Genotoxicity in cells may occur in different ways, direct interaction, production of electrophilic metabolites, and secondary genotoxicity via oxidative stress. Chloroform, dichloromethane, and toluene are primarily metabolized in liver by CYP2E1, producing reactive electrophilic metabolites, and may also produce oxidative stress via the uncoupled CYP2E1 catalytic cycle. Additionally, GSTT1 also participates in dichloromethane activation. Despite the oxidative metabolism of these compounds and the production of oxidative adducts, their genotoxicity in the bone marrow micronucleus test is unclear. The objective of this work was to analyze whether the oxidative metabolism induced by the coexposure to these compounds would account for increased micronucleus frequency. We used an approach including the analysis of phase I, phase II, and antioxidant enzymes, oxidative stress biomarkers, and micronuclei in bone marrow (MNPCE) and hepatocytes (MNHEP). Rats were administered different doses of an artificial mixture of CLF/DCM/TOL, under two regimes. After one administration MNPCE frequency increased in correlation with induced GSTT1 activity and no oxidative stress occurred. Conversely, after three-day treatments oxidative stress was observed, without genotoxicity. The effects observed indicate that MNPCE by the coexposure to these VOCs could be increased via inducing the activity of metabolism enzymes.
Genotoxic compounds are known to exert their effects on DNA either in a direct way or through their metabolites after going through an enzymatic transformation. Some compounds, however, have been described as being genotoxic via alternative pathways, like the production of ROS.
That is the case of DCM, which is primarily metabolized by CYP2E1 [
CLF is another compound of this kind, capable of increasing malondialdehyde deoxyguanosine (M1dG) adducts and lipid peroxidation in HepG2 cells [
TOL biotransformation also occurs through CYP2E1 [
In spite of the described oxidative metabolism of chloroform, dichloromethane, and toluene, and the formation of oxidative adducts produced by them, their genotoxicity in the micronucleus test, as many studies demonstrate, is not clear (Table
Acute toxic effects in rodent i.p. exposed to dichloromethane (DCM), chloroform (CLF) or toluene (TOL).
VOC | Dose (mmol/kg) | P-450 | CYP2E1 | Lipid peroxidation | GSH | MNPCE | CA | Reference |
---|---|---|---|---|---|---|---|---|
DCM | 2.5 | 0 | 0 | 0 | 0 | Pilot study | ||
CLF | 2.6 | 0 | +0.48 | 0 | 0 | Pilot study | ||
TOL | 8 | −0.48 | +0.50 | 0 | 0 | Pilot study | ||
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DCM | 1.2–2.4 | 0 | 0 | [ | ||||
4.8–9.5 | +0.4–0.50 | +0.35 | [ | |||||
6 | 0 | [ | ||||||
5–20 | 0 | [ | ||||||
1.2–23.5 | 0 | [ | ||||||
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CLF | 0.1 | 0 | [ | |||||
1.3 | 0 | 0 | 0 | [ | ||||
1.7 | −0.07 | [ | ||||||
2.0–8.0 | 0 | [ | ||||||
0.01 | +3.75 | [ | ||||||
0.1–1.0 | +7.75 | [ | ||||||
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TOL | 5 | 0 | +1.16 | [ | ||||
5.4 | 0 | [ | ||||||
10.8 | +0.30 | [ | ||||||
16.2 | +0.17 | [ | ||||||
20 | 0 | [ | ||||||
1.2 | 0 | 0 | [ | |||||
2.4 | +0.32 | 0 | [ | |||||
4.7 | 0 | +2.71 | [ |
Data represent significant fold increases (+) or decreases (−) with respect to control animals; zero means no change.
Cytochrome P450 (P-450), cytochrome 2E1 (CYP2E1), lipid peroxidation and glutathione (GSH) were determined in liver, micronuclei (MNPCE) and chromosomal aberrations (CA) were determined in bone marrow.
