2,4-Dichlorophenol induces apoptosis in primary hepatocytes of grass carp (Ctenopharyngodon idella) through mitochondrial pathway

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2,4-Dichlorophenol induces apoptosis in primary hepatocytes of grass carp (Ctenopharyngodon idella) through mitochondrial pathway Hui Li, Xiaoning Zhang, Qian Qiu, Zhen An, Yongmei Qi, Dejun Huang ∗ , Yingmei Zhang ∗ School of Life Sciences, Lanzhou University, Lanzhou 730000, China

a r t i c l e

i n f o

Article history: Received 6 February 2013 Received in revised form 15 May 2013 Accepted 16 May 2013 Keywords: 2,4-DCP Apoptosis Mitochondria-dependent pathway Grass carp (Ctenopharyngodon idella)

a b s t r a c t 2,4-Dichlorophenol (2,4-DCP), a major type of chlorophenols, has been widely used to produce some herbicides and pharmaceuticals, yet due to its incomplete degradation and bioaccumulation characteristics, it is toxic to aquatic organisms. Apoptosis is one of the most severe outcomes of cell poisoning and injury. So far, the potential molecular mechanism of 2,4-DCP-induced apoptosis has not been reported. This study showed that 2,4-DCP significantly induced apoptosis in primary hepatocytes of grass carp (Ctenopharyngodon idella). 2,4-DCP exposure upregulated mRNA of caspase-3, reduced the mitochondrial membrane potential (␺m), increased intracellular reactive oxygen species (ROS) and the Bax/Bcl-2 ratio, while protection of mitochondria with acetyl-l-carnitine hydrochloride (ALC) rescued 2,4-DCP-induced apoptosis, restored the ␺m and reduced the Bax/Bcl-2 ratio. Taken together, this is the first study that has identified that 2,4-DCP exposure induced apoptosis through the mitochondria-dependent pathway in primary hepatocytes of grass carp. © 2013 Elsevier B.V. All rights reserved.

1. Introduction 2,4-Dichlorophenol (2,4-DCP) is a widely used chemical for producing some herbicides and pharmaceuticals and is regarded as one of the most abundant phenolic compounds in some aquatic environments (House et al., 1997). The high concentration of 2,4DCP has been detected in surface water (Zhong et al., 2010), ground water (Varank et al., 2011), and even in the human body (Bukowska, 2003). 2,4-DCP has been defined as a priority pollutant in the aquatic environment in the USA (USEPA, 1979). Moreover, its accumulation (Kondo et al., 2005) and toxicity (Amer and Aly, 2001; Aoyama et al., 2005) in aquatic organisms have also been major concerns. The effects of 2,4-DCP have been studied on diverse biological systems. For example, it resulted in chromosome aberrations in germ cells in mice (Amer and Aly, 2001), reproductive toxicity in rats (Aoyama et al., 2005), endocrine disruption in zebrafish and H295R cells (Ma et al., 2012), impairment of vision and injury of the respiratory tract in humans (USEPA, 2000). Also, 2,4-DCP has been reported to induce oxidative stress in the marine diatom (Skeletonema costatum) (Yang et al., 2002), goldfish (Carassius auratus) (Zhang et al., 2004) and human erythrocytes (Bors et al., 2011; Bukowska, 2003). Several recent studies have demonstrated that

∗ Corresponding authors. Tel.: +86 139 19123067; fax: +86 931 8913631. E-mail addresses: [email protected] (D. Huang), [email protected] (Y. Zhang). 0166-445X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquatox.2013.05.015

