2,5-Hexanedione (2,5-HD) is the toxic metabolite of n-hexane which is widely used as solvent in numerous industries. The present study elucidated the precise mechanism of 2,5-HD in hepatorenal toxicity by determining the involvement of oxidative stress in rats. Adult male Wistar rats were exposed to 0, 0.25, 0.5, and 1% 2,5-HD in drinking water for 21 days. Exposure to 2,5-HD caused liver and kidney atrophy evidenced by significant elevation in serum aminotransferases, alkaline phosphatase, albumin, bilirubin, urea, creatinine, and electrolytes levels compared with control. The marked dose-dependent increase in total cholesterol (TC), triglyceride (TG), and low-density lipoprotein (LDL) was accompanied with significant decrease in high-density lipoprotein (HDL) levels in 2,5-HD-exposed animals when compared with the control. Administration of 2,5-HD significantly diminished glutathione (GSH) level but increased the activities of superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), and glutathione-S-transferase (GST) concomitantly with marked elevation in hydrogen peroxide (H2O2) and malondialdehyde (MDA) levels in liver and kidney of the treated groups compared with control. These findings suggest that undue exposure to 2,5-HD at environmentally relevant levels may impair liver and kidney functions through induction of oxidative stress.
2,5-Hexanedione (2,5-HD) is the main toxic metabolite of n-hexane, an organic solvent widely used in chemical engineering and pharmaceutical and cosmetic industries [
The adverse effects resulting from exposure to xenobiotics could occur via several mechanisms leading to significant alterations in the levels of biomolecules such as enzymes and metabolic products, normal functioning, and histomorphology of the organs. The paucity of information in the literature about 2,5-HD-induced hepatorenal toxicity accentuates the need to undertake a detailed study evaluating the antioxidant status of the kidney and liver in rats exposed to 2,5-HD. It is well known that oxidative stress is involved in the pathogenesis of several diseases following exposure to environmental contaminants. The liver is particularly vulnerable to toxicity produced by reactive metabolite because it is the major site of xenobiotic metabolism. The kidney is a highly specialized organ that maintains the internal environment of the body by selectively excreting or retaining various substances according to specific body needs [
To delineate the mechanism of action of 2,5-HD at subcellular levels in liver and kidney, we investigated its effect on the antioxidant enzymes, oxidative stress indices, and biomarkers of renal and hepatic functions following subchronic exposure in the treated rats.
2,5-Hexanedione (98.99%), epinephrine, glutathione (GSH), 5,5′-dithio-bis-2-nitrobenzoic acid (DTNB), hydrogen peroxide (H2O2), thiobarbituric acid (TBA), and 1-chloro-2,4-dinitrobenzene (CDNB) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other reagents were of analytical grade and were obtained from the British Drug Houses (Poole, Dorset, UK). Kits for serum biochemistry were purchased from Randox Laboratory Limited, United Kingdom.
A total of thirty-two healthy adult male Wistar rats (10 weeks old, ≈170 g) purchased from the Department of Biochemistry, University of Ibadan, Ibadan, Nigeria, were used for this study. They were housed in plastic cages placed in a well-ventilated rat house, provided rat pellets and water
All the rats were sacrificed by cervical dislocation 24 hours after the last intake of 2,5-hexanedione, and blood was collected by cardiac puncture. The livers and kidneys were quickly removed, weighed, and placed on an ice bath. The blood was allowed to clot and centrifuged at low speed (3000 ×g) at room temperature for 15 minutes. The body weights of rats were taken before exposure to various treatments and before killing.
Serum activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were determined by the method of Reitmann and Frankel [
Levels of serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) were determined using commercially available diagnostic kits (Randox Laboratories Limited, UK).
Remaining portions of liver and kidney were homogenized in 50 mM Tris-HCl buffer (pH 7.4) containing 1.15% potassium chloride. Following the centrifugation of the homogenate at 10,000 ×g for 15 minutes at 4°C, the supernatant was collected for the estimation of superoxide dismutase (SOD) activity by the method of Misra and Fridovich [
Liver and kidney biopsies were processed for histology according to Songur et al. [
Statistical analyses were carried out using one-way analysis of variance (ANOVA) to compare the experimental groups followed by Bonferroni’s test to identify significantly different groups (SPSS for Windows, version 17).
The rats in all the experimental groups remained active and vigorous throughout the treatment period. The body weight and relative organ weights of control rats and those exposed to 2,5-HD are presented in Table
Body weights and organ weights of rats exposed to 2,5-hexanedione for 21 days.
Control | 0.25% 2,5-HD | 0.50% 2,5-HD | 1.0% 2,5-HD | |
---|---|---|---|---|
Body weight gain (g) | 31.56 ± 6.64 | 12.81 ± 4.54* | 3.71 ± 0.04* | 1.07 ± 3.48* |
RLW (g/100 g bw) | 2.27 ± 0.13 | 2.31 ± 0.11 | 2.56 ± 0.89* | 2.75 ± 0.93* |
RKW (g/100 g bw) | 0.53 ± 0.08 | 0.54 ± 0.07 | 0.59 ± 0.04* | 0.71 ± 0.08* |
RLW: relative liver weight; RKW: relative kidney weight. The data are expressed as mean ± SD for eight animals per group. *
To investigate the impact of 2,5-HD treatment on liver function, the levels of AST, ALT, ALP, albumin, and total and conjugated bilirubin were determined in the serum of control and 2,5-HD-treated rats after 21 days. Table
Biomarkers of hepatic dysfunction in rats exposed to 2,5-hexanedione for 21 days.
