (A)) and toluene (300 ppm).

Sample collection

At the end of the experiment, the rats were anesthetized with ketamine chlorhydrate (50 mg/kg) and euthanized by decapitation. Blood samples were collected in heparinized tubes for biochemical plasma analysis, centrifuged at 3,000 × g for 10 min at 4 °C, and stored. The kidneys were promptly excised, rinsed with 0.1 M phosphate buffer saline (pH 6.8), and bisected. One portion was fixed in 10% neutral formalin for subsequent histological analysis, while the other was stored at −80 °C for biochemical analysis.

Biochemical assays in plasma

Biochemical analyses of renal function parameters, including urea and creatinine, were conducted on plasma samples. The assays were performed using the COBAS INTEGRA 400 Plus automated analyzer (Switzerland), following standardized protocols to ensure accuracy and reliability.

Histological study

For histological examination, rat kidneys were perfused and immersed in a fixative solution (10% neutral buffered formalin) for 48 h, dehydrated through a graded series of ethanol, embedded in paraffin, sectioned with a microtome (Leica Biosystems RM2245, Great Britain) to obtain 4–5 μm-thick paraffin sections, and subsequently stained with hematoxylin and eosin (H&E).

Oxidative stress markers determination

A portion of the kidney was weighed and washed with ice-cold saline immediately and kept at −80 °C until analysis. The kidney tissue was homogenized (ULTRA-TURRAX® IKA-WERK, Germany) in ice-cold PBS buffer (pH =7.2) and centrifuged for 20 min (10,000 rpm at 4 °C). The supernatants were collected and maintained at −80 °C until subsequent biochemical analysis.

Lipid peroxidation determination

The determination of lipid peroxidation (LPO) was based on the detection of malondialdehyde (MDA), which was determined using the Buege and Aust¹⁵ method. Samples were mixed with a BHT-TCA solution (1%-20%, v/v) and centrifuged at 1,000 rpm for 10 min. The supernatant was blended with TBA-Tris solution (120 mM-26 mM, v/v) in an acidic solution (0.6 M HCl). The mixture was heated at 100 °C for 15 min and then cooled at room temperature. Absorbance was measured at 532 nm using a UV-spectrophotometer (Labomed, UV-2650, Inc, USA) and expressed in nmol of MDA/mg of protein.

Catalase activity

Catalase (CAT) activity was measured according to the method of Aebi et al.¹⁶ This assay is based on the ability of this enzyme to degrade hydrogen peroxide (H₂O₂), which results in a decrease in the absorption of the reaction mixture at 240 nm. Catalase activity was expressed in μmoles/min/mg of protein.

Superoxide dismutase (SOD) activity

Superoxide dismutase (SOD) activity was evaluated as described by Beyer and Fridovich¹⁷ using epinephrine (50 mM carbonate buffer, pH 10.2) and bovine catalase (0.4 U/mL). This method is based on the inhibition of the auto-oxidation of epinephrine to adrenochrome in the presence of SOD. Absorbance was measured at 480 nm, and SOD activity was expressed in UI/min/mg of protein.

Total protein level determination

The protein concentration in lung samples was determined using the Bradford¹⁸ method with bovine serum albumin (BSA) as the standard. Absorbance readings were measured at 595 nm, and the total protein content was calculated and reported in mg/ml.

Statistical analyses

Results were expressed as means ±SD. Statistical analyses were performed using Biostat software for Windows. Significant differences between all groups (n = 6) were determined using the Kruskal-Wallis test followed by the U-Mann–Whitney test for multiple comparisons with a statistical significance of P < 0.05.

Result

Identification of toluene by GC-FID analysis

The presence of toluene in the exposure chamber atmosphere was verified using gas chromatography with flame ionization detection (GC-FID) analysis. Air samples were collected through activated charcoal adsorption, and subsequent analysis revealed a distinct chromatographic peak at a retention time of 4.859 min, consistent with that of a certified toluene standard, thereby confirming the compound’s identity (see Fig. 2).

Fig. 2.

