Evaluation of the effect of 2,4-dichlorophenol on oxidative parameters and viability of human blood mononuclear cells (in vitro)

Abstract

2,4-Dichlorophenol (2,4-DCP) is formed in drinking water as a result of its chlorination, and it is created in the environment during transformation of various xenobiotics such as triclosan or herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The molecular mechanism depicting the action of phenolic compounds on nucleated blood cells has been insufficiently studied, and therefore, we have assessed the effect of 2,4-DCP on the structure and viability of human peripheral blood mononuclear cells (PBMCs). We have evaluated necrotic, apoptotic, and morphological changes (alterations in the size and granulation) in PBMCs incubated with 2,4-DCP in the concentration ranging from 10 to 500 µg mL⁻¹ for 4 h at 37°C. Moreover, we have estimated changes in reactive oxygen species (ROS) formation, lipid peroxidation, and protein carbonylation in the incubated cells. We have noted that 2,4-DCP increased ROS formation and lipid peroxidation (from 10 µg mL⁻¹) and oxidized proteins (from 50 µg mL⁻¹) in PBMCs. The compound studied also provoked apoptotic (from 50 µg mL⁻¹), necrotic (from 100 µg mL⁻¹) and alterations in the size and granulation (from 50 µg mL⁻¹) in the incubated cells. The analysis of quinolinium 4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(trimethyl-ammonio)-propyl]-diiodide/propidium iodide staining revealed that 2,4-DCP (50–250 µg mL⁻¹) more strongly increased the number of apoptotic than necrotic cells, which suggests that this cell death type is mainly provoked by this compound in PBMCs. The observed changes were caused by relatively high concentrations of 2,4-DCP, which cannot influence human organism during environmental exposure and thus may only occur as a result of acute or subacute poisoning with this compound.

Keywords

Apoptosis 2 4-DCP 4-D peripheral blood mononuclear cells cell morphology oxidative stress necrosis protein carbonylation lipid peroxidation reactive oxygen species

Introduction

2,4-Dichlorophenol (2,4-DCP) is formed in drinking water as a result of its chlorination.¹ The 2,4-DCP may also be formed in the environment during transformation of various pesticides, mainly phenoxyherbicides, such as 2,4-dichlorophenoxyacetic acid (2,4-D),^1,2 as well as the antibacterial and antifungal agent triclosan.³ Chlorophenols are also biosynthesized by anaerobic microorganisms as a result of transformation of biogenic chlorinated hydroquinone metabolite.⁴ 2,4-DCP is also formed as a major metabolite of 2,4-D in animals. Moreover, 2,4-DCP is used as a precursor for the synthesis of 2,4-D, and it is also the main contaminant in the preparation of this herbicide.⁵

Due to widespread occurrence of 2,4-DCP in the environment, and thus in human organism, the mechanism of action of this substance is still being investigated in vitro and in vivo.^6
–8 Up to now, it has been shown that phenolic compounds including 2,4-DCP may reveal genotoxic, mutagenic, and carcinogenic activities.^9,10

The presence of chlorophenols in blood and urine of the general population has been proven in some studies. For instance, in the population of the United States, chlorophenols have been detected in the concentrations ranging from 3 to 65 µg L⁻¹ of blood serum.¹¹ Other studies have shown that workers permanently exposed to chlorophenols had high urinary levels of these substances. For instance, workers employed in sawmills, where chlorophenols were used to prevent fungal growth of lumber had high concentrations of these compounds (from 196 to 2320 µg L⁻¹) in blood and urine.¹² In another study, it was observed that workers permanently exposed to pentachlorophenol (PCP) had enormous PCP amounts up to 84.9 mg L⁻¹ in blood serum.¹³

Peripheral blood mononuclear cells (PBMCs, mainly lymphocytes) are very important cells of the immune system because they are responsible for the organism’s defense against pathogens as well as maintaining homeostasis in the body, having the so-called function immunological supervision.¹⁴ Moreover, PBMCs are potentially exposed to numerous toxicants entering human organism.^10,15 Nutrients, drugs, and toxic compounds are transported by blood in living organisms, which leads to numerous interactions between various xenobiotics and PBMCs, and thus changes in structure and functionality of these cells. Therefore, PMBCs have been repeatedly used as model cells in the investigation of xenobiotics toxicity on nucleated cells.^16
–18

Up to now, insufficient information has been obtained concerning the mechanism of phenolic compounds’ action on nucleated blood cells, and no study has been conducted to analyze the effect of 2,4-DCP on PBMCs. Therefore, the purpose of this study was to assess the effect of 2,4-DCP on necrotic, apoptotic, and morphological (size and granulation) alterations in PMBCs. Moreover, the effect of 2,4-DCP on lipid and protein damage as well as free radical formation in the cells studied was assessed. The 2,4-DCP was used in the concentrations that can enter human organism as a result of occupational exposure or acute (accidental) poisoning with this compound.