Endpoints | Control | 0.25% 2,5-HD | 0.50% 2,5-HD | 1.0% 2,5-HD |
---|---|---|---|---|
AST (U/L) | 21.50 ± 0.71 | 32.50 ± 0.71* | 39.50 ± 2.89* | 41.08 ± 1.41* |
ALT (U/L) | 19.33 ± 1.15 | 24.50 ± 3.11* | 26.73 ± 1.15* | 27.50 ± 0.71* |
ALP (U/L) | 18.33 ± 2.08 | 23.69 ± 1.15 | 25.50 ± 4.95* | 29.03 ± 6.68* |
Albumin (g/L) | 26.50 ± 1.29 | 27.10 ± 2.00 | 28.04 ± 0.82 | 28.40 ± 1.67 |
Conjugated bilirubin (mmol/L) | 1.75 ± 0.89 | 1.87 ± 0.53 | 2.63 ± 0.55* | 2.40 ± 0.55* |
Total bilirubin (mmol/L) | 3.75 ± 0.96 | 5.75 ± 0.96* | 7.20 ± 1.00* | 7.75 ± 1.71* |
ALT: alanine aminotransferase; AST: aspartate aminotransferase; ALP: alkaline phosphatase. The data are expressed as mean ± SD for eight animals per group. *
Table
Lipid profile in male rats exposed to 2,5-hexanedione for 21 days.
Endpoints | Control | 0.25% 2,5-HD | 0.50% 2,5-HD | 1.0% 2,5-HD |
---|---|---|---|---|
HDL (mmol/L) | 1.15 ± 0.13 | 0.88 ± 0.03* | 0.85 ± 0.13* | 0.83 ± 0.12* |
LDL (mmol/L) | 0.67 ± 0.19 | 0.93 ± 0.17* | 1.19 ± 0.28* | 1.35 ± 0.33* |
TG (mmol/L) | 1.21 ± 0.14 | 1.49 ± 0.16* | 1.52 ± 0.13* | 1.57 ± 0.18* |
TC (mmol/L) | 1.67 ± 0.13 | 1.99 ± 0.18* | 2.33 ± 0.21* | 2.37 ± 0.23* |
Atherogenic index | 0.58 ± 0.05 | 1.05 ± 0.08* | 1.40 ± 0.07* | 1.62 ± 0.06* |
HDL: high-density lipoprotein; LDL: low-density lipoprotein; TG: triglyceride; TC: total cholesterol; atherogenic index (LDL/HDL). The data are expressed as mean ± SD for eight animals per group. *
To investigate the integrity of the kidney following 2,5-HD exposure to rats, the concentrations of biomarkers of renal dysfunction were determined. The effects of 2,5-hexanedione on urea, creatinine, Na+, K+,
Biomarkers of renal dysfunction in rats exposed to 2,5-hexanedione for 21 days.
Endpoints | Control | 0.25% 2,5-HD | 0.50% 2,5-HD | 1.0% 2,5-HD |
---|---|---|---|---|
Na+ (mmol/L) | 126.15 ± 1.71 | 139.67 ± 1.53* | 140.33 ± 2.31* | 147.20 ± 1.41* |
K+ (mmol/L) | 3.78 ± 0.33 | 4.03 ± 0.33 | 4.63 ± 0.61* | 5.05 ± 0.66* |
|
20.60 ± 1.04 | 24.70 ± 1.92* | 28.50 ± 1.09* | 34.64 ± 1.13* |
Cl− (mmol/L) | 83.30 ± 1.63 | 88.67 ± 3.21 | 94.50 ± 1.91* | 96.50 ± 2.22* |
Urea (mmol/L) | 14.30 ± 0.62 | 18.03 ± 1.03* | 18.86 ± 1.67* | 19.46 ± 0.57* |
Creatinine (mmol/L) | 89.75 ± 3.30 | 135.33 ± 3.79* | 151.04 ± 2.00* | 161.80 ± 2.65* |
The data are expressed as mean ± SD for eight animals per group. *
Following exposure to 2,5-HD, the antioxidant statuses of the liver and kidney of the treated rats were determined using a panel of assays including enzymatic and nonenzymatic antioxidant levels along with hydrogen peroxide and lipid peroxidation levels. Figures
Comparison of hepatic and renal SOD and CAT activities among the control and treated rats (
Comparison of hepatic and renal GPx and GST activities among the control and treated rats (
Comparison of hepatic and renal GSH, H2O2 generation, and MDA levels among the control and treated rats. Data are described as mean ± SD of eight animals per group. Asterisk indicates statistical difference from control (
The representative photomicrographs of the control and 2,5-HD-treated liver and kidney are presented in Figures
Influence of 2,5-HD on liver morphology after treatment for 21 consecutive days in rats. Representative photomicrographs of liver samples of rats in control (a) and 0.25% 2,5-HD (b) appear normal whereas mild central venous congestion (green arrow) and cellular infiltration (black arrow) by neutrophils were identified in 0.5% 2,5-HD (c) and 1% 2,5-HD (d).