chlorophenols can induce apoptosis both in vivo and in vitro (Dong ´ 2009), et al., 2009; Folch et al., 2009; Michałowicz and Sicinska, which involves the activation of a series of molecular events, such as regulation of Bcl-2 family, release of cytochrome c and activation of caspases (Ghobrial et al., 2009). However, no reports have focused on 2,4-DCP and mitochondria-mediated apoptosis until now. Apoptosis, also named programmed cell death, is characterized by cell shrinkage, chromatin condensation, formation of cytoplasmic blebs and apoptotic bodies (Ziegler, 2004). There are two main apoptotic pathways, the mitochondrial pathway and the death receptor pathway (Elmore, 2007). Apoptosis through the mitochondrial pathway is preceded by the overproduction of reactive oxygen species (ROS) and regulation of Bcl-2 family, which results in mitochondrial outer membrane permeabilization, thus leading to the release of pro-apoptotic proteins and caspase activation (Kuwana and Newmeyer, 2003; Spierings et al., 2005). Apoptosis via the death receptor pathway is triggered by the Fas death receptor, a member of the tumor necrosis factor (TNF) receptor superfamily (Zapata et al., 2001). The pathways of apoptosis have been studied extensively, but the signaling pathway of 2,4-DCP-induced apoptosis remains undefined. The present study aims to explore the effect of 2,4-DCP on apoptosis and its potential mechanism. 2,4-DCP can bioaccumulate in the fish body, and the liver is the main detoxification organ, suffering high concentrations of 2,4-DCP. Therefore, apoptosis in primary hepatocytes of grass carp (Ctenopharyngodon idella) was measured by flow cytometric analysis (Annexin V-FITC/PI staining), the mRNA levels of caspase-3, Bax and Bcl-2 were monitored

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by qPCR (quantitative PCR), ROS and the mitochondrial membrane potential (␺m) were detected by a fluorescence spectrometer. Furthermore, acetyl-l-carnitine hydrochloride (ALC), a mitochondria protecting agent (Haorah et al., 2011), was used in the present study to investigate the mechanisms involved in apoptosis by 2,4DCP in primary hepatocytes of grass carp. 2. Materials and methods 2.1. Chemicals 2,4-DCP (99% purity) was obtained from A Johnson Matthey company. ALC (>98.0% purity) was purchased from Tokyo Chemical Industry Co., Ltd. All other chemicals used in this study were of analytical grade and obtained from local companies. 2.2. Fish and isolation of hepatocytes A batch of grass carp (C. idellus) (≥20) with similar weight (700–800 g) and age (9–10 months old) were purchased from a local fish farm and maintained in the laboratory for 2 weeks prior to use to allow for acclimatization and evaluation of their health, and only three healthy fish were used for the hepatocytes isolation in each independent experiment. The cellular suspension from one fish was used as one sample for detection of all the parameters and at least two repeats of each sample were done. Overall, nine fish were used in three independent experiments (n = 3). Hepatocytes were isolated from grass carp following the method by Wan et al. (2004), with some modifications. Briefly, fish skin was sterilized by alcohol and cleared of blood from the tail vein. Then the abdomen was dissected, and liver tissue was excised and rinsed twice with sterilized phosphate buffered saline (PBS). The liver tissue was then minced into small pieces and transferred to a 50 mL conical tube, to which a solution of 0.25% trypsin was added (1:20, w/v). The mixture was shaken sharply for 10 min to obtain the cell suspension, which was then filtered through a 200-mesh sieve and centrifuged at 1000 rpm for 2 min. The cells were washed twice and resuspended in the culture medium. The isolated cells were used for experiments when viability was >90%. The hepatocytes were cultured in D-MEM/F-12 (GIBCO, USA) basal medium (pH 7.0–7.2) supplemented with 15% (v/v) FBS (Hyclone, USA), 10 ␮g/mL insulin (Sigma, German) and 1% (v/v) penicillin–streptomycin (10,000 IU/mL to 10,000 ␮g/mL), and were incubated in a 5.0% CO2 incubator (Heal Force, Hong Kong) at 27 ◦ C. 2.3. Cell culture and exposure experiment Cells were seeded in a culture plate at 1.5 × 106 cells/mL. After hepatocytes were attached to the wells and formed a monolayer of 70–80% confluence within 24 h, the culture medium was replaced with different concentrations of 2,4-DCP (0.10, 0.50 and 1.00 mM) alone or co-incubated with ALC (5.00 mM) for 12 h. After exposure, cells were harvested for detection. 2.4. Apoptosis assay The apoptosis assay was performed with Alexa Fluor 488 annexin V/Dead Cell Apoptosis Kit (Invitrogen, Carlsbad, CA) by flow cytometry (Beckman Coulter Epics XL) following the manufacturer’s instructions. After treatments, cells were collected and washed with cold PBS, gently resuspended in Annexin V-FITC binding buffer, incubated with Annexin V-FITC/PI in the dark for 15 min at room temperature, and analyzed by flow cytometry using Expo 32 software. The percentages of apoptotic and necrotic cells for each sample were accounted.

