A Battery of Tests for Ecotoxicological Evaluation of Pentachlorophenol on Common Carp

ABSTRACT

Pentachlorophenol (PCP) is a highly toxic contaminant of chlorophenols. Due to its slow and incomplete biodegradation, it can be found in surface, groundwater, and in soils. In this study, immunotoxicity, defensive toxicity, cytotoxicity, reproductive toxicity, and mitochondrial toxicity test of common carp exposed to PCP for 24 and 72 h were examined. The results showed that the lymphocyte and granulocyte counts were significantly decreased in comparison with the control groups under the higher PCP exposure (P <.05), no significant reduction in lymphocyte and granulocyte counts in the lower exposure groups compared with the control (P <.05). EROD activities after exposure for 24 and 72 h increased with PCP concentration increasing, and ethoxyresorufin O-deethylase (EROD) activities for 72-h treatment were higher than those of 24-h exposure in all treatment groups (P <.05). GSH activities after 24 and 72 h of exposure decreased with PCP concentration and incubation time. There was a significant increase in cellular MDA concentration after PCP treatments. And PCP induced increases in malondialdehyde (MDA) levels with incubation time. PCP-induced the increase in Δ ψ _m in liver mitochondrial compared to control levels when treated with PCP. The Δ ψ _m decreased significantly compared to control levels when liver mitochondria were treated with PCP (2.0, 4.0, 6.0 mg/L, 24 h) (P <.05). It presents a dose-dependent manner and the Δ ψ _m in common carps greatly decreased with the incubation time under the higher PCP exposure. The dose of PCP in lower concentration has no effect on serum testosterone in male common carp. But a significant decrease in serum testosterone concentration was observed in male common carps exposure to 2.0, 4.0, and 6.0 mg/L when compared to control within 24 and 72 h (P <.05). Relatively high concentrations of vitellogenin (VTG) were found in females exposed to a nominal concentrations of 0.1 and 1.0 mg/L PCP. VTG concentrations decreased within 24 and 72 h under high dose of PCP exposure in female common carp than those in control groups (P <.05).

KEYWORDS:

• common carp
• ecotoxicological evaluation
• pentachlorophenol

INTRODUCTION

Pentachlorophenol, a well-known pesticide, fungicide, and nonselective contact herbicide mainly used as wood preservative, was applied to check the performance of the proposed battery as a whole. Due to its proven carcinogenicity and toxicity and improper disposal, as well as the existence of a large number of known PCP-contaminated sites, PCP has become an environmental pollutant and is now considered to be ubiquitous.^{[ 1 ]} Acute and chronic poisoning may occur by dermal absorption, inhalation, or digestion. PCP has been designated as a “priority toxic pollutant” by the United States Environmental Protection Agency (EPA) under the guidance of the Clean Water Act in 1972. The general population may be exposed to PCP primarily through the ingestion of water and food.^{[ 2 , 3 ]} Previous studies indicate that PCP toxicity seems to be related to the uncoupling of oxidative phosphorylation in mitochondria, and the generation of reactive oxygen species (ROS) would be the principal mechanism of action.^{[ 4 ]} Moreover, cumulative data from in vivo and in vitro experiments have suggested that PCP is a promoter of carcinogenesis in rodents,^{[ 5 ]} an endocrine disruptor, and a probable carcinogen in humans.^{[ 4 , 6 ]} Exposures to PCP have been shown to affect the endocrine system of vertebrate life forms and may lead to immune system dysfunction, and disruption of normal sexual, cognitive, physical, and emotional development. The aim of this study was to design an ecotoxicological test systems and indicators able to detect different effects using a variety of endpoints. Such a battery of test systems and indicators would be representative of a wide range of organisms. The systems studied included immunotoxicity test, defensive toxicity test, cytotoxicity test, reproductive toxicity test, and mitochondrial toxicity test in common carp. Moreover, we probed the toxicity of PCP in sublethal concentration levels and different incubation time scales by exposing common carp, a species of freshwater fish widely distributed in China. The changes of leukocytes counts, EROD, GST activity, and MDA contents were detected in order to evaluate the toxicity effect and endocrine disruption activities of PCP, as well as its effect on mitochondrial membrane potential (Δ ψm).