Influence of 2,5-HD on kidney morphology after treatment for 21 consecutive days in rats. Representative photomicrographs of control (a) show normal renal architecture. Kidney histology revealed progressive degeneration of the proximal tubules characterized by mild hemorrhage (green chevron) at interstitium in tubular epithelial cells in 0.25% 2,5-HD (b) and severe vacuolation and renal tubular necrosis (blue chevron) in 0.5% 2,5-HD (c) and 1% 2,5-HD (d).
The current trend in toxicology entails investigating the effects of toxic chemicals at environmentally relevant concentrations, which is the situation normally encountered by the population in industrialized countries. The concentrations of 2,5-HD used in the present study are within the range of human exposures and allow identification of mechanisms of its biological effects on the liver and kidney at low versus high concentrations [
In the present study, exposure to 2,5-HD caused a significant decrease in the body weights but increased the relative liver and kidney weights of the treated rats, thus indicating an overt general and organ toxicity in the rats. Moreover, relative organ weight is an important index of swelling, atrophy, or hypertrophy [
Alkaline phosphatase (ALP) is a marker enzyme for the integrity of the hepatobiliary system and the flow of bile into the small intestine. The increase in hepatic ALP activity indicates obstructive event or cholestatic effect following 2,5-HD treatment. Furthermore, the elevation in total and conjugated bilirubin levels observed in the present study indicates posthepatic toxicity possibly caused by an interruption to the drainage of bile in the biliary system following exposure to 2,5-HD. Evaluation of serum albumin level is a good criterion for assessing the secretory ability of the liver [
Kidney damage is associated with decline in renal function which could lead to renal failure. The decrease in renal function evidenced by significant increase in plasma levels of urea and creatinine in rats treated with 2,5-HD was clearly demonstrated in the present investigation. While an increase in serum urea may indicate decrease in reabsorption at the renal epithelium, an increase in serum creatinine reflects impairment in the kidneys, particularly for glomerular filtration rate [
The histopathological report revealed that oral exposure to 2,5-HD at environmentally relevant concentrations produced remarkable dose-dependent damaging effect to both liver and kidney of the treated rats, hence supporting the observed biochemical observations. The treatment-related lesions such as mild central venous congestion and cellular infiltration by neutrophils identified in the liver of rats exposed to 2,5-HD suggest its deleterious effect on the structure and function of the liver of the treated animals. Also, the progressive degeneration of the proximal tubules of 2,5-HD-treated animals was characterized by mild hemorrhage at interstitium in tubular epithelial cells with severe vacuolation and renal tubular necrosis. The proximal susceptibility of the tubular epithelium to toxicants has been attributed to the intense filtration of substances from the blood, their transport, and the high energy requirement of these functions [
In an attempt to delineate the mechanism of action of 2,5-HD at subcellular levels in the liver and kidney, we investigated its influence on the hepatic and renal antioxidant status. Normally the deleterious effects of oxidative stress are counteracted by the natural antioxidant defense mechanisms to protect the biological system against reactive oxygen species. Antioxidant statuses are regulated by multiple factors. The oxidative status of the cell is the primary factor regulating gene expression and the activity of antioxidant enzymes [
In the present study, the activities of hepatic and renal SOD, CAT, GPx, and GST were markedly increased in rats treated with 2,5-HD. The dose-dependent induction of these antioxidant enzymes may indicate an adaptive response to counter the damaging effect of oxidative stress possibly generated during 2,5-HD metabolism. Glutathione plays a pivotal role in the scavenging of hydroxyl radical and singlet oxygen directly as well as in the detoxification of hydrogen peroxides and lipid hydroperoxides by the activity of GPx. Further, GST is involved in the biochemical conjugation of electrophilic oxidants with GSH to form water-soluble compound products that are readily excreted from the system [
The present study obviously demonstrates that an uncontrolled exposure to toxic industrial solvent 2,5-HD is capable of inflicting biological damage leading to the pathology of many conditions including liver and kidney damage. The differential detrimental effects of 2,5-HD on the liver and kidney in rats are strongly associated with biochemical changes including impairment of function, metabolic disorders, oxidative stress, and histological alteration. The data presented herein are novel and show, for the first time, that the hepatorenal toxicity of 2,5-HD in the experimental rats is presumably by increased generation of ROS which led to perturbation of antioxidant defense systems. Hence, the extrapolation of the present animal study to human indicates that 2,5-HD has potential health risk in exposed individuals.
The authors declare that there is no conflict of interests.
This work was supported in part by the Multidisciplinary Research Grants under the Staff Training and Research Capacity Building Programme of the John D. and Catherine T. Mac-Arthur Foundation Grant (USA) endowment from the University of Ibadan, Nigeria, awarded to Professor E. O. Farombi.