GC-FID chromatogram of the air sample collected by charcoal adsorption from the exposure chamber showing retention time of toluene 4.859: Toluene retention time (min).

Additionally, the chromatogram exhibited a secondary peak corresponding to carbon disulfide (CS₂), the solvent used for desorption, which further validated the sampling and extraction procedure. Importantly, no extraneous peaks were detected, indicating the absence of other volatile organic compounds and confirming that the atmosphere within the chamber contained pure toluene without contamination. This analytical profile ensured the reliability of the exposure conditions used in the experimental protocol.

Effects of noise and toluene exposure on renal function parameters: Urea and creatinine

Table 1 illustrates the plasma concentrations of urea and creatinine in rats exposed to noise, toluene, or their combination compared to the control group.

Table 1.

Renal function parameters across experimental groups.

	Urea (mmol/L)	Creatinine (μmol/L)
Control C	5.12 ± 0.7	51.20 ± 4
Noise group N (85 dB)	6.20 ± 0.12*	54.31 ± 2*
Toluene group T (300 ppm)	6.96 ± 0.5*	61.45 ± 0.05*
Noise & toluene group NT (85dB + 300 ppm)	7.86 ± 0.5*	63.45 ± 0.05*

The results were expressed as mean ± SD of six animals. The different pairs of groups were compared using the Mann–Whitney U test. ^*P < 0.05 vs control group.

In the control group, the mean plasma urea concentration was 5.12 ± 0.7 mmol/L. Exposure to noise (N _{(85 dB)}) significantly increased urea levels to 6.20 ± 0.12 mmol/L (P < 0.05), while exposure to toluene (T _{(300 ppm)} led to an even greater increase, with values reaching 6.96 ± 0.5 mmol/L (P < 0.05). Notably, co-exposure to noise and toluene NT _{(85dB + 300 ppm)}) resulted in a pronounced elevation in plasma urea levels to 7.86 ± 0.5 mmol/L, demonstrating a marked difference compared to the control group (p < 0.05 for all comparisons).

Similarly, the mean plasma creatinine concentration in the control group (C) was 51.20 ± 4 μmol/L. Exposure to noise led to a significant increase in creatinine levels, reaching 54.31 ± 2 μmol/L (P < 0.05), while exposure to toluene caused an even greater rise, with values of 61.45 ± 0.05 μmol/L (P < 0.05). The highest creatinine concentration was observed in the co-exposure group, with levels climbing to 63.45 ± 0.05 μmol/L, indicating a significant difference from the control group (P < 0.05).

These findings underscore the nephrotoxic effects of noise and toluene exposure, with co-exposure amplifying the impact on renal function parameters, such as urea and creatinine.

Impact of noise, toluene, and combined exposure on oxidative stress markers and antioxidant enzyme activities in kidney tissues

Table 2 presents the results of the MDA assay, along with the activities of superoxide dismutase (SOD) and catalase (CAT) in kidney tissues. This analysis aimed to evaluate the effects of noise (85 dB) and toluene (300 ppm) exposure, both individually and in combination, on oxidative stress markers. Significant changes were observed in all experimental groups, emphasizing the impact of these environmental stressors.

Table 2.

Effect of exposure to noise (85 dB (A) and/or toluene (300 ppm) on rat kidney oxidative parameters.

	Malondialdehyde (MDA) (nmol/mg prot)	Catalase (CAT) (μmol/min/mg prot)	Superoxide dismutase (SOD) (U SOD/min/mg prot)
Control C	2.513 ± 0.055	3.135 ± 0.040	5.27 ± 0.214
Noise group N (85 dB)	3.92 ± 0.129*§	6.149 ± 0.015*§	9.101 ± 0.663*§
Toluene group T (300 ppm)	5.64 ± 0.258**§	1.039 ± 0.003**§	3.677 ± 0.489**§
Noise & toluene group NT (85dB + 300 ppm)	6.75 ± 0.138**	0.934 ± 0.009**	2.012 ± 0.663**

Results are expressed as means ± SD of six animals. Different pairs of groups were compared using the Mann–Whitney U test. ^*P < 0.05 vs control group, ^**P < 0.01 vs control group (N = 6), §P < 0.05 vs NT_{(85dB + 300 ppm)} group (N = 6).