Table 1 Primers of detected genes. Genes

Primers

caspase-3

Forward: 5 -ATACAGCCTAAACTACCCCAA-3 Reverse: 5 -CAAATGAAGCACAGCGACT-3

Bax

Forward: 5 -AGTGGCAATGACCAGATAC-3 Reverse: 5 -GTCGGTTGAAGAGCAGAG-3

Bcl-2

Forward: 5 -AAAATGGAGGTTGGGATG-3 Reverse: 5 -GCACTTTCGTTAGGTATGAC-3

␤-Actin

Forward: 5 -GGCACTGCTGCTTCCTC-3 Reverse: 5 -ACCGCAAGACTCCATACCC-3

2.5. Measurement of intracellular ROS generation The level of intracellular ROS was measured by the alteration of fluorescence resulting from oxidation of DCFH-DA according to our previous study (Huang et al., 2008). Following exposure to the 2,4-DCP, the cells were harvested and washed with cold PBS, suspended in 1 mL PBS containing 10 ␮M DCFH-DA, and incubated at 27 ◦ C for 30 min in darkness. The fluorescence was analyzed by flow cytometry (BD Biosciences), with an excitation filter of 485 nm and an emission filter 535 nm. The ROS level was calculated as the percentage of that in control group cells. 2.6. Mitochondrial membrane potential determination Mitochondrial membrane potential was monitored using Rhodamine 123 (Rh 123), which is a lipophilic cationic fluorescent dye that is incorporated into the mitochondria in a transmembrane potential-dependent manner. It is selectively taken up by mitochondria, and its accumulation is directly proportional to the ␺m of cells (Scaduto and Grotyohann, 1999). Cells were collected, washed, and incubated with 1 ␮M Rh 123 in PBS at 27 ◦ C for 30 min. Rh 123 fluorescence was subsequently monitored using a fluorescence spectrometer (Perkin Elmer LS-5) with excitation and emission wavelengths of 507 and 530 nm, respectively. 2.7. Gene expression analysis Total RNA was isolated using RNAiso Plus regent (TaKaRa, Japan) and reverse-transcribed to cDNA using the RNA LA PCR TM Kit (TaKaRa, Japan). qPCR was performed with the qPCR System instrument (BioRad, USA) using SYBRs PrimeScript TM Kit (TaKaRa, Japan) according to the manufacturer’s instructions. The expression levels of apoptosis-related genes (caspase-3, Bax, Bcl-2) were analyzed with the following primers shown in Table 1. Expression levels of ␤-actin were used as endogenous controls within each sample and the related genes expression levels were calculated. Each sample was run in three parallel reactions. 2.8. Statistical analysis Data are presented as mean ± SD. Statistical comparisons were made using one-way analysis of variance (ANOVA) followed by LSD post hoc test. Differences were considered significant at P < 0.05. 3. Results 3.1. 2,4-DCP induced apoptosis Grass carp hepatocytes that were exposed to 2,4-DCP for 12 h were observed by fluorescence microscopy to detect apoptosis. Treatment with 0.10, 0.50 and 1.00 mM 2,4-DCP caused some typical features of apoptosis such as nuclear shrinkage and apoptotic

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Fig. 1. 2,4-DCP-induced apoptosis in primary hepatocytes of grass carp (C. idellus). The cells were exposed to 2,4-DCP (0.10, 0.50 and 1.00 mM) for 12 h and then the apoptosis was detected with Annexin V-FITC/PI staining by flow cytometry. The results were expressed as dot plots representing one of the three independent experiments. D1, necrotic cells; D2, late apoptotic cells; D3, live cells; D4, early apoptotic cells.