Due to the fact that cytochrome P-450-isoform CYP2E1 (CYP2E1) is mainly responsible for the oxidative metabolism of these VOCs [
A pilot study was conducted where each compound was administered for three days in doses equivalent to the 10% of the LD50 in rats to analyze whether they would induce oxidative stress and, consequently, micronuclei in bone marrow polychromatic erythrocytes (MNPCE). No genotoxic effects were found with any of the compounds, and CLF and TOL induced CYP2E1 activity. Oxidative stress, measured by the levels of GSH in liver homogenate, was not detected under any treatment (Table
Therefore, in the present study a rat model was used to analyze the hepatic xenobiotic metabolism response (P-450 levels, CYP2E1, GST, and GSTT1 activities), the antioxidant response (antioxidant enzymes activity, GSH/GSSG, and TBARS), and whether there would be a relationship of these responses with the genotoxic damage in liver or in bone marrow that could be produced by the coexposure to the three compounds.
HPLC grade chloroform (CAS: 67-66-3), dichloromethane (CAS: 75-09-2), and toluene (CAS: 108-88-3) were purchased from Honeywell Burdick & Jackson (Muskegon, MI, USA); protein assay dye reagent and acetylacetone were purchased from Bio-Rad (CA, USA). All other reagents were purchased from Sigma-Aldrich.
Three-week-old male Wistar rats (89.5 g ± 14.5) were maintained under controlled temperature
Toluene, chloroform, and methylene chloride, regardless of the route of exposure, are distributed widely throughout the body (toluene in liver and brain) and their metabolism occurs mainly in the liver [
The genotoxic potential, induction of P-450, and reduced glutathione levels for separated treatments with CLF, DCM, or TOL, at i.p. doses of 2.5, 2.6, and 8.1 mmol/kg/day/3 days, respectively (corresponding to 1/10 LD50 of each compound, based on the LD50 reported in the Merck Index), were tested in rats, five animals/compound, in order to approximate the doses that would be used in the mixture.
Due to the wide variability of the environmental levels reported, our protocol was based on LD50 in order to obtain the maximum biological response in the shortest time (according to MNPCE protocols, by Krishna and Hayashi [
The exposure regime was based on protocols for the rodent micronucleus assay [
Doses administered per treatment.
Group of treatment | Doses |
|
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Neg. control (corn oil)* | 125 |
5 |
TOL/DCM/CLF |
||
Low-mix* | 2.0/0.6/0.65 | 5 |
Mid-mix* | 4.0/1.2/1.3 | 5 |
High-mix* | 8.0/2.5/2.6 | 5 |
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Positive controls |
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tSBO | 2.0 | 3 |
CCl4 | 10.0 | 3 |
BEN | 12.0 | 3 |
DEN | 1.0 | 3 |
BEN and DEN were administered for two days; CCl4 and tSBO, for three days.
Additionally, four groups of rats were treated with different chemicals that served as positive controls: trans-Stilbene oxide (tSBO) (2 mmol/kg, i.p., xenobiotic metabolism inducer), carbon tetrachloride (CCl4) (9.75 mmol/kg, i.p., oxidative stress damage), benzene (BEN) (12.8 mmol/kg, p.o., BM genotoxic damage), and diethylnitrosamine (DEN) (0.97 mmol/kg, i.p., hepatic genotoxic damage). The number of animals used for each treatment is presented in Table
The animals were euthanized by cervical dislocation 24 h after the last dose of the corresponding treatment. Livers were freshly excised and washed in cold 0.15 M KCl. Two small pieces (0.25 g approximately) of each liver were obtained to assess micronucleus in hepatocytes (MNHEP), proliferation (mitotic index), and glutathione (GSH/GSSG) levels. The fragment of liver designated for the evaluation of genotoxicity and proliferation was placed in 7 mL of 10% buffered formalin, and the fragment for quantification of GSH/GSSG was frozen in dry ice. Half of the liver from each animal was homogenized in 0.1 M phosphate buffer pH 7.0, with 0.1% Triton X-100, and centrifuged at 19,000 g for 10 min; the supernatant was used for the determination of the following antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GRed). The remaining liver was stored at −80°C until preparation of microsomal and cytosolic fractions (for no more than 2 weeks). Additionally, both femurs were removed to assess micronucleus in bone marrow polychromatic erythrocytes (MNPCE) and cytotoxicity (%PCE) in bone marrow.