Experimental

Chemicals

2,4-DCP, Histopaque, Roswell Park Memorial Institute (RPMI) medium with l-glutamine, calcein-AM, propidium iodide (PI), valinomycine, and camptothecin were obtained from Sigma (St Louis, Missouri, USA). Quinolinium 4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(trimethyl-ammonio)-propyl]-diiodide (YO-PRO-1) and 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) were bought from Molecular Probes (Eugene, Oregon, USA). All other chemicals were from POCh (Poland) and were of analytical grade.

Human PBMCs isolation

Human PBMCs were obtained from leukocyte–platelet buffy coat taken from healthy donors in the Blood Bank of Łódź, Poland. The cells were isolated using Histopaque gradient by centrifugation at 1500 r min⁻¹ for 30 min at 20°C. The cells were suspended in RPMI medium with l-glutamine, centrifuged, and washed with phosphate-buffered saline (PBS) without calcium and magnesium at 1500 r min⁻¹ for 15 min at 4°C and then resuspended in RPMI medium with l-glutamine. The cells were counted in a hemocytometer. The final PBMCs density (with 2,4-DCP) used in the experiments was adjusted to 1 × 10⁶ cells mL⁻¹ by adding RPMI with l-glutamine to the cells suspension. The 2,4-DCP was dissolved in PBS (pH 7.4). The cells that were incubated only with PBS were used as controls. The cells were incubated with 2,4-DCP in the final concentrations ranging from 10 to 500 µg mL⁻¹ for 4 h at 37°C in total darkness.

Analytical methods

Cell viability

Cell viability was quantified by measuring acetomethyl derivative of calcein (calcein-AM; evaluation of viable cells) and PI (evaluation of necrotic cells) entering PBMCs.^19,20

Calcein-AM penetrates the cells that maintain plasma membrane integrity and accumulates in the cytoplasm, while PI penetrates cells in which membrane lost its integrity and binds to DNA.

After incubation, the samples were centrifuged at 1000 r min⁻¹ for 5 min at 4°C, the supernatant was discarded, and the cells were supplemented with RPMI. Then, the markers were added, and the samples were incubated for 15 min at 37°C in total darkness. The final concentration of calcein-AM and PI was 1 µM and 0.1 µM, respectively. The flow cytometry (FMC) gate on PBMCs has been established for data acquisition, and the fluorescence was measured with excitation/emission of 494/517 nm for calcein and 535/617 nm for PI, respectively.

Measurement of cell morphology

Cell morphology was analyzed using a flow cytometer (LSR II; Becton Dickinson, East Rutherford, New Jersey, USA). The FMC gate on PBMCs has been established for data acquisition, and cell size and granularity were evaluated with simultaneous separate detection of low-angle (forward scatter pulse area (FSC-A)) and right-angle (side scatter pulse area (SSC-A)) light scattering. The data obtained were displayed in the form of a diagram of cell number versus light scatter and were analyzed using the standard computer program WinMDI2.8. The light scattered near the forward direction (low angle) is expected to be proportional to particle size (volume), whereas scattering at the right angle depends on cell granulation (internal properties of the scattered particles).²¹

Analysis of apoptosis

Apoptosis was quantified using the fluorescence probes YO-PRO-1 (quantification of apoptotic cells) and PI (quantification of necrotic cells).²² Living cells exhibited a low level of green fluorescence, apoptotic cells exhibited incrementally higher level of green fluorescence, and necrotic cells revealed both red and green fluorescence. Percentage of apoptotic cells was defined as the percent of YO-PRO-1 stained cells within the cell population of interest. The positive control contained the cells in which the apoptosis was induced with 20 µM of camptothecin. The samples were incubated for 4 h at 37°C in total darkness. Then, the mixture of YO-PRO-1 and PI (0.1 µM each) was added to the samples. After incubation for 20 min on ice in total darkness, the samples were analyzed by flow cytometry (LSRII; Becton Dickinson) with excitation at 488 nm to visualize the YO-PRO-1 green fluorescence (520/30 bandpass filter) and PI red fluorescence (610/20 bandpass filter).