body formation (Supplemental data Fig. 1). 2,4-DCP-induced apoptosis was further quantified by flow cytometry (Fig. 1), and the results showed that 2,4-DCP at 0.10, 0.50 and 1.00 mM caused significant degrees of apoptosis (P < 0.05, Fig. 2). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.aquatox.2013.05.015. 3.2. 2,4-DCP caused intracellular ROS overproduction To explore whether 2,4-DCP induced overproduction of ROS in hepatocytes of grass carp, we detected intracellular ROS by flow cytometry. The results showed that intracellular ROS level was much higher than that in control group with 2.0- and 2.8-fold increase when cells were treated by 0.10 and 0.50 mM 2,4-DCP for 5 h (P < 0.05, Fig. 3), respectively. However, there were no significant differences (P > 0.05) between control and 2,4-DCP treatment groups (0.10 and 0.50 mM) after cells were exposed for 12 h (Fig. 3). 3.3. ALC rescued 2,4-DCP-induced ␺m collapse and apoptosis

Fig. 3. 2,4-DCP induced ROS generation in primary hepatocytes of grass carp (C. idellus). The cells were exposed to 2,4-DCP (0.10, 0.50 mM) for 5 h and 12 h and then intracellular ROS was monitored by flow cytometry. Data are presented as mean ± SD, n = 3. *P < 0.05 compared with control.

To address whether the apoptosis induced by 2,4-DCP is related to the mitochondrial pathway, the ␺m was detected. ALC was used to protect mitochondria. As shown in Fig. 4, the ␺m was decreased to 79.50% and 77.50% after grass carp hepatocytes were exposed to 0.10 and 0.50 mM concentrations of 2,4-DCP for 12 h (P < 0.05). While the decrease of ␺m was significantly inhibited

by ALC (P < 0.05), namely, 5.00 mM ALC increased the ␺m induced by 0.10 mM 2,4-DCP by about 12%. To confirm the role of mitochondrial pathway in 2,4-DCPinduced apoptosis, we compared the apoptosis rates in primary

Fig. 2. ALC decreased 2,4-DCP-induced apoptosis in primary hepatocytes of grass carp (C. idellus). The cells were exposed to 2,4-DCP alone (0.10, 0.50 mM) or in combination with ALC (5.00 mM) for 12 h and then the apoptosis was detected with Annexin V-FITC/PI staining by flow cytometry. Data are presented as mean ± SD, n = 3. *P < 0.05 compared with control.

Fig. 4. The mitochondrial membrane potential in primary hepatocytes of grass carp (C. idellus). The cells were exposed to 2,4-DCP alone (0.10 and 0.50 mM) or in combination with ALC (5.00 mM) for 12 h. The fluorescence was measured by a fluorescence spectrometer. Data are presented as mean ± SD, n = 3. *P < 0.05 compared with control, # P < 0.05 compared with the corresponding 2,4-DCP treatment alone.

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Fig. 5. The mRNA expression of caspase-3 (A) and the Bax/Bcl-2 ratio (B) in primary hepatocytes of grass carp (C. idellus). The cells were exposed to 2,4-DCP (0.50 mM) alone or in combination with ALC (5.00 mM) for 12 h. Data are presented as mean ± SD, n = 3. *P < 0.05 compared with control, # P < 0.05 compared with 0.50 mM 2,4-DCP treatment.

Table 2 The fold increase of Bax and Bcl-2 mRNA expression. Groups

Bax

Bcl-2

0 0.50 mM 2,4-DCP 0.50 mM 2,4-DCP + 5.00 mM ALC

1.00 ± 0.00 1.95 ± 0.19∗ 1.55 ± 0.07#

1.00 ± 0.00 1.05 ± 0.05 0.97 ± 0.04

The cells were exposed to 2,4-DCP (0.50 mM) alone or in combination with ALC (5.00 mM) for 12 h. Data are presented as mean ± SD. n = 3. * P < 0.05 compared with control. # P < 0.05 compared with 0.50 mM 2,4-DCP treatment.