The MDA assay revealed a notable increase in lipid peroxidation in rats exposed to noise, toluene, and their co-exposure, relative to the control group. This increase reflects the oxidative damage caused by these exposures, with the most substantial effect occurring under combined exposure conditions. Specifically, MDA levels were significantly higher in all exposed groups than in the controls (2.513 ± 0.055 nmol/mg protein, P ≤ 0.01). Noise exposure alone elevated MDA levels to 3.92 ± 0.129 nmol/mg protein, while toluene exposure resulted in MDA levels of 5.64 ± 0.258 nmol/mg protein. The highest MDA levels were observed in the co-exposed group (6.75 ± 0.138 nmol/mg protein), highlighting the cumulative effect of both stressors on lipid peroxidation.

Catalase activity significantly decreased in the toluene-exposed (1.039 ± 0.003 μmoL/min/mg protein) and co-exposed groups (0.934 ± 0.009 μmoL/min/mg protein) compared to the controls (3.135 ± 0.040 μmoL/min/mg protein), with p-values <0.01. In contrast, noise exposure alone led to a substantial increase in catalase activity (6.149 ± 0.015 μmoL/min/mg protein).

Similarly, SOD activity was significantly reduced in both the toluene-exposed and co-exposed groups compared to that in the controls (p ≤ 0.01). While noise exposure alone enhanced SOD activity (9.101 ± 0.663 U SOD/min/mg protein), the toluene (3.677 ± 0.489 U SOD/min/mg protein) and co-exposed groups (2.012 ± 0.663 U SOD/min/mg protein) showed marked reductions in SOD activity relative to controls (5.27 ± 0.214 U SOD/min/mg protein).

These results indicate that both noise and toluene exposure, individually or in combination, significantly altered oxidative stress markers in the kidney tissues.

Serum inflammatory cytokine assessment

Quantitative analysis of the pro-inflammatory cytokines TNF-α and IL-1β was performed to evaluate the systemic inflammatory response following exposure. As shown in Table 3, the serum cytokine concentrations in the control group remained within the physiological range and exhibited no significant alterations.

Table 3.

Serum concentrations of the proinflammatory cytokines TNF-α, and IL-1β in Wistar rats.

	TNF-α (pg/mL)	IL-1β (pg/mL)
C	28.16 ± 2.04	68.83 ± 9.49
N	54.5 ± 1.81*§	205.5 ± 19.36**§
T	71.5 ± 2.81**§	235.5 ± 11.16**§
NT	79.66 ± 6.80**	257.83 ± 16.82**

The results are expressed as the means ± SDs of six animals, with the mean concentrations in pg/mL of cytokines. The different pairs of groups are compared via the Mann–Whitney U test. ^*  p < 0.05 vs control group, ^**p < 0.01 vs control group, § p < 0.05 vs NT(85dB +300 ppm) group.

However, rats subjected to environmental stressors, including noise (N), toluene (T), and their combination (NT), displayed a marked and statistically significant elevation in both TNF-α and IL-1β levels compared to controls. TNF-α concentrations increased to 54.5 ± 1.81 pg/mL in the N group (P ≤ 0.05), 71.5 ± 2.81 pg/mL in the T group (P ≤ 0.01), and 79.66 ± 6.80 pg/mL in the NT group (P ≤ 0.01) versus 28.16 ± 2.04 pg/mL in controls. Similarly, IL-1β levels rose significantly in the exposed groups, with values of 205.5 ± 19.36 pg/mL (N), 235.5 ± 11.16 pg/mL (T), and 257.83 ± 16.82 pg/mL (NT), in contrast to 68.83 ± 9.49 pg/mL in the control group (P ≤ 0.01 for all).

These results clearly demonstrate that both individual and combined exposure to noise and toluene induces a robust proinflammatory state. Notably, the combined exposure group (NT) exhibited the most pronounced cytokine response, suggesting a possible enhanced effect of these two environmental factors.

