Effects of Chronic Exposure to Microcystin-LR on Hepatocyte

Feb 27, 2015 - ... of Medicine, Nanjing, Jiangsu 210093, People's Republic of China .... from irrigation water: Implications for public health and foo...
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Effects of Chronic Exposure to Microcystin-LR on Hepatocyte Mitochondrial DNA Replication in Mice Xinxiu Li,†,‡ Qingya Zhao,†,‡ Wei Zhou,†,‡ Lizhi Xu,†,‡ and Yaping Wang*,†,‡ †

Department of Medical Genetics, and ‡Jiangsu Key Laboratory of Molecular Medicine, Nanjing University School of Medicine, Nanjing, Jiangsu 210093, People’s Republic of China S Supporting Information *

ABSTRACT: Microcystins (MCs) are produced by cyanobacterial blooms, and microcystin-LR (MC-LR) is the most toxic among the 80 MC variants. Data have shown that the liver is one of the specific target organs for MC-LR, which can cause mitochondrial DNA (mtDNA) damage, resulting in mitochondrial dysfunction. However, the underlying mechanism is still unclear. In the present study, we evaluated the genetic toxicity of MC-LR in mice drinking water at different concentrations (1, 5, 10, 20, and 40 μg/L) for 12 months. Our results showed that long-term and persistent exposure to MCLR increased the 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels of DNA in liver cells, damaged the integrity of mtDNA and nuclear DNA (nDNA), and altered the mtDNA content. Notably, MC-LR exposure can change the expression of mitochondrial genes and nuclear genes that are critical for regulating mtDNA replication and repairing oxidized DNA. They also further impaired the function of mitochondria and liver cells.

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INTRODUCTION

through ROS generation, condensed chromatin, and interference of the DNA repair processes.7−9 Mitochondria are a major intracellular source of ROS generation, and the mitochondria become vulnerable targets of various toxins by inducing excessive ROS. The activities of ATP synthase as one of the novel intracellular targets for MCLR were decreased in rat kidneys and zebrafish embryos after MC-LR treatment.10−12 The decrease of ATP synthase activity would affect the structure and function of mitochondria.13 Chen et al.10 showed that MC-LR exposure affected the transcriptional levels of mitochondrial genes. These results indicated that MC-LR impaired not only the structure but also the function of the mitochondria. Mitochondrial function is strongly associated with the mitochondrial DNA (mtDNA) content and integrity.14 A sufficient mtDNA content is necessary for maintaining the stability of cells, and the mtDNA content is related with mitochondrial inheritance and mitochondrial biogenesis.15 Lee and Wei14 showed that cells can synthesize more copies of mtDNA and increase the mitochondrial abundance to compensate for dysfunctional mitochondria. Taanman and Tang et al.16,17 reported that the mtDNA content could affect mitochondrial gene expression and was closely associated with the regulation of mtDNA replication. However, few studies have focused on the effects of

Microcystins (MCs) are a family of cyclic heptapetide endotoxins that are mainly produced by cyanobacterial blooms in various eutrophic inland waters worldwide. More than 80 different structural analogues of MCs have been identified, of which microcystin-LR (MC-LR) is the most common endotoxic variant.1 Experimental studies have found that MCLR accumulates in the liver, kidney, heart, brain, skeletal muscle, lung, gastrointestinal, and gonads secondary to drinking contaminated water and eating seafood. However, the liver has been regarded as the specific target organ of MC-LR because of the presence of the liver-specific organic anion-transporting polypeptide (rodent, Oatps; human, OATPs) membrane transport system.2 MC-LR-induced hepatotoxicity occurs by specifically inhibiting serine/threonine protein phosphatases 1 and 2A (PP1 and PP2A), which results in the hyperphosphorylation of many cellular proteins and eventually leads to cytoskeletal damage, necrosis, and loss of cell morphology.2,3 MC-LR could cause mitochondrial membrane rupture and ultrastructural damage of mitochondria and induce the changes of mitochondrial permeability transition (MPT) and mitochondrial membrane potential (MMP).4,5 Meanwhile, oxidative stress has also been shown to play a crucial role in MC-LR toxicity, mainly because of an excessive formation of reactive oxygen species (ROS).6 The genotoxicity of MC-LR is related to excessive ROS, which causes DNA strand breaks either directly through mutagenesis or indirectly © 2015 American Chemical Society