hepatocytes with or without ALC co-treatment. The apoptosis rates increased 27.04- and 35.69-fold after cells were exposed to 0.10 and 0.50 mM concentrations of 2,4-DCP (P < 0.05, Fig. 2) compared to those of the control group, respectively. However, 5.00 mM ALC decreased the apoptosis rates by 23.93% and 23.28% compared to the cells treated with 0.10 and 0.50 mM 2,4-DCP alone (Fig. 2). Taken together, these data suggested that 2,4-DCP-induced apoptosis was associated with the mitochondrial pathway. 3.4. 2,4-DCP elevated the mRNA expression of caspase-3, Bax and Bcl-2 To uncover the molecular mechanism of apoptosis induced by 2,4-DCP, the mRNA expression of caspase-3, Bax and Bcl-2 was measured after exposure of grass carp hepatocytes to 2,4-DCP for 12 h by qPCR. The results showed that 2,4-DCP at 0.50 mM elevated the mRNA expression of caspase-3 (Fig. 5A) and Bax/Bcl-2 ratio (Table 2 and Fig. 5B) by 44.79% and 86.53%, respectively (P < 0.05). Meanwhile, the combination of 5.00 mM ALC caused significant reduction in caspase-3 expression and Bax/Bcl-2 ratio by about 43% and 26%, respectively, compared with 2,4-DCP treatment alone (P < 0.05). 4. Discussion Several studies have shown that apoptosis was induced by chlorophenols including monochlorophenol, dichlorophenol, trichlorophenol and pentachlorophenol (PCP) in human hepatoma HepG2 cells (Jiang et al., 2004), rat cerebellar granule neurons (Folch et al., 2009), and Vero cells (Fernández Freire et al., 2005). PCP induced apoptosis by the generation of ROS (Dong et al., 2009), reduction of mitochondrial transmembrane poten´ tial and induction of caspase-3 activity (Michałowicz and Sicinska, 2009), dysfunction of mitochondria and fragmentation of nuclei (Fernández Freire et al., 2005) as well as induction of classical

p53 apoptotic pathway (Folch et al., 2009). However, the detailed mechanism of dichlorophenol-induced apoptosis is still poorly understood. In this study, we found that 2,4-DCP could induce apoptosis in primary hepatocytes of grass carp. Caspase-3, Bax mRNA expression, and Bax/Bcl-2 ratio were increased, and mitochondrial membrane potential was decreased, while the combination of ALC and 2,4-DCP rescued apoptosis by reducing the Bax/Bcl-2 ratio. 2,4-DCP-induced apoptosis in this study was consistent with the research by Chen et al. (2004) who also found that 2,4-DCP induced apoptosis in rat connective tissue fibroblast L929 cells. The results by flow cytometry showed that the cells in early phase apoptosis were much more than those in late phase apoptosis after hepatocytes being exposed to 2,4-DCP, which might be due to the short exposure time (12 h) or the cell type (Nevoie et al., 2011; ´ Srdic-Raji c´ et al., 2011). Caspase-3 activation serves as a final common channel for both pathways of apoptosis (Ghobrial et al., 2009). The increased apoptotic rate and caspase-3 mRNA level induced by 2,4-DCP were tested in this study, while ALC decreased the apoptotic rate and the up-regulation of caspase-3 mRNA induced by 2,4-DCP. Mitochondrium is a major cellular source of ROS, and the overproduction of ROS will disrupt ␺m (Zhang et al., 2012). Studies have demonstrated that 2,4-DCP can lead to free radical generation and oxidative stress (Luo et al., 2005a,b). The present study showed that 2,4-DCP induced overproduction of intracellular ROS at an early time point (5 h) before cells went to apoptosis (12 h), suggesting that ROS were involved in 2,4-DCP induced apoptosis. The dissipation of ␺m is an early event of apoptosis and it is a pointof-no-return of apoptosis (Kroemer, 2003). Our results showed that 2,4-DCP caused the decrease of ␺m, suggesting that the mitochondrial pathway was involved. These results were similar to the report by Dong et al. (2009) who found that PCP could induce apoptosis in primary hepatocytes of goldfish (Carassius carassius), which might be because these chlorophenols have same target points. ALC, an acetylated derivative of l-carnitine, has been shown to protect cells from oxidative injury (Rump et al., 2010), suppress the oxidative stress in and around mitochondria, and finally prevent the mitochondrial signaling pathway which leads to apoptosis (Zhu et al., 2008). The present study showed that ALC inhibited the mitochondrial depolarization induced by 2,4-DCP, implying that the apoptosis induced by 2,4-DCP was related with mitochondria. The mitochondria-mediated pathway of apoptosis is regulated by the Bcl-2 family of pro-apoptotic proteins (Bax, Bad, and Bak) and anti-apoptotic proteins (Bcl-2, Bcl-xl, Mcl-1), and they regulate apoptosis by controlling the permeabilization of the outer