Microscopic analysis of kidney tissues

In the control group, the renal parenchyma, tubules, and glomeruli exhibited a normal histological architecture with well-preserved structural integrity, an intact tubular epithelium, and a typical glomerular organization (Fig. 3A, a). In contrast, the noise-exposed group showed several atypical proximal tubules that had lost their characteristic brush borders, along with evident tubular degeneration and vascular congestion (Fig. 3B, b1, b2). The toluene-exposed group displayed an altered glomerular structure characterized by glomerular cell shrinkage, numerous atypical proximal tubules lacking their brush borders, as well as vascular congestion and prominent leukocyte infiltration in the interstitial areas (Fig. 3C, c1, c2). The co-exposure group (noise and toluene) exhibited the most pronounced histopathological alterations, including significant glomerular structural abnormalities with marked glomerular cell shrinkage, extensive proximal tubular degeneration with complete loss of brush borders, widespread tubular degeneration within the renal parenchyma, severe vascular congestion, and prominent leukocyte infiltration in the interstitial areas (Fig. 3D, d1, d2).

Fig. 3.

Microscopic analysis of kidney tissue (H&E) in control and noise- and/or toluene-exposed rats (85 dB (A), 300 ppm).Histological analysis showed normal renal architecture in the control group (fig. 3A, a). The noise-exposedgroup (fig. 3B, b1, b2) exhibited proximal tubular alterations (yellow arrows), tubular degeneration, andvascular congestion (green arrows). The toluene-exposed group (fig. 3C, c1, c2) glomerular cell shrinkage(black star), proximal tubular damage (yellow arrows), vascular congestion (green arrows), and leukocyteinfiltration (red circle). The co-exposure group (fig. 3D, d1, d2) displayed the most severe alterations, withmarked glomerular shrinkage (black star), extensive tubular degeneration (yellow arrows), severe vascularcongestion (green arrows), and prominent leukocyte infiltration (red circle).

Discussion

Occupational diseases associated with environmental stressors such as chronic noise and toluene exposure constitute a significant threat to workers’ health. This study aimed to investigate the combined effects of noise (85 dB) and toluene (300 ppm) on kidney tissue under subchronic exposure conditions. Through the analysis of biochemical renal plasma parameters, oxidative stress markers, including lipid peroxidation and antioxidant enzyme activities, and histological assessments, we sought to elucidate the extent of the damage caused by these stressors on renal function.

Kidneys play a pivotal role in maintaining homeostasis by regulating the balance of electrolytes, fluids, and waste products in the body. This includes the filtration of blood to excrete metabolic byproducts such as urea and creatinine. These parameters serve as crucial indicators of renal function, reflecting the capacity of the kidneys to filter and eliminate waste. In this study, the plasma concentrations of urea and creatinine were assessed in rats exposed to noise, toluene, or their combination. The results revealed significant alterations in these renal parameters following exposure to environmental stressors, emphasizing the nephrotoxic effects of both noise and toluene, particularly when combined.

Urea levels, an important marker of renal function, were significantly elevated in all exposed groups compared with the controls. Similarly, creatinine levels, another key indicator of kidney function, increased significantly in both noise- and toluene-exposed groups, with the highest concentrations observed in the co-exposure group. These observations are consistent with findings from previous studies, such as Alizadeh et al.,³ who reported elevated creatinine levels in rats exposed to 100 dB of noise for 4 h per day over 14 days. Additionally, Salihu et al.¹⁹ demonstrated increased urea and creatinine levels in rats receiving 4.5 mL/kg of toluene for 7 days. Abouee-Mehrizi et al.⁵ further corroborated these findings, noting disruptions in renal parameters under combined exposure to noise (100 dB) and toluene (1,000 ppm) for 14 days. Their study observed that noise exposure elevated albumin, uric acid, and creatinine levels, while reducing urea levels. Toluene exposure decreased albumin, uric acid, and urea levels but increased creatinine levels. Simultaneous exposure exacerbated these effects, leading to reduced albumin, uric acid, and urea levels, and increased creatinine levels.