Microsomal and cytosolic fractions were prepared according to the procedure described by Guengerich [
Protein concentrations in the microsomal and cytosolic fractions were determined using the protein assay dye reagent (Bio-Rad) according to supplier’s instructions.
The cytochrome P-450 (P-450) content in the hepatic microsomal fraction was determined from the spectrum of the ferrous-carbon monoxide complex, using the molar extinction coefficient of 91 mM−1 cm−1 at 450/490 nm [
CYP2E1 enzyme activity was determined by measuring the hydroxylation of 4-nitrophenol (4-NP) to 4-nitrocatechol (4-NCC) as described by Koop [
Total GST activity was measured using the method described by Habig and Jakoby [
The glutathione-
Frozen liver samples were homogenized in 5 mL of 5-sulfosalicylic acid/g of tissue, using sonication (30 sec, 4.5 intensity, 4°C). The homogenates were centrifuged at 15,000 ×g for 3 min at room temperature and the acid supernatants were recovered.
Total glutathione was quantified in the acid supernatants using the enzymatic recycling assay of Anderson [
Quantification of oxidized glutathione (GSSG) was performed by derivatization of the reduced glutathione (GSH) present in the sample with 2-vinylpyridine prior to the enzymatic recycling assay, thus preventing GSH from participating in the reaction. Derivatization reaction contained 300
The amount of GSH in the sample was calculated by subtracting the amount of GSSG from the amount of total glutathione. The results were expressed in nmol GSH or GSSG/g liver.
TBARS were quantified using the method of Janero and Burghardt [
CAT activity was determined following the enzymatic decomposition of H2O2 [
SOD activity was measured by a competitive inhibition assay using xanthine-xanthine oxidase system to reduce nitroblue tetrazolium (NBT) [
GPx activity was assayed by a coupled reaction with glutathione reductase (GRed) [
GRed activity was spectrophotometrically assayed using GSSG as substrate and measuring the disappearance of NADPH at 340 nm [
Evaluation of MNPCE was performed according to the procedure of Romagna and Staniforth [
Two smears were made per animal and slides were stained with undiluted Wright-Giemsa (Sigma). A total of 2,000 polychromatic erythrocytes (PCE) from each rat were evaluated for the micronucleus frequency. Additionally, BM cytotoxicity was evaluated by recording the %PCE present in 2,000 erythrocytes per animal.
Formalin-fixed tissue was used according to the method of Parton and Garriott [
Two smears were made per animal and slides were stained with undiluted Wright-Giemsa. A total of 2,000 hepatocytes with good morphology from each rat were evaluated for the micronucleus frequency.
In the same slides used for micronucleus determination, one thousand hepatocytes were counted per animal, enumerating the amount of mitotic figures. The mitotic index was calculated as the number of mitosis observed/one thousand cells observed.
All experiments described were done by triplicate and data were captured and analyzed using Stata 7.0 software. Values were expressed as mean ± s.d. and group comparisons were assessed using Kruskal-Wallis test. Pearson correlations were explored among data after one-day treatment or three-day treatments, and linear regression was used to analyze the correlations found. Differences between negative and positive controls were calculated by the Student’s
Effects on the parameters studied were clearly different between one-day and three-day treatments (Table
(a) Result of the one-day treatments. (b) Result of the three-day treatments.