MATERIALS AND METHODS

Common Carp and PCP Treatment

The test common carps (Cyprinus carpio) were collected from a noncontaminated areas of Lianyungang Aquafarm; mean weight of the fish (n = 60) was 100.4 ± 36.5 g. Some common carps were analyzed beforehand so as to confirm the absence of PCP. The fish were transferred to the laboratory 2 days before the experiments and maintained at room temperature. A group of common carps, from the same offspring, were reared in 80-L glass tanks in a recirculation system. Water temperature was adjusted to 25°C and the fish were fed with commercial pellets at a daily feeding rate of 1% of body weight. Treatment was at concentrations of 0, 0.1, 1.0, 2.0, 4.0, and 6.0 mg/L PCP at 24 and 72 h. During the experiment, the fish were kept under the standardized laboratory conditions (temperature 25°C, oxygen level 5–7 mg L^{− 1}, photoperiod 15-h light/9-h dark). Because of these standardized laboratory conditions, it is assumed that all the fish had been subjected to common environmental effects. Finally, all experiments were performed in duplicate.

Leukocyte Purification from Head Kidney and Relative Leukocyte Differential Counts

Leukocytes were purified from head kidney using hypotonic lysis, as modified from Crippen et al.^{[ 7 ]} A relative differential for lymphocytes and granulocytes was performed using flow cytometry, as modified from Inoue et al.^{[ 8 ]}

Isolation of Hepatocytes and Hepatic Enzyme Activities Assay

Liver microsomal fractions were prepared as described previously.^{[ 9 ]} Microsomal protein concentrations were determined using a protein assay kit (Bio-Rad, Richmond, CA) with bovine serum albumin (BSA) as the standard. Hepatic microsomal ethoxyresorufin O-deethylase (EROD) activity was assayed using the method of DeVito et al.^{[ 9 ]} All substrate concentrations were 1.5 nM. EROD values were calculated as pmol resorufin per mg protein per minute and graphically represented as a percentage of control activity.

Determination of Glutathione (GSH) and Malondialdehyde (MDA)

GSH levels in the primary hepatocytes were measured according to the method described by Dringen et al.^{[ 10 ]} The protein content of liver suspensions was determined by the method described by Lowry et al.^{[ 11 ]} Samples containing 500 mg protein were employed in the determination of MDA, according to the method reported by Girotti et al.^{[ 12 ]} MDA has been identified as a product of lipid peroxidation, it reacts with thiobarbituric acid (TBA) to give a complex substrate absorbing at 535 nm. The TBA assay is most frequently used although other cellular components, such as sugars, also react with TBA to produce a chromophore absorbing at 535 nm.^{[ 12 , 13 ]} These interfering substances were almost totally removed from the suspensions in the course of preparation.

Determination of Serum Testosterone and Vitellogenin (VTG)

After exposure to PCP for 24 and 72 h, the blood of common carp was collected into Eppendorf tubes using the Watson method,^{[ 14 ]} and the serum was separated from blood by centrifugation at 9000 × g for 5 min and stored at −20°C. Serum testosterone concentrations were detected using a radioimmunology assay (Testosterone RIA KIT-125I; Tianjin Jiuding Parmaceutic and Bioengineering, Tianjin, China). The serum testosterone was extracted using dichloromethane before determination (5 mL dichloromethane:1 mL serum). The dichloromethane was volatilized at 37°C before adding pure ethanol to a total volume of 200 μ L. All plasma samples were assayed in duplicate and values are reported as pg/mL of plasma. VTG concentrations in plasma of common carps were quantified by direct enzyme-linked immunosorbent assay (ELISA). VTG was first purified by anion-exchange chromatography (LMB VTG 102396B) and its protein concentration determined by the Bradford method for use as a standard.^{[ 15 ]}

Measurement of Mitochondrial Membrane Potential (Δ ψm)

The mitochondrial membrane potential (Δ ψm) was estimated by measuring the fluorescence of rhodamine 123–labeled mitochondria as described.^{[ 16 ]} In brief, the cells were seeded at 105 cells/well in 6-well plates (2 mL/well). After different treatments, the cells were washed twice with phosphate-buffered saline (PBS) (pH 7.4), and incubated in 1 mL rhodamine 123 solution (10 μ M) for 30 min at room temperature. Fluorescent signal was recorded using a fluorescence microscopy (505 nm filter; OLYMPUS IX-71, Japan). The Δ ψm was assessed by measurement of fluorescence intensity with WinView32 software.

Data Analyses

Statistical analysis was conducted using Origin7.5. The data were analyzed with analysis of variance (ANOVA) and Tukey's test for normality. Differences were considered significant if P <.05.