Received: Revised: Accepted: Published: 4665

December 6, 2014 February 22, 2015 February 27, 2015 February 27, 2015 DOI: 10.1021/es5059132 Environ. Sci. Technol. 2015, 49, 4665−4672

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Environmental Science & Technology

Information). The results of 8-OHdG contents were normally expressed in monogram and converted to 8-OHdG/106 as described by Sun et al.20 mtDNA Content Quantification. DNA concentrations were precisely measured by the PicoGreen double-stranded DNA (dsDNA) binding agent, and each sample was diluted with TE buffer (Tris-hydrochloride buffer at pH 8.0) to 2 ng/ μL as described by Guo et al.21 The obtained DNA was kept at −20 °C until polymerase chain reaction (PCR) analysis. The mtDNA content quantification was carried out on a StepOne real-time system with FastStart Universal SYBR Green Master mix (Roche, Switzerland). The primer pair22 for the 117 base pair (bp) fragment from the mtDNA was used to quantify the mtDNA (see Table S1 of the Supporting Information). The real-time PCR was performed in triplicate for each sample. All PCR products were estimated by melting curve analysis and 2% agarose gel electrophoresis. Long-Range Quantitative PCR. The long-range quantitative PCR assays performed with an 8.7 kilobase (kb) fragment22 from β-globin (X14061) and a 10 kb fragment22 from mtDNA were designed to assess the nuclear DNA (nDNA) and mtDNA. A TaKaRa LA Taq PCR kit (TaKaRa, Japan) was used, and long-range PCRs were performed as described (see Protocol S2 of the Supporting Information) by Guo et al.21 The amount of PCR products was quantified using a PicoGreen dsDNA binding agent. The long-range PCR products from mtDNA were normalized by the effect of the mtDNA content. All of the PCR reactions were performed in triplicate for each sample. RNA Isolation and Reverse Transcription (RT)-PCR. The liver tissues were broken with a homogenizer (Retsch, Germany), and the total RNA was extracted with TRIZOL reagent (TaKaRa, Japan) according to the protocol of the manufacturer (see Protocol S3 of the Supporting Information). A total of 1 μg of the purified total RNA was reversetranscribed with a first-strand cDNA synthesis kit (TaKaRa, Japan). The obtained cDNA was kept at −20 °C until PCR analysis. We tested the mRNA expression of the cytochrome c oxidase (COX3), Cytochrome b (CYTB), ATP synthase 6 (ATP6), and 16S rRNA (RNR2) genes to assess mtDNA transcriptional activity and the mRNA levels of the DNA polymerase γ (POLG), mitochondrial single-stranded DNAbinding protein (mtSSB), mitochondrial helicase (TWINKLE), and mitochondrial transcription factor (TFAM), which were nuclear genes associated with the regulation of the mtDNA content, to evaluate mtDNA replication function of the liver cells. Quantitative reverse transcription PCR (qRT-PCR) was performed to amplify target cDNA using the FastStart Universal SYBR Green Master mix kit (Roche, Switzerland) and ABI StepOne real-time system (Applied Biosystems) as directed by the manufacturer. Each sample was tested in triplicate for each PCR performance. Results of the transcript RNA quantification were normalized with the β-actin gene expression level and calculated using the 2−ΔΔCT method. The sequences of mRNA primer pairs used in this procedure are listed in Table S1 of the Supporting Information. Isolation of Proteins and Western Blot Analyses. Proteins from liver tissue were isolated with a total protein extraction kit (KeyGen Biotech, China). Briefly, liver tissues were isolated and homogenized, followed by a brief oscillation for 5 min in lysis buffer, including phenylmethanesulfonyl fluoride (PMSF), phosphates, and protease inhibitors. Then,