Treatment | Phase I and phase II xenobiotic metabolism | ||||
---|---|---|---|---|---|
|
[CYP] (nmol/mg) | CYP2E1 (nmol/min/mg)** | GST (mmol/min/mg) | GSTT1 (nmol/min/mg)** | |
CT | 5 |
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Low Dose | 5 |
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Mid Dose | 5 |
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High Dose | 5 |
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Treatment | Antioxidant enzymes | ||||
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SOD† | GPx (U/mg) | Gred (U/mg) | ||
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CT | 5 |
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Low Dose | 5 |
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Mid Dose | 5 |
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High Dose | 5 |
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Treatment | Oxidative stress | ||||
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[GSH] (mmol/g) | [GSSG] (mmol/g) | [GSH]/[GSSG] | TBARS (nmol/mg) | |
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CT | 5 |
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Low Dose | 5 |
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Mid Dose | 5 |
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High Dose | 5 |
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Treatment | Genotoxicity and proliferation | ||||
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MNPCE ( |
%PCE | MNHEP/1000 | Metaphase ( |
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CT | 5 |
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Low Dose | 5 |
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Mid Dose | 5 |
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High Dose | 5 |
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Ben/DEN | 3 |
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Treatment | Phase I and phase II xenobiotic metabolism | ||||
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CYP (nmol/mg)** | CYP2E1 (nmol/min/mg)** | GST (mmol/min/mg) | GSTT1 (nmol/min/mg)** | |
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CT | 5 |
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Low Dose | 5 |
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Mid Dose | 5 |
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High Dose | 5 |
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tSBO | 3 |
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Treatment | Antioxidant enzymes | ||||
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SOD†** | GPx (U/mg)** | Gred (U/mg)** | CAT (k/mg) | |
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CT | 5 |
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Low Dose | 5 |
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Mid Dose | 5 |
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High Dose | 5 |
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CCl4 | 3 |
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Treatment | Oxidative stress | ||||
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[GSH] (mmol/g)** | [GSSG] (mmol/g)** | [GSH]/[GSSG]** | TBARS (nmol/mg)** | |
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CT | 5 |
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Low Dose | 5 |
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Mid Dose | 5 |
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High Dose | 5 |
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CCl4 | 3 |
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Treatment | Genotoxicity and proliferation | ||||
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MNPCE ( |
%PCE** | MNHEP/1000 | Metaphase ( |
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CT | 5 |
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Low Dose | 5 |
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Mid Dose | 5 |
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High Dose | 5 |
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Ben/DEN | 3 |
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Mean values plus standard deviations are presented for all the parameters.
*Positive controls significantly different from negative controls (Student
**Parameters where a difference due to the treatment was found at least in one dose. Kruskal-Wallis rank test,
†Relative units.
The simultaneous exposure to the three compounds at different concentrations after one-day treatment resulted in the increased activity of metabolic enzyme GSTT1 with each treatment (Pearson coefficient = 0.76;
Correlations found between parameters after one-day treatments.
Treatments | GSTT1 (nmol/min/mg) | TBARS (nmol/mg) | |
---|---|---|---|
GSTT1 (nmol/min/mg) | 0.76 |
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MNPCE ( |
0.57 |
0.49 |
|
MNHEP ( |
0.44 |
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CYP2E1 (nmol/min/mg) | 0.61 |
0.48 |
−0.48 |
Pearson coefficients.
*Close to significance.
One-day treatment. (a) GSTT1 metabolic enzyme showed increased activity with the dose. Boxes represent the first and the third quartile and the median value. The line behind the bars represents the overall mean for this enzyme activity. (b) GSTT1 induced activity correlated with MNPCE,
Micronuclei in PCE showed increased frequencies with the higher doses with a maximal increase of 2.7-fold and a Pearson correlation with the treatments with a coefficient value of 0.57 and
Increased MNHEP were observed in the liver related with the dose with a maximal increase of 11-fold which was close to significance (Pearson coefficient = 0.44;
A summary of the correlations found with this regime of treatment is presented in Table
Contrary to what was observed in the single-day treatment, three-day treatments with the mixture produced significant responses in the metabolic enzymes at the low dose. Total P-450 were induced in the low dose and then a significant reduction with the dose was observed (Kruskal-Wallis chi value 18.7,
Antioxidant enzyme GPx showed significantly decreased activity with the low and high dose (Kruskal-Wallis chi value 11.5,
Correlations found between parameters after three-day treatments.