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mitochondrial membrane (Brunelle and Letai, 2009). The ratio of Bax/Bcl-2 is a decisive factor and plays an important role in determining whether cells will undergo apoptosis (Gupta et al., 2002). Our study revealed that the Bax/Bcl-2 ratio was increased significantly after exposure to 2,4-DCP. On the contrary, ALC was shown to inhibit the 2,4-DCP-induced apoptosis via downregulating expression of Bax. These results suggested that 2,4-DCP-induced apoptosis was regulated by the Bax/Bcl-2 ratio. All together, these results suggested that 2,4-DCP-induced apoptosis was mitochondria-dependent. Nevertheless, we found that ALC only partly reversed the 2,4-DCP-induced apoptosis, thus speculating that there were other pathways that lead to apoptosis. We found that 2,4-DCP increased the mRNA expression of TNF␣ (tumor necrosis factor-␣) and the increase was inhibited by ALC (data not shown). 5. Conclusion In conclusion, the present study demonstrated that 2,4-DCP could induce apoptosis in primary hepatocytes of grass carp (C. idellus). This process was accompanied by disruption of ␺m and the increase of the Bax/Bcl-2 ratio, while the combination of ALC and 2,4-DCP rescued the apoptosis ratio by reducing the Bax/Bcl-2 ratio. This suggests that 2,4-DCP-induced apoptosis in primary hepatocytes occurs through the mitochondrial pathway. Other pathways of 2,4-DCP-induced apoptosis need further study. Conflict of interest statement The authors declare that there is no potential conflict of interest involved in this study. Acknowledgements This work was supported by National Natural Science Foundation of China (20907019), the Specialized Research Fund for the Doctoral Program of Higher Education (20110211110032), the Program for New Century Excellent Talents in University (NCET10-0464) and the Fundamental Research Funds for the Central Universities (Dr. Huang DJ). We thank Ailing Li and Amber Harris (Department of Psychology, University of Texas at Arlington) for the critical reading of the manuscript. References Amer, S.M., Aly, F.A.E., 2001. Genotoxic effect of 2,4-dichlorophenoxy acetic acid and its metabolite 2,4-dichlorophenol in mouse. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 494, 1–12. Aoyama, H., Hojo, H., Takahashi, K.L., Shimizu, N., Araki, M., Harigae, M., Tanaka, N., Shirasaka, N., Kuwahara, M., Nakashima, N., 2005. A two-generation reproductive toxicity study of 2,4-dichlorophenol in rats. Journal of Toxicological Sciences 30, 59–78. ´ Bors, M., Bukowska, B., Pilarski, R., Gulewicz, K., Oszmianski, J., Michałowicz, J., Koter-Michalak, M., 2011. Protective activity of the Uncaria tomentosa extracts on human erythrocytes in oxidative stress induced by 2,4-dichlorophenol (2,4DCP) and catechol. Food and Chemical Toxicology 49, 2202–2211. Brunelle, J.K., Letai, A., 2009. Control of mitochondrial apoptosis by the Bcl-2 family. Journal of Cell Science 122, 437–441. Bukowska, B., 2003. Effects of 2,4-D and its metabolite 2,4-dichlorophenol on antioxidant enzymes and level of glutathione in human erythrocytes. Comparative Biochemistry and Physiology – Part C 135, 435–441. Chen, J., Jiang, J., Zhang, F., Yu, H., Zhang, J., 2004. Cytotoxic effects of environmentally relevant chlorophenols on L929 cells and their mechanisms. Cell Biology and Toxicology 20, 183–196. Dong, Y., Zhou, P., Jiang, S., Pan, X., Zhao, X., 2009. Induction of oxidative stress and apoptosis by pentachlorophenol in primary cultures of Carassius carassius hepatocytes. Comparative Biochemistry and Physiology – Part C 150, 179–185. Elmore, S., 2007. Apoptosis: a review of programmed cell death. Toxicologic Pathology 35, 495–516. Fernández Freire, P., Labrador, V., Pérez Martín, J.M., Hazen, M.J., 2005. Cytotoxic effects in mammalian Vero cells exposed to pentachlorophenol. Toxicology 210, 37–44.

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