MC-LR on the mtDNA content and regulation of mtDNA replication in the liver cells. MC-LR is an environmental toxin that can cause DNA damage and mitochondrial dysregulation through additional ROS. Previous studies were primarily designed to evaluate its toxicological effects in acute or sub-acute tests1,10,18 that were generally observed for less than 6 months. In addition, most of the acute experiments were defined as a one-time exposure by intraperitoneal injection. This exposure mode was commonly used with the high concentrations for the investigated toxin and to show relative acute toxicity. To better assess human health risk to the environmental toxin, we established a long-term exposure mode for MC-LR and observed a concentration− effect relationship for the response of the body to the toxin in the chronic exposure. Here, we tested the MC-LR toxicity at different concentrations by oral exposure and investigated the concentration effects of MC-LR on the mtDNA content, the regulation of mtDNA replication, and the expression of mitochondrial genes in liver cells. Our results indicated that the alteration of the mtDNA content, integrity, and mitochondrial gene expression could be one of the basic mechanisms for the hepatotoxicity induced by MC-LR.

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MATERIALS AND METHODS Chemicals. MC-LR [purity of ≥95%, by high-performance liquid chromatography (HPLC)] was purchased from Alexis, Inc. (Switzerland). Dimethyl sulfoxide (DMSO) (purity of ≥99.7%) was obtained from Sigma-Aldrich (St. Louis, MO). All other chemicals were of analytical grade and were provided by common commercial suppliers. Animals. The experiments were performed on normal male C57BL/6 mice (6−8 weeks old and 20−22 g in size) that had been provided by the Model Animal Research Center of Nanjing University. All of the mice were acclimated to the environmental conditions for 7 day before the toxicological experiment was performed and were housed in a temperatureand humidity-controlled room (21 ± 2 °C, 55 ± 5% humidity, and specific pathogen-free) with a 12 h light/dark cycle. A total of 30 animals were randomly divided into the following six groups (n = 5): five test groups [exposure to MC-LR at 1, 5, 10, 20, and 40 μg/L in 0.08% (v/v) DMSO for drinking water, respectively] and one solvent control group. Water and rodent chow were freely available to mice. After a 12 month exposure, all animals were sacrificed by CO2. Liver samples were immediately excised and weighed, followed by being fixed with 4% (w/v) paraformaldehyde in phosphate-buffered saline (PBS) (4% PFA), and the residual livers were kept at −80 °C until use. All of the procedures of animal administration were approved by the Ethics Committee for Animal Research of Nanjing University. DNA Isolation and Analysis of 8-Hydroxy-2′-deoxyguanosine (8-OHdG) Contents in Genomic DNA of the Liver Cells by an Enzyme-Linked Immunosorbent Assay (ELISA). DNA was isolated from the liver cells by the slightly modified salting out procedure.19 Initial DNA concentrations were determined by a biophotometer (Eppendorf, Germany). DNA was kept at −80 °C before use. The 8-OHdG content of the genomic DNA was detected by an ELISA kit (highly sensitive 8-OHdG check, JaICA, Fukuroi, Shizuoka, Japan). According to the kit instructions, a typical layout for loading in triplicate for each sample was used to detect the 8-OHdG content and the processes (see Protocol S1 of the Supporting 4666

DOI: 10.1021/es5059132 Environ. Sci. Technol. 2015, 49, 4665−4672

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Environmental Science & Technology

Figure 1. Levels of 8-OHdG and MUTYH and OGG1 protein expression in the DNA of liver cells exposed to different MC-LR concentrations. The 8-OHdG content in liver at different groups was shown in panel A. The protein levels of (B and D) MUTYH and (C and E) OGG1 were measured with a western blot. β-Actin was used as a loading control. The data were showed as the mean ± SDM (n = 5). p < 0.05 was considered statistically significant.

and MC-LR-treated groups. One-way analysis of variance (ANOVA) was applied for calculating the significance of the difference among the MC-LR-treated groups. The relationships between two continuous variables were evaluated by the Pearson correlation coefficient. p < 0.05 was considered statistically significant.