Treatments | TBARS (nmol/mg) | GSTT1 (nmol/min/mg) | CYP2E1 (nmol/min/mg) | P-450 (nmol/mg) | CAT (k/mg) | |
---|---|---|---|---|---|---|
GRed (U/mg) | −0.61 |
−0.87 |
||||
P-450 (nmol/mg) | −0.48 |
0.62 |
||||
GSH/GSSG (ratio) | −0.51 |
0.55 |
0.55 |
0.68 |
||
TBARS (nmol/mg) | 0.82 |
|||||
SOD | −0.75 |
−0.72 |
0.56 |
|||
GSTT1 (nmol/min/mg) | 0.38 |
|||||
GPx (activity) | −0.55 |
Pearson coefficients.
Three-day treatment. TBARS were increased in a dose-related manner, probably as the result of the reduced activity of antioxidant enzymes like GRed which showed an inverse correlation with it (a),
The oxidative stress was not reflected in micronucleus production in the BM, whereas in the liver MNHEP were increased in the high dose (3-fold), although at a lower level than with the one-day treatment. A greater variability in %PCE was observed in the BM with significant reductions at the low and high doses (Kruskal-Wallis, chi value 8.6,
In order to gain insight into the relationship of metabolism, oxidative stress, and micronucleus production related with the coexposure to CLF, TOL, and DCM, our study considered two different regimes of exposure in a rat model: a single-day treatment and a three-day treatment (one dose/day). The two regimes produced a different pattern of response (Table
No change in the antioxidant response was observed under the single-day treatment and oxidative stress biomarkers such as TBARS and the GSH/GSSG ratio were not altered. Phase I and phase II enzymatic activity, in turn, exhibited induction; that is, CYP2E1 and GSTT1 activities were induced (Table
Diagram representing how the combined metabolism of the three compounds could induce the responses observed. After one-day treatments, GSTT1 and CYP2E1 induction could contribute to the generation of metabolites and ROS to produce increased MNPCE. After three-day treatments, still induced CYP2E1 and reduced activity of antioxidant enzymes (empty arrows) lead to the accumulation of H2O2 and the superoxide anion, damaging membranes and allowing an oxidative stress.
MNPCE showed an increase related with the dose and interestingly, they were correlated with GSTT1 activity. This was the only parameter measured in the liver that showed a correlation with BM MNPCE, which could be explained in two possible ways: (1) The exposure to the mixture of pollutants could induce GSTT1 activity in the erythroid line, this process could increase bioactivation of DCM on the bone marrow, leading to genotoxicity [
The three-day regime exerted a more intensive oxidative effect than the single-day treatment, reducing the activity of the antioxidant enzyme GPx (which reduces H2O2 into water), inducing CYP2E1 (whose activity generates H2O2 and superoxide anion), and producing damage to lipids (TBARS) in all doses, which was inversely correlated with GRed (which has the function of recovering GSH from GSSG and making it available to protect the cell from oxidation) and SOD (which conjugates superoxide anion) activities. The ratio GSH/GSSG was first induced and then decreased with increasing doses in a similar manner as CYP2E1; these two parameters showed a significant correlation and a similar correlation was found with induced GSTT1 (Table
Previous studies showed little evidence of oxidative stress produced by the individual compounds when administered
Our results are comparable to those obtained by Bird et al. [
P-450 levels under the three-day treatment were significantly induced in the low dose, but then they decreased with the dose, being significantly reduced only in the high-dose treatment (Table
CYP2E1 activity, in turn, was significantly increased at the low and mid-doses. When individually administered in the pilot study, CLF and TOL also produced an induction (Table
In relation with the type of interaction of the three compounds, it depends on the doses used and on the biomarkers taken as a reference. Based on previous studies (Table
Given that an oxidative stress was induced with the treatments, an increase in MN was expected either in the BM or in the liver. Individual compounds had been analyzed for their genotoxicity and the results were inconsistent, indicating some clastogenic activity for the three compounds but not in every test [