Hoechst 33342/PI staining

Morphological changes in PBMCs treated with 2,4-DCP were assessed by double staining with Hoechst 33342 and PI. The cells were treated with 2,4--DCP and incubated for 4 h at 37°C in total darkness. After incubation, the cells were centrifuged at 200g for 3 min at 4°C, and then the supernatant was discarded. The cells were suspended in 0.5 mL PBS, and then the mixture of 1 µL of Hoechst 33342 and 1 µL of PI (1 mg mL⁻¹ each) was added to cells suspension. After 1 min incubation at 37°C in total darkness, the cells were analyzed by fluorescence microscopy (Olympus IX70, Japan) at 400× magnification. The cells were classified on the basis of their morphological staining characteristics: viable (blue fluorescence), early apoptotic (intensive bright blue fluorescence), late apoptotic (blue–violet fluorescence), and necrotic (red fluorescence).²³

The oxidation of H₂DCFDA

The rate of H₂DCFDA oxidation was measured by flow cytometry. This fluorescence probe is widely used for the detection of intracellular oxidants production.²⁴ Bartosz suggested that 2′,7′-dichlorodihydrofluorescein was originally introduced to detect “ultramicro quantities” of hydroperoxides, that is, hydrogen peroxide. The samples were incubated for 4 h at 37°C in total darkness. Then, fluorescence probe was added to the samples, which were incubated for 15 min at 37°C in total darkness. The final concentration of the probe was 10 µM. FMC gate on the PBMCs has been established for data acquisition, and fluorescence was measured by flow cytometer (LSR II, Becton Dickinson) with the excitation and emission maxima of 488 and 530 nm, respectively.

Lipid peroxidation

Lipid peroxidation is associated with oxidative damage to lipids by xenobiotics and their transformation products, that is, organic radicals and reactive oxygen species (ROS). Evaluation of lipid peroxidation in PBMCs was conducted by measuring cis-parinaric acid (PnAC) fluorescence at excitation/emission of 320/432 nm. The method is based on initial incubation of the cells with PnAC, which leads to the incorporation of this substance into cellular membrane. Then, the cells are incubated with individual xenobiotic. Damage to lipids results in a decrease of PnAC fluorescence. The analysis was performed in 96-well plates using a microplate reader (Cary Eclipse, Varian, Palo Alto, California, USA).²⁵

Protein carbonylation

Protein carbonylation was determined spectrophotometrically by the method of Levine et al.²⁶ 2,4-Dinitrophenylhydrazine was used to determine the concentration of carbonyl groups in PBMCs. As a result of the reaction, a yellow product, hydrazine, was formed. The content of carbonyl groups was calculated from the absorbance measurement of the sample at 370 nm, and the absorption coefficient for hydrazone was e = 21.01 mmol⁻¹ cm⁻¹.

2.4. Statistical analysis

Data are expressed as mean ± SD. Multiple comparisons among group mean differences were analyzed by one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. Differences were considered to be statistically significant when p value was less than 0.05. All statistical analyses were done using STATISTICA software (StatSoft Inc., Tulusa, Oklahoma, USA). The mean value was calculated from three to five individual experiments, (3–5 blood donors), whereas for each individual an experimental point was a mean value of three replications.

Results

Cells viability and morphology

It was observed that 2,4-DCP in the concentrations of 250 µg mL⁻¹ and 500 µg mL⁻¹ induced statistically significant decrease in PBMCs viability (F5;77 = 72.80; p < 0.001; Figure 1). The 2,4-DCP at the highest concentration of 500 µg mL⁻¹ decreased cell viability up to 58.32 ± 12.74%.

Figure 1.

Changes in the viability of human blood mononuclear cells exposed to 2,4-DCP in the concentrations ranging from 10 to 500 µg mL⁻¹. *Significant differences compared with controls (one-way ANOVA and a posteriori Tukey test). 2,4-DCP: 2,4-dichlorophenol; ANOVA: analysis of variance.

The analysis of FSC-A parameter allowed obtaining information about the size of PBMCs (Figure 2). It was observed that 2,4-DCP at 50 and 100 µg mL⁻¹ caused statistically significant increase in PBMCs size, but in the highest concentration of 500 µg mL⁻¹, it induced statistically significant decrease in FSC parameter (F(5, 98) = 98.10; p < 0.001).

Figure 2.

Alterations (expressed as %) in the size (FSC-A) and granulation (SSC-A) of human blood mononuclear cells (exposed to 2,4-DCP at concentrations ranging from 10 to 500 µg mL⁻¹. *Significant differences compared with controls (one-way ANOVA and a posteriori Tukey test). 2,4-DCP: 2,4-dichlorophenol; ANOVA: analysis of variance; FSC-A: forward scatter pulse area; SSC-A: side scatter pulse area.