RESULTS

Immunotoxicity Test of PCP—Leukocyte Differential Counts

The primary leukocytes were exposed to various concentrations of PCP for 24 and 72 h (Figure 1). The lymphocyte and granulocyte counts were significantly decreased in comparison with the control groups under the higher PCP exposure (P <.05), there were no significant reduction in lymphocyte and granulocyte counts in the lower exposure groups (0.1 and 1.0 mg/L) compared with the control (P <.05). With increasing concentrations, the lymphocyte and granulocyte counts were decreased to 40.0% ± 6.8%, 64.3% ± 3.9%, and 71.3% ± 5.2% at 2.0, 4.0, and 6.0 mg/L of PCP, respectively, within 24 h, and to 53.1% ± 8.9%, 73.5% ± 9.6%, and 80.5% ± 8.3% at 2.0, 4.0, and 6.0 mg/L of PCP, respectively, within 72 h. Thus, exposure of common carp from 2.0 to 4.0 and 6.0 mg/L PCP for 24 and 72 h resulted in a decrease in lymphocyte and granulocyte counts in a dose-dependent manner. Moreover, it can be seen that exposure time exerted significant effect on the lymphocyte and granulocyte counts in common carps exposed to PCP.

FIGURE 1 Relative head kidney lymphocyte and granulocyte counts in common carp following PCP exposure for 24 and 72 h. Values are given as mean ± SEM. P values provided are the probability that there are no differences between control and treatment groups. *P <.05.

Defensive Toxicity Test of PCP—EROD Activity

Liver microsomes EROD activities in common carp exposed to PCP for 24 and 72 h have significantly different induction compared with the controls (P <.05) in the 2.0, 4.0, and 6.0 mg/L groups (Figure 2). Percent induction of EROD activity relative to controls were 122%, 183%, and 195% in 24-h and 176%, 243%, and 261% in 72-h 2.0, 4.0, and 6.0 mg/L PCP treatment groups, respectively. EROD activities after exposure for 24 and 72 h increased with PCP concentration, and EROD activities for the 72-h treatment were higher than those of the 24-h exposure in all treatment groups (P <.05). Also EROD activities in analyzed liver microsomes from all PCP-exposed common carps were consistently high. Moreover, the EROD activities in common carps greatly increased with the incubation time. Thus it can be seen that exposure time exerted significant effect on the activity of EROD in common carps exposed to PCP, it presents a dose-dependent manner and the EROD activities in common carps greatly increased with the incubation time.

FIGURE 2 Mean EROD activity of common carp exposed to PCP for 24 and 72 h. Error shown is stranded error of the mean. A, control; B–F, 0.1, 1.0, 2.0, 4.0, and 6.0 mg/L PCP, respectively. *Significantly different from control value (P <.05).

Cytotoxicity Test of PCP—GSH and MDA Content

We analyzed the changes of GSH levels in primary hepatocytes (Figure 3). The analysis of GSH activities showed that GSH activities after 24 and 72 h of exposure decreased with PCP concentration and incubation time. Under PCP exposure, there was no significant difference in the GSH contents at 0.1 and 1.0 mg/L of PCP compared with the control groups within 24 h (P <.05). But, all common carp exposed to higher concentration PCP for 24 h have significantly different inhibition in the 2.0, 4.0, and 6.0 mg/L groups (P <.05) and percent induction of GSH activity versus controls were 92.3%, 130.7%, and 163.2%, respectively. However, GSH activities were inhibited remarkably after 72 h of exposure in all dose groups (P <.05), percent inhibitions versus controls were 31.8%, 45%, 81.3%, 190%, and 243%, respectively. GSH activities were lower after 72 h of exposure than those after 24 h of exposure (P <.05).

FIGURE 3 Mean GSH content of common carp exposed to PCP for 24 and 72 h. Error shown is stranded error of the mean. A, control; B–F, 0.1, 1.0, 2.0, 4.0, and 6.0 mg/L PCP, respectively. *Significantly different from control value (P <.05).

Malondialdehyde (MDA) is indicative of lipid peroxidation. Cellular MDA was measured after cell treatment with various concentrations of PCP for 24 and 72 h. There was a significant increase in cellular MDA concentration after PCP treatments (Figure 4). Also PCP induced increases in MDA levels with incubation time, incubation for longer times produced significant increases in cellular MDA. When the cells were incubated with 2.0, 4.0, and 6.0 mg/L, intracellular MDA increased approximately to 26.6%, 36.8%, and 80.2% of control values, respectively.

FIGURE 4 Mean MDA content of common carp exposed to PCP for 24 and 72 h. Error shown is stranded error of the mean. A, control; B–F, 0.1, 1.0, 2.0, 4.0, and 6.0 mg/L PCP, respectively. *Significantly different from control value (P <.05).