These findings indicate that noise and toluene, individually or in combination, impair renal filtration capacity and disrupt homeostasis. The effect of co-exposure underscores the compounded risk posed by environmental stressors on the kidney function. This highlights the critical need for protective measures to protect workers from occupational hazards.

Oxidative stress plays a pivotal role in mediating the toxic effects of environmental stressors such as noise and toluene. This condition arises from an imbalance between excessive production of reactive oxygen species (ROS) and the body’s antioxidant defense mechanisms. ROS are highly reactive molecules capable of causing oxidative damage to cellular components including lipids, proteins, and DNA, ultimately leading to cellular dysfunction, inflammation, and tissue injury.²⁰ Kidneys, which are highly vascularized and metabolically active, are particularly susceptible to oxidative damage, rendering them a primary target for the deleterious effects of these stressors.²¹

In this study, we examined oxidative stress markers in renal tissue to evaluate the extent of oxidative damage and its contribution to renal injury. Our results revealed a significant increase in malondialdehyde (MDA) levels, a well-established marker of lipid peroxidation, in all exposure groups. The highest MDA levels were observed in the co-exposed group, indicating a pronounced effect of noise and toluene in exacerbating oxidative damage to the cochlea. This suggests that combined exposure amplifies the generation of reactive oxygen species (ROS), overwhelming the antioxidant defenses of the kidney and accelerating tissue injury. Notably, our findings align with recent studies that demonstrated that isolated or combined exposure to noise and toluene significantly increased lipid peroxidation in multiple organs, including the heart, lungs, kidneys, and liver.^{5,11–13,22,23} These findings further underscore the pervasive impact of these environmental stressors on systemic oxidative damage and elucidate the critical role of oxidative stress in their toxicological effects.

To gain deeper insight into oxidative stress dynamics, we examined the activity profiles of key antioxidant enzymes, superoxide dismutase (SOD) and catalase. Notably, enzymatic activity levels varied depending on the type of exposure. In rats exposed to noise alone, the activities of SOD and catalase were significantly upregulated, indicating an adaptive response to increased oxidative stress. This upregulation likely reflects a compensatory mechanism aimed at neutralizing ROS accumulation to maintain cellular redox homeostasis. Such adaptive induction of antioxidant enzymes under moderate stress conditions is well documented in the renal system, where the kidney must frequently counter oxidative challenges arising from both endogenous and exogenous sources.²⁴

In contrast to the adaptive response observed under noise exposure, toluene exposure led to a marked suppression of superoxide dismutase (SOD) and catalase activity in renal tissue. This enzymatic downregulation is likely attributable to oxidative inactivation or depletion resulting from the persistent overproduction of reactive oxygen species (ROS) during toluene metabolism. Notably, the biotransformation of toluene via the cytochrome P450 and aldehyde dehydrogenase pathways generates highly reactive intermediates, including benzyl radicals and hydroperoxides.^25–27 The resulting oxidative burden appears to overwhelm the renal antioxidant defense system, as evidenced by significantly reduced SOD and catalase activities. This impaired enzymatic capacity to neutralize ROS may lead to enhanced lipid peroxidation, as reflected by elevated malondialdehyde (MDA) levels. Remarkably, the group subjected to combined noise and toluene exposure exhibited the most severe biochemical alterations. In this group, antioxidant enzyme activities were drastically reduced, alongside the highest MDA levels, indicating an interaction between the two stressors that aggravates oxidative damage beyond the renal capacity for adaptation to stress. This pattern of oxidative exhaustion reflects the inability of renal tissues to sustain prolonged defense against simultaneous physical and chemical injuries.