The SSC-A parameter provided the information about granulation of PBMCs (Figure 2). A statistically significant increase in SSC parameter was noted after incubation of the cells with 2,4-DCP in the concentrations ranging from 50 to 500 µg mL⁻¹ (F(5, 93) = 25.21; p < 0.001).

Apoptotic changes, photos of cells

The 2,4-DCP induced apoptosis of PBMCs, which was associated with changes in cell membrane permeability, and thus penetration of YO-PRO-1 probe into the cells (Figure 3(a)). The exemplary dot plots illustrating necrotic and apoptotic PBMCs measured by flow cytometry are shown in Figure 3(b). The 2,4-DCP in the concentration of 50 µg mL⁻¹ provoked a strong increase in the number of apoptotic cells (F(4, 55) = 110; p < 0.001) but in the concentration of 100 µg mL⁻¹ provoked statistically significant increase in the number of necrotic cells (F(4, 55) = 42.81; p < 0.001).

Figure 3.

(a) Changes in the percentage of apoptotic and necrotic cells exposed to 2,4-DCP at concentrations ranging from 10 to 250 µg mL⁻¹. *Significant differences compared with controls. (b) Quantification of viable (quadrant Q3), apoptotic (quadrant Q4), and necrotic (quadrant Q2) mononuclear blood cells after 2,4-DCP treatment. The analysis was performed by flow cytometry using YO-PRO-1 and PI stains. Control cells (1), cells treated with 2,4-DCP at 50 μg mL⁻¹ (2), cells treated with 2,4-DCP at 250 μg mL⁻¹ (3). 2,4-DCP: 2,4-dichlorophenol; YO-PRO-1: quinolinium 4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(trimethyl-ammonio)-propyl]-diiodide; PI: propidium iodide.

Figure 4 shows representative photomicrographs of Hoechst 33324/PI-stained PBMCs pretreated with PBS (control) and 2,4-DCP at 10, 50, and 250 µg mL⁻¹. In control sample, mainly viable cells were observed. In samples treated with 2,4-DCP at 10 µg mL⁻¹ mostly viable and a single apoptotic cells were noticed. In samples incubated with 2,4-DCP at 50 µg mL⁻¹, several viable, a single necrotic and a few apoptotic cells appeared, while at 250 µg mL⁻¹, mostly apoptotic and a single necrotic cell were observed.

Figure 4.

The representative photomicrographs of Hoechst 33324/PI-stained PBMCs pretreated with PBS (control) and 2,4-DCP at 10, 50, and 250 µg mL⁻¹. Viable (blue fluorescence), early apoptotic (intensive bright blue fluorescence), late apoptotic (blue-violet fluorescence) and necrotic (red fluorescence). PI: propidium iodide; PBMC: peripheral blood mononuclear cell; PBS: phosphate-buffered saline; 2,4-DCP: 2,4-dichlorophenol.

The level of ROS, lipid peroxidation and protein carbonylation

It was observed that 2,4-DCP in the concentrations ranging from 50 µg mL⁻¹ to 500 µg mL⁻¹ induced a statistically significant increase in H₂DCFDA oxidation in the cells studied (F(5, 51) = 36.78; p < 0.001) (Figure 5(a)). The exemplary dot plots obtained by flow cytometry are shown in Figure 5(b).

Figure 5.

(a) Oxidation of H₂DCFDA in control human blood mononuclear cells exposed to 2,4-DCP in the concentrations ranging from 2 to 500 µg mL⁻¹. *Significant differences compared with controls. (b) Histogram of changes in the intracellular ROS levels in human blood mononuclear cells after 2,4-DCP treatment; control and treated cells were stained with H₂DCFDA; the fluorescence of the stain intensified in chlorophenol-treated cells. Count (relative cells number)/fluorescence of DCF. H₂DCFDA: 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate; 2,4-DCP: 2,4-dichlorophenol; ROS: reactive oxygen species; 2,4-DCP: 2,4-dichlorophenol; H₂DCFDA: 6-carboxy-2’,7’-dichlorodihydrofluorescein diacetate; DCF: 2′,7′-dichlorofluorescein.

We observed that 2,4-DCP at 10 µg mL⁻¹ strongly induced lipid peroxidation (F(5, 121) = 224.87; p < 0.001) in the incubated cells (Table 1).

Table 1.

Lipid peroxidation and protein carbonylation in control cells and cells exposed to 2,4-DCP in the concentrations ranging from 10 to 500 µg mL⁻¹.