Mitochondrial Toxicity Test of PCP—Membrane Protential

We further studied if PCP induced apoptosis coincides with disrupted mitochondrial function, the Δ ψ _m collapse is a critical step that occurs in all cell types undergoing apoptosis, regardless of the inductive signal. Δ ψ _m was monitored using Rh-123 staining (Figure 5). PCP-induced increase in Δ ψ _m in liver mitochondrial compared to control levels when treated with PCP (0.1 and 1.0 mg/L, 24 h) (P <.05), the Δ ψ _m of liver mitochondria slightly increased, by 15.2% and 23.4%, respectively, compared with control groups. However, when liver mitochondria were treated with PCP (2.0, 4.0, 6.0 mg/L, 24 h), the Δ ψ _m decreased significantly compared to control levels (P <.05). When the liver mitochondria were incubated 72 h with 2.0, 4.0, and 6.0 mg/L, the Δ ψ _m decreased approximately to 50.6%, 80.9%, and 120.2% of control values, respectively. It presents a dose-dependent manner and the Δ ψ _m in common carps greatly decreased with the incubation time under the higher PCP exposure.

FIGURE 5 Effects of PCP on the mitochondrial transmembrane potential. Data are presented as means ± SD (n = 6). A, control; B–F, 0.1, 1.0, 2.0, 4.0, and 6.0 mg/L PCP, respectively. *Significantly different from control value (P <.05).

Reproductive Toxicity Test of PCP—Testosterone and VTG

We selected 24 and 72 h as the standard incubation times for the experiments. Effects of sublethal concentration PCP on serum testosterone level in male common carps were shown in Figure 6. No significant differences were observed in serum testosterone at 0.1 and 1.0 mg/L of PCP compared with the control groups within 24 and 72 h (P <.05). This indicated that the lower dose of PCP has no effect on serum testosterone in male common carp. But a significant decrease in serum testosterone concentration was observed in male common carps exposed to 2.0, 4.0 and 6.0 mg/L PCP when compared to control within 24 and 72 h (P <.05). Also, after exposure to PCP for 72 h, serum testosterone levels in male common carp decreased significantly (P <.05) with the 2.0, 4.0, and 6.0 mg/L for 72 h treatments. Prolonged exposure time decreased testosterone concentration further at 6.0 mg/L.

FIGURE 6 Mean concentration of testosterone in male common carp exposed to PCP for 24 and 72 h. Error shown is stranded error of the mean. A, control; B–F, 0.1, 1.0, 2.0, 4.0, and 6.0 mg/L PCP, respectively. ast;Significantly different from control value (P <.05).

Vitellogenin (VTG), the precursor of livetin produced by the mature female common carps, was determined only in female common carp. Levels of VTG in plasma from common carp from the PCP exposure study are presented in Figure 7. Relatively high concentrations of VTG were found in females exposed to nominal concentrations of 0.1 and 1.0 mg/L PCP. VTG concentrations decreased approximately by 83%, 121%, and 189% within 24 h of PCP exposure, and declined approximately by 125%, 187%, and 252% within 72 h of PCP exposure (2.0, 4.0, 6.0 mg/L) in female common carp as compared to those in control groups (P <.05).

FIGURE 7 Plasma VTG in female common carp with 24 and 72 h of PCP exposure. Error shown is stranded error of the mean. A, control; B–F, 0.1, 1.0, 2.0, 4.0, and 6.0 mg/L PCP, respectively. *Significantly different from control value (P <.05).

DISCUSSION

Due to the concerns about increasing use of PCP and its prevalence in the environment, in the current study we evaluated the ecotoxicological effects of pentachlorophenol on common carp using a battery of indicators. We found that the lymphocyte and granulocyte counts were significantly decreased in comparison with the control groups under the higher PCP exposure (2.0, 4.0, 6.0 mg/L) (P <.05), there were no significant reduction in lymphocyte and granulocyte counts in the lower exposure groups (0.1 and 1.0 mg/L) compared with the control (P <.05), and that exposure time exerted significant effect on the lymphocyte and granulocyte counts in common carps exposed to PCP. Reactive oxygen species (ROS) produced inside macrophages or granulocytes during respiratory burst can leak out into surrounding tissues and uncontrolled or excessive production of ROS can result in oxidative stress and tissue damage such as lipid peroxidation, necrosis, or cancer.^{[ 17 ]} The predisposition of pesticide-exposed leukocytes to produce excessive amounts of oxygen radicals following antigen stimulation might cause damage to tissues other than the immune system, thus lymphocyte function may be more important for clearance of this organism. Although PCP use has been significantly curtailed in recent years, due to its persistence and continued use in some areas of the world, environmental concentrations still remain elevated and water concentrations of 2.94 mg/L have been reported recently, which is within the range used in the experiment that resulted in changes to the immune system.^{[ 18 , 19 ]}