These enzymatic disturbances are consistent with previous reports demonstrating redox imbalance under similar exposure conditions. Our findings are in agreement with those of Gannouni et al.,²³ who reported a redox imbalance in the cardiac tissue of rats exposed to 70 dB and 85 dB noise levels for three months. This oxidative stress response was characterized by elevated lipid peroxidation, as indicated by increased MDA levels, and dysregulated antioxidant enzyme activity, with a significant upregulation of SOD and a reduction in catalase activity. Additionally, studies conducted by Abouee-Mehrizi et al.^5,22 have shown that combined exposure to noise and toluene results in a disruption of the redox equilibrium in rabbits, particularly in vital organs such as the liver and kidneys. This disruption is manifested by significant alterations in oxidative stress biomarkers, including increased MDA levels, as well as changes in antioxidant enzyme activities, notably a decrease in glutathione peroxidase and superoxide dismutase activity.

The oxidative disturbances identified in our study not only compromise cellular homeostasis but also appear to initiate a broader pathological cascade involving multiple organ systems. In addition to oxidative stress, systemic inflammation emerged as a key pathological feature across all exposed groups, as demonstrated by elevated serum levels of the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β), reflecting the widespread activation of inflammatory signaling pathways.

The mechanistic crosstalk between oxidative stress and inflammation is well-established, particularly in renal pathophysiology.²⁸ Reactive oxygen species (ROS) activate redox-sensitive transcription factors, such as nuclear factor kappa B (NF-κB), which promote the transcription of multiple pro-inflammatory mediators. Once released into the systemic circulation, these cytokines further amplify oxidative stress through immune cell recruitment and enhanced ROS generation, thereby establishing a self-perpetuating cycle of oxidative and inflammatory injuries.²⁹ Furthermore, excessive ROS levels can initiate apoptosis in various organs by upregulating pro-apoptotic genes and simultaneously suppressing anti-apoptotic signaling cascades.³⁰ Consistent with these biochemical disruptions, histopathological examination of renal tissues revealed pronounced structural abnormalities in all exposed groups. These include tubular degeneration, glomerular distortion, and inflammatory cell infiltration, which are hallmarks of inflammation-induced renal damage.

In agreement with our findings, Abouee-Mehrizi et al.⁵ reported significant histopathological alterations in the kidneys of New Zealand white rabbits exposed to noise and toluene, including glomerular cell shrinkage and cytoplasmic vacuolization. Additional lesions, such as capsular thickening, glomerular tuft contraction, and increased connective tissue deposition in the renal interstitium, were also observed. Similarly, Ahmadizadeh et al. [31] demonstrated that intraperitoneal administration of toluene at 900 mg/kg in Wistar rats led to extensive renal tissue disorganization and vascular congestion in the kidney.

Taken together, our findings support the hypothesis that combined exposure to noise and toluene triggers a cascade of renal injury initiated by ROS-driven lipid peroxidation and antioxidant enzyme depletion, which subsequently activates systemic inflammatory responses. This oxidative-inflammation axis appears to play a central role in mediating renal structural and functional deterioration in the presence of environmental co-exposure. These data highlight the heightened vulnerability of renal tissue to both physical and chemical stressors and underscore the pathological significance of these interactions. Further investigations into the molecular mediators of this interaction are warranted, with the aim of identifying potential therapeutic strategies to preserve renal function in chronically exposed populations.

Conclusion

This study demonstrates that co-exposure to noise and toluene significantly exacerbates oxidative stress, antioxidant enzyme depletion, systemic inflammation, and renal dysfunction in Wistar rats. The combined exposure intensifies lipid peroxidation and disrupts redox homeostasis more severely than individual insults, leading to marked structural kidney damage. These findings highlight the critical interplay between physical and chemical environmental stressors, underscoring the importance of considering their combined impact in risk assessments. Our results advocate stricter regulatory policies and targeted preventive strategies to mitigate the compounded renal risks posed by such environmental exposures.

Author contributions

Conceptualization: TBA, AM, EALM; Methodology: TBA, AM, EALM, LBK, SG; Project administration: AM; Resources: AM, EALM; Supervision: AM, Writing—original draft: TBA, EALM; Writing/review the draft: TBA, AM, EALM. All authors have read and agreed to the published version of the manuscript.

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Conflicts of interest: The authors declare that they have no conflict of interest.

Data availability statement

All data and materials are available upon request.