Concentration (µg mL⁻¹)	Fluorescence of PnAC (%)	Carbonyl groups (%)
0	100	100
10	81.1 ± 14.8^a	106.1 ± 21.7
50	58.4 ± 11.8^a	121.2 ± 26.5^a
100	48.4 ± 11.9^a	140.9 ± 23.0^a
250	23.7 ± 7.4^a	137.1 ± 25.1^a
500	15.6 ± 6.5^a	—
ANOVA and A posteriori Tukey test	F(5, 121) = 224.87; p < 0.001	F(4, 61) = 9.01; p < 0.001

2,4-DCP: 2,4-dichlorophenol; ANOVA: analysis of variance; PnAC: cis-parinaric acid.

^aSignificant differences compared with controls (one-way ANOVA and a posteriori Tukey test).

The measurement of carbonyl group content proved that 2,4-DCP from 50 µg mL⁻¹ caused statistically significant damage to proteins in human PBMCs (2,4-DCP; F(4, 61) = 9.01; p < 0.001; Table 1).

Discussion

Phenolic compounds have been proven to provoke oxidative changes in living cells.^27

–32 In this study, fluorescent probe H₂DCFDA was used to detect oxidative potential of 2,4-DCP in human PBMCs. It was shown that 2,4-DCP at 10 µg mL⁻¹ enhanced oxidation of the probe (Table 1). Those findings are in agreement with our previous results obtained on human erythrocytes, which showed that 2,4-DCP significantly increased ROS level in this cell type.⁷

It is known that various ROS (hydroxyl radical, hydrogen peroxide, etc.) may induce lipid peroxidation. Lipid peroxidation can be defined as the oxidative deterioration of lipids containing any number of carbon–carbon double bonds. Destruction of membrane lipids induces alteration of its integrity, fluidity, and permeability as well as causes functional loss of biomembranes and modification of low-density lipoprotein (LDL) to proatherogenic and proinflammatory forms.³³

We found that 2,4-DCP at 10 µg mL⁻¹ strongly induced lipid peroxidation (Table 1). The effect of 2,4-DCP on lipid peroxidation in mammalian model was observed by Clerhata et al.³⁴ who administrated this compound to guinea pigs. They observed that 2,4-DCP significantly increased lipid peroxidation in the liver of the animals studied. Similarly, Luo et al.³⁵ noticed that 2,4-DCP increased the content of malondialdehyde (the marker of cellular lipid damage) in liver of Carassius auratus. Moreover, Duchnowicz et al., observed that chlorinated aromatics induced lipid peroxidation in human erythrocytes. They suggested that it may be an additive effect of primary processes, that is, direct generation of free radicals by these xenobiotics and secondary processes like generation of ROS by oxyhemoglobin released during hemolysis of the erythrocytes.³¹

Protein damage is associated with oxidation of some amino acids residues (e.g. lysine, arginine, and proline) and oxidative cleavage of the peptide backbone by way of the α-amidation pathway, which results in carbonyl derivative formation.^36,37 Our research shows much stronger oxidation of lipids than proteins by 2,4-DCP (Table 1). 2,4-DCP at 50 µg mL⁻¹ decreased fluorescence of PnAC by 19%, while statistically significant changes in the oxidation of proteins occurred from the concentration of 50 µg mL⁻¹ of this compound. 2,4-DCP at 250 µg mL⁻¹ increased lipid peroxidation in PBMCs by 76%, while it depleted carbonyl groups level only by 37%. The oxidation of proteins is likely to be a secondary effect of 2,4-DCP action, which is a consequence of lipid peroxidation and increased ROS level induced by this chlorophenol.

Cellular membranes are efficient barriers between the intracellular and extracellular environment; thus, they have to be overcome by toxic compounds entering the cell. As the consequence, morphological changes in permeability and fluidity of those compartments occur.³⁸

Changes in membrane structure and organization can be analyzed by flow cytometry that is cell-by-cell qualitative and quantitative measurement technique, in which free cells in suspension are assessed for cytosolic or surface constituents.³⁹

In this study, human PBMCs morphology was determined based on cell size and granulation as measured by FSC and SSC light scattering characteristics.

The results showed that 2,4-DCP changed PBMCs morphology. It was observed that 2,4-DCP at lower concentrations (up to 50 µg mL⁻¹) increased SSC and FSC values, while at its highest concentration of 500 µg mL⁻¹ it decreased cell size and increased cell granulation. The observed changes in cell morphology might have been associated with significant cytotoxic effect of 2,4-DCP on PBMCs (Figure 1). It may be supposed that at the beginning, the cell was swelling, which resulted in an increase of its size, followed by the occurrence of cell shrinking, which led to a decrease of the PBMCs size. Simultaneously, gradual increase in PBMCs granulation was observed.