Hepatic enzymes play a key role in detoxication and elimination of PCP in both vertebrates and invertebrates. Their presence and activity determine potential biological effects of contaminant exposure. Exposure to many organic PCP can lead to the induction of these enzymes.^{[ 20 ]} In the present study, the results demonstrated that PCP caused a significant change in hepatic EROD and GSH activities in common carp. EROD activities after exposure for 24 and 72 h increased with PCP concentration, and EROD activities for 72-h treatment were higher than those of 24-h exposure in all treatment groups (P <.05). GSH is the substrate for glutathione peroxidase, an enzyme that reduces hydrogen peroxide to water. The present study shows that GSH activities after 24 and 72 h of exposure decreased with PCP concentration and incubation time. GSH has been shown to cause a reduction in the initiation of lipid peroxidation.^{[ 21 , 22 ]} Lipid peroxidation(LPO)in lipoproteins is known ultimately to generate various aldehydes. Malondialdehyde (MDA), a major end product of lipid degradation, represents a well-established parameter for LPO. The study result shows an increase in MDA levels after PCP treatments. MDA are known to bind strongly to oxygen, nitrogen, and sulfur atoms. High affinity of aromatic pesticides to sulfhydryl groups and disulfide bonds may cause damage in the secondary structure of proteins and affect the enzyme activities, leading to the disturbance of various metabolic pathways.^{[ 23 ]} The role of oxidative stress in apoptosis may include high levels of ROS directly increasing caspase activity, disrupting intracellular function, and resulting in the ATP depletion due to the close relationship between ROS, MDA, and mitochondria.^{[ 24 , 25 ]} In our study, PCP induced the increase of Δ ψ _m in liver mitochondria compared to control levels when treated with PCP (0.1 and 1.0 mg/L, 24 h) (P <.05). However, when liver mitochondria were treated with PCP (2.0, 4.0, 6.0 mg/L, 24 h), the Δ ψ _m decreased significantly compared to control levels (P <.05). It is possible to hypothesize that the mitochondria is involved in apoptosis induced by PCP.^{[ 26 ]}

Previous studies have documented that polychlorinated biphenyls (PCBs),^{[ 27 ]} halogenate aromatic hydrocarbons,^{[ 28 ]} alkylphenols,^{[ 29 ]} and chlorobenzenes^{[ 30 ]} may alter serum testosterone and 17β -estradiol in fish and could produce reproductive and endocrine effects. The changes of sex steroids (e.g., testosterone, estradiol, progesterone) are known to be sensitive biomarkers for screening and evaluating the endocrine-disrupting activities of xenobiotic chemicals.^{[ 31 , 32 ]} No effect on serum testosterone in male common carp at lower PCP concentration was found. But a significant decrease in serum testosterone concentration was observed in male common carps exposed to 2.0, 4.0, and 6.0 mg/L PCP when compared to control within 24 and 72 h (P <.05). Decreased serum testosterone levels in this study indicated that PCP may have endocrine-disrupting activity in common carp and may have profound effects on the reproductive success of this species. Serum testosterone concentrations decreased with increase in EROD and decrease GSH activities. This is likely due to the role of these enzymes in the maintenance of steroid hormone levels in these organisms. The measurement of VTG in female fish have emerged recently as a key exposure biomarker for endocrine-disrupting chemicals.^{[ 33 , 34 ]} Relatively high concentrations of VTG were found in females exposed to nominal concentrations of 0.1 and 1.0 mg/L PCP. VTG concentrations decreased approximately by 83%, 121%, and 189% within 24 h of PCP exposure, and declined approximately by 125%, 187%, and 252% within 72 h of PCP exposure, in female common carp than those in control groups (P <.05).

In conclusion, the results in this study showed that PCP contributed to the effect of immune system and disturbance of steroid hormone levels with a significant increase PCP concentration and resulted in significant changes in hepatic EROD and GSH activities, as well as liver mitochondria function in common carp. These results demonstrated that PCP may have endocrine-disrupting activity and affect the endocrine system of vertebrate life forms and may lead to immune system dysfunction and disruption of normal sexual and physiological functions.

Acknowledgments

This research was supported by the Foundation of Social Development Planning of Lianyungang City of Jiangsu province, China (Project No. SH0809). The authors thank Jia-Hong Wang and Wen Zhou Lv for exemplary technical assistance and Shao Cong Han for valuable support on statistical analysis.