Micronuclei in hepatocytes had not been reported before for these compounds. They are weakly genotoxic to the BM and according to our results their genotoxicity in a coexposure would depend on the induction of metabolic enzymes like GSTT1 and hence to the production of genotoxic metabolites. The observed effect on hepatocytes was 5- to 11-fold higher than in controls after only one-day treatment; however, proliferation in the liver was reduced at the same time. Hepatic proliferation is considerably lower than in the BM and the proliferation of cells is necessary for the formation of MN, and even though young rats were used and an increase with the dose was found (Table
The toxic effects of xenobiotics depend on the dose and on the time of exposure. In the present study, three doses of the mixture under two types of xenobiotic exposure regimen (single and repeated doses) were tested. This type of experimental design was useful for understanding the toxicological behavior of the mixture of VOCs in different scenarios. The single exposure regime was used to evaluate the first responses of the organism when exposed to a mixture of VOCs, while the regimen of repeated doses was used to assess the accumulation of damage.
The single dose protocol revealed that the biomarkers of oxidative stress and cytotoxicity were maintained at normal levels, whereas the biomarkers of xenobiotic metabolism and genotoxicity increased with the dose. These results can be interpreted as follows: with a single dose, defense systems are not exceeded and are able to maintain cell homeostasis; however the organism is able to sense the presence of xenobiotics and activates the metabolism to accelerate detoxification; the increase in metabolism also increases the levels of reactive metabolites and biomolecular damage (micronuclei) is more probable.
With the repeated dose protocol, biomarkers of oxidative stress, membrane damage, cytotoxicity, and xenobiotic metabolism were increased with the dose, whereas genotoxicity was decreased. This result indicates that damage to macromolecules accumulated and the defense system was completely exceeded, leading to cell injury or death. Since the formation of micronuclei depends on cell proliferation, cell arrest or cytotoxicity would explain the decrease in micronuclei frequency. These results are in contrast to what was found with individual compounds in the pilot study which did not induce genotoxicity, cytotoxicity, or oxidative stress; however, in a coexposure like this, the outcome was synergistic and even overpassed the antioxidant defense of the organism causing visible liver damage comparable to what has been described about alcohol-dependent liver injury [
In summary, the use of two exposure regimes allowed us to propose scenarios where the cellular response is sufficient to maintain the viability even if sustaining a genotoxic effect that could translate in subtle alterations on the long term, or when the response is completely exceeded, compromising cellular integrity that could lead to tissue illness in a short period of time.
The coexposure to CLF, DCM, and TOL induced the activity of metabolism enzyme GSTT1 and it was correlated with the micronucleus frequency in the bone marrow, after only one treatment. Even though the micronuclei induction was not as high as it is with benzene or other well-established clastogenic agents, these lesions could be of relevance in a prolonged exposure regime or in a combined exposure with a clastogenic agent which is possible in a polluted environment scenario. At the same time ROS could have been produced by the induced activity of CYP2E1, generating genotoxicity, but in levels not affecting the activity of antioxidant enzymes or GSH levels, opening the possibility that a lower and sustained exposure over time could produce significant chromosomal damage in both tissues. Future experiments would help dilucidate this matter.
Sustained exposure for three days under this regime led to oxidative stress at all doses without affecting the survival of the animals but producing fat liver.
The authors declare that they have no conflict of interests.
The authors express their gratitude to Pedro Medina for his technical assistance in the preparation and staining of hepatocytes and to Bernardino Huerta for his technical assistance in the determination of TBARS and antioxidant enzymes. This work was supported by Consejo Nacional de Ciencia y Tecnología (CONACYT) Grant no.