Bukowska et al.⁴⁰ noticed that chlorophenols were capable of interacting with proteins and lipids near the surface of the cellular membrane and between the lipid chains of membrane hydrophobic core. They also observed that chlorophenols induced changes in the content of erythrocyte membrane proteins including spectrin, band 3 and 6 proteins as well as low–molecular-weight proteins. In another work, Marczak et al. proved that each change in the content of spectrin altered cell shape, thereby decreased cellular deformability and cell survival.⁴¹

It must also be remembered that hydrophobicity and ionization of each substance, which is expressed by their log P and pK _a values, determine its behavior in the cellular environment.^42,43 It was proven that the increase in log P value and a decrease in pK _a value enhance toxicity of weak acids like phenolic compounds. 2,4-DCP has relatively high log P _o/w value of 3.23 and moderate pK _a value of 7.81.

Changes in cell morphology induced by chlorophenols were also observed by other scientists. Biberstein and Braunbeck observed that 3,5-dichlorophenol (3,5-DCP) caused alterations in shape of fibrocyte-like fish cell line (RTG-2) from rainbow trout.⁴⁴ They noticed that 3,5-DCP provoked the appearance of surface blebs as well as progressive retraction of extensions of the cells studied. In another study, Bukowska observed that 2,4,5-trichlorophenol (2,4,5-TCP) caused significant morphological changes in human erythrocytes.⁴⁵

Changes in cell size are often associated with apoptotic alterations, in which cytoplasmic condensation occurs with simultaneous maintenance of cell membrane integrity. Moreover, alterations in cell granulation (observed in our work) that are associated with degradation of nucleus and other organelles may be characteristic feature of apoptotic cell death. Apoptosis is an essential event in the organism development and homeostasis. Apoptosis is characterized by various biochemical changes such as increase in cell membrane permeability and the occurrence of number of corrugations on the cell surface. In order to analyze apoptosis, we used YO-PRO-1 dye, which enters the cells with disturbed membrane permeability characteristic for this process. It was found that 2,4-DCP increased the number of apoptotic PBMCs (from 50 µg mL⁻¹) (Figure 4(b)).

The studies of Bironaite et al. showed that phenol was capable of inducing apoptosis in HL-60 cells and human bone marrow endothelial cells.⁸ Other study, conducted by Jiang et al., revealed apoptotic alterations in fibroblast L929 and human liver cancer HepG2 cells incubated with chlorophenols.⁴⁶ Recently, Li et al. observed that 2,4-DCP induced apoptosis in primary hepatocytes of grass carp by mitochondrial pathway.⁴⁷

Based on the obtained data, we can suggest the following order of toxicity of 2,4-DCP in PBMCs: increase in ROS level and lipid peroxidation (10 µg mL⁻¹), protein carbonylation, apoptotic alterations and decrease in cell size, an increase in cell granulation (50 µg mL⁻¹), and finally an increase of necrotic changes (100 µg mL⁻¹). In summary, our results indicate that 2,4-DCP toxicity in PBMCs is associated with significant oxidative properties of this compound, which are responsible for cell death mostly through the induction of apoptosis.

The observed changes were induced by relatively high concentrations of 2,4-DCP, which cannot influence human organism during environmental exposure. Nevertheless, similar chlorophenols concentration has been determined in the blood serum of workers poisoned with these substances,⁴⁸ which may suggest that some toxic effects such as ROS formation or damage to lipids (observed in our study) may occur during acute or subacute exposure to 2,4-DCP.

Footnotes

Acknowledgement

We are thankful to Professor Aneta Koceva-Chyła (Department of Termobiology) for allowing us to work on the fluorescence microscope (Olympus IX70, Japan).

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was granted from the Research 505/387/R admitted for Department of Biophysics of Environmental Pollution University of Lodz.

References

Michalowicz

Stufka-Olczyk

Milczarek

. Analysis of annual fluctuations in the content of phenol, chlorophenols and their derivatives in chlorinated drinking waters. Environ Sci Pollut Res 2011; 18: 1174–1183.

Garabrant

Philibert

. Review of 2,4-dichlorophenoxyacetic acid (2,4-D) epidemiology and toxicology. Crit Rev Toxicol 2002; 32: 233–257.

Latch

Packer

Stender

. Aqueous photochemistry of triclosan: formation of 2,4-dichlorophenol, 2,8-dichlorodibenzo-p-dioxin, and oligomerization products. Environ Toxicol Chem 2005; 24(3): 517–525.

Milliken

Meier

Sowers

. Chlorophenol production by anaerobic microorganisms: transformation of a biogenic chlorinated hydroquinone metabolite. Appl Environ Microbiol 2004; 70(4): 2494–2496.

Różański

. Przemiany pestycydów w organizmach żywych i środowisku. Poznań: AGRA-ENVIRO LAB, 1998.

Heck

. A review and assessment of menthol employed as a cigarette flavoring ingredient. Food Chem Toxicol 2010; 48: 1–38.

Bukowska

Michałowicz

Krokosz

. Comparison of the effect of phenol and its derivatives on protein and free radical formation in human erythrocytes (in vitro). Blood Cells Mol Dis 2007; 39: 238–244.

Bironaite

Siegel

Moran

. Stimulation of endothelial IL-8 (eIL-8) production and apoptosis by phenolic metabolites of benzene in HL-60 cells and human bone marrow endothelial cells. Chem Biol Interact 2004; 139; 37–49.

Cavalieri

. Catechol ortho-quinones: the electrophilic compounds that form depurinating DNA adducts and could initiate cancer and other diseases. Carcinogenesis 2003; 23: 1071–1077.

10.

Michalowicz

Majsterek

. Chlorophenols, chlorocatechols and chloroguaiacols induce DNA base oxidation in human lymphocytes (in vitro). Toxicology 2010; 268: 171–175.

11.

Hill

Head

Baker

. Pesticides residues in urine of adults living in the United States: reference range concentrations. Environ Res 1995; 71: 99–108.

12.

Pekari

Luotamo

Jarisalo

. Urinary excretion of chlorinated phenols in saw-mill workers. Arch Occup Environ Health 1991; 63: 57–62.

13.

Cline

Hill

Philips

. Pentachlorophenol measurements in body fluids of people in log homes and workplaces. Arch Environ Health 1989; 18: 475–478.

14.

Kersey

Gajl-Peczalska

. T and B lymphocytes in humans. A review. Am J Pathol 1975; 81: 446–457.

15.

Feng

Kong

Wang

. Assessing the genotoxicity of imidacloprid and RH-5849 in human peripheral blood lymphocytes in vitro with comet assay and cytogenetic tests. Ecotoxicol Environ Saf 2005; 61(2): 239–246.

16.

Singh

Kumar

Nada

. Evaluation of copper toxicity in isolated human peripheral blood mononuclear cells and it’s attenuation by zinc: ex vivo. Mol Cell Biochem 2006; 282: 13–21.

17.

Nowak

Krokosz

Rodacka

. Study on the effect of polyhydroxylated fullerene, C₆₀(OH)₃₆, on X-ray irradiated human peripheral blood mononuclear cells. Radiat Phys Chem 2014; 97: 325–331.

18.

Blasiak

Kowalik

Małecka-Panas

. DNA damage and repair in human lymphocytes exposed to three anticancer platinum drugs. Teratog Carcinog Mutagen 2000; 20(3): 119–131.

19.

Papadopoulos

Dedoussis

Spanakos

. An improved fluorescence assay for determination of lymphocyte—mediated cytotoxicity using flow cytometry. J Immunol Methods 1994; 177: 101–109.

20.

Sędek

Mazur

. The application of monoclonal and policlonal antibodies in flow cytometry. Adv Cell Biol 1995; 35(24): 17–34.

21.

De Grooth

Terstapeen

Pupples

. Light-scattering polarization measurements as a new parameter in flow cytometry. Cytometry 1987; 8: 539–544.

22.

Idziorek

Estaquier

De Bels

. YOPRO-1 permits cytofluorometric analysis of programmed cell death (apoptosis) without interfering with cell viability. J Immunol Methods 1995; 185: 249–258.

23.

Rogalska

Gajek

Szwed

. The role of reactive oxygen species in WP 631-induced death of human ovarian cancer cells: a comparison with the effect of doxorubicin. Toxicol In Vitro 2011; 25:1712–1720.

24.

Bartosz

. Use of spectroscopic probes for detection of reactive oxygen species. Clin Chim Acta 2006; 368: 53–76.

25.

Miró

Alonso

Jarreta

. Smoking disturbs mitochondrial respiratory chain function and enhances lipid peroxidation on human circulating lymphocytes. Carcinogenesis 1990; 20(7): 1331–1336.

26.

Levine

Garland

Oliver

. Determination of carbonyl content in oxidatively modified proteins. Met Enzymol 1990; 186: 464–478.

27.

Zhang

, Qiu Q, et al. 2,4-Dichlorophenol induces apoptosis in primary hepatocytes of grass carp (Ctenopharyngodon idella) through mitochondrial pathway. Aquat Toxicol 2013; 140-141: 117–122.

28.

Bors

Michałowicz

Pilarski

. Studies of biological properties of Uncaria tomentosa extracts on human blood mononuclear cells. J Ethnopharmacol 2012; 142: 669–678.

29.

Luo

Sui

, Wang X, et al. 2-Chlorophenol induced hydroxyl radical production in mitochondria in Carassius auratus and oxidative stress—An electron paramagnetic resonance study. Chemosphere 2008; 71(7): 1260–1268.

30.

Han

Ichikawa

Utsumi

. Quantitative analysis for the enhancement of hydroxyl radical generation by phenols during ozonation of water. Water Res 1998; 11: 3261–3266.

31.

Duchnowicz

Koter

Duda

. Damage of erythrocyte by phenoxyacetic herbicides and their metabolites. Pest Biochem Physiol 2002; 74: 1–7.

32.

Bukowska

Chajdys

Duda

. Catalase activity in human erythrocytes: effect of phenoxyherbicides and their metabolites. Cell Biol Int 2000; 24(10): 705–711

33.

El-Beltagi

Mohamed

. Reactive oxygen species, lipid peroxidation and antioxidative defense mechanism. Not Bot Horti Agrobo 2013; 41(1): 44–57.

34.

Clerhata

Kováciková

Veningerová

. The effect of 2,4-dichlorophenol on lipid peroxidation in tissues of guinea pigs with different ascorbic acid intake. Ind Health 1996; 34(4): 415–419.

35.

Luo

Shi

Wang

. Free radical generation and lipid peroxidation induced by 2,4-dichlorophenol in liver of Carassius auratus . Huang Jing Ke Hue 2005; 26: 29–32.

36.

Uchida

Kato

Kawakishi

. A novel mechanism for oxidative damage of propyl peptides induced by hydroxyl radicals. Biochem Biophys Res Commun 1990; 169: 265–271.

37.

Garrison

. Reaction mechanisms of radiolysis of peptides, polypeptides, and proteins. Chem Rev 1987; 87: 381–398.

38.

Nath

. Membrane biology. Nature 2005; 438: 577.

39.

Webster

Bowles

Karim

. Flow cytometric analysis of peripheral blood lymphocyte subset light scatter characteristic as a means of monitoring the development of rat small bowel allograft rejection. Clin Exp Immunol 1995; 100: 536–542.

40.

Bukowska

Marczak

Michałowicz

. The effect of phenol, catechol, chloro- and methylphenol on the human erythrocyte membrane (in vitro). Pol J Environ Stud 2009; 18: 569–577.

41.

Marczak

Walczak

Jóźwiak

. The combined effect of IDA and glutaraldehyde on the erythrocyte membrane proteins. Int J Pharm 2007; 335: 154–162.

42.

Moridani

Siraki

Chevaldina

. Quantitative structure toxicity relationships for catechols in isolated rat hepatocytes. Chem Biol Interact 2004; 147: 297–307.

43.

Moridani

Siraki

O’Brien

. Quantitative structure toxicity relationships for phenols in isolated rat hepatocytes. Chem Biol Interact 2003; 145: 213–223.

44.

Bieberstein

Braunbeck

. Light scanning electron microscopic cytopathology of 3,5-dichlorophenol in the permanent fish cell line RTG-2. Ecotoxicol Environ Saf 1998; 41: 298–306.

45.

Bukowska

. 2,4,5-T and 2,4,5-TCP induce oxidative damage in human erythrocytes: the role of glutathione. Cell Biol Int 2004; 28(7): 557–563.

46.

Jiang

Chen

. Quantitative structure activity relationship and toxicity mechanisms of chlorophenols on cells in vitro. Chin Sci Bull 2004; 49: 562–566.

47.

Zhang

Qiu

. 2,4-Dichlorophenol induces apoptosis in primary hepatocytes of grass carp (Ctenopharyngodon idella) through mitochondrial pathway. Aquat Toxicol 2013; 141-142: 117–122.

48.

DHHS/CDC. Morbidity and mortality weekly report (MMWR) 49 23: 516–518, http://www.cdc.gov/mmwr/preview/mmwrhtml/mm4923a3.htm (2000)

Evaluation of the effect of 2,4-dichlorophenol on oxidative parameters and viability of human blood mononuclear cells (in vitro)

Abstract

Keywords

Introduction

Experimental

Chemicals

Human PBMCs isolation

Analytical methods

Cell viability

Measurement of cell morphology

Analysis of apoptosis

Hoechst 33342/PI staining

The oxidation of H2DCFDA

Lipid peroxidation

Protein carbonylation

2.4. Statistical analysis

Results

Cells viability and morphology

Apoptotic changes, photos of cells

The level of ROS, lipid peroxidation and protein carbonylation

Discussion

Footnotes

Acknowledgement

Declaration of Conflicting Interests

Funding

References

The oxidation of H₂DCFDA