the lysate was centrifuged at 12000g for 5 min, and the supernatant was denaturated and saved at −80 °C. Western blot was used to checked the protein expressions of the base excision repair (BER) pathway, the MutY glycoslase homologue (MUTYH), 8-oxoguanine DNA glycosylase (OGG1), the mitochondrial genes involved in the electron transport chain (ETC) and oxidative phosphorylation (OXPHOS) systems, COX3, CYTB, and ATP6, and the nuclear genes associated with mtDNA replication, POLG, mtSSB, TWINKLE, and TFAM. Total proteins were separated on 8−12% polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes (0.45 μM, Millipore, Billerica, MA). After blockage in phosphate-buffered saline with Tween 20 (PBST) with 5% skim milk and 0.5% bovine serum albumin (BSA), the PVDF membranes were incubated with the specific primary antibodies of the checked proteins (see Protocol S4 of the Supporting Information) overnight at 4 °C. The membrane was washed with PBST 6 times and incubated with the appropriate horseradish-peroxidase (HRP)conjugated secondary antibodies for 2 h at room temperature. The signal was detected using a chemiluminescent HRP substrate (Millipore, Billerica, MA). Statistical Analysis. The results were analyzed using SPSS 18.0 (SPSS, Inc., Chicago, IL). All of values were expressed as the mean ± standard deviation of the mean (SDM). Continuous variables were analyzed with the independentsample t test to assess significant difference between the control

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RESULTS MC-LR Increases the 8-OHdG Levels of Genomic DNA in the Liver Cells. MC-LR can cause DNA damage through the excessive generation of ROS. 8-OHdG is a typical biomarker of oxidative stress. Therefore, the 8-OHdG levels in the genomic DNA of liver cells were measured. Figure 1A showed that MC-LR exposure increased the 8-OHdG level of the genomic DNA isolated from the liver cells of the mice, although a statistically significant difference was only found between the 10 μg/L group and the control group (p = 0.037). MC-LR Damaged the DNA Integrity and Changed the Content of mtDNA in the Mouse Liver Cells. To identify the damage of nDNA and mtDNA in liver cells further, longrange PCR was performed. As shown in panels A and D of Figure 2, the products of long-range PCR for nDNA showed a weakly negative correlation with the MC-LR concentration (Pearson correlation coefficient = −0.305; p = 0.107). However, there was an obvious fluctuation between the exposed MC-LR concentration and the products of long4667

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Figure 2. Relative amplifications of nDNA and mtDNA with long-range PCR and mtDNA contents among the liver cells exposed to different MCLR concentrations. Long-range PCR was performed for both (A) nuclear β-globin fragment and (B) mtDNA in the liver cells. (C) mtDNA contents of the liver cells in different groups. (D) Correlation between MC-LR concentrations and products of long-range PCR for nDNA in the liver cells. The data were showed as the mean ± SDM (n = 5). p < 0.05 was considered statistically significant.

Figure 3. Transcriptional changes and protein levels of mitochondrial genes in liver cells of the mice exposed to different MC-LR concentrations. (A) Transcripts of COX3, CYTB, ATP6, and RNR2 in the liver cells were examined by real-time PCR. The protein levels of (B) COX3, (C) CYTB, and (D) ATP6 were measured with a western blot. β-Actin was used as a loading control. p < 0.05 was considered statistically significant.

compared to that in the 20 μg/L group, although the difference did not reach the statistically significant level (Figure 2B). These results showed that the integrity of nDNA and mtDNA were damaged, regardless of whether the content of mtDNA was changed. To compare the liver mtDNA content from the mice with different concentrations of MC-LR, a 117 bp fragment was amplified with quantitative PCR (qPCR). A

range PCR for mtDNA. In general, a downward trend of the relative amplification of mtDNA was observed with the increased MC-LR concentration, but the lowest products of long-range PCR for mtDNA were detected in the liver cells of the 20 μg/L group (p = 0.003; Figure 2B). Meanwhile, the one notable exception was that the products of long-range PCR increased in the liver cells of the 40 μg/L group, when 4668

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Figure 4. Expressions of POLG, mtSSB, TWINKLE, and TFAM in liver cells of the mice exposed to different MC-LR concentrations. (A and B) Transcripts of POLG, mtSSB, TWINKLE, and TFAM of liver cells were examined by real-time PCR. The protein expressions of (C) POLG, (D) mtSSB, (E) TWINKLE, and (F) TFAM were measured with a western blot. β-Actin was used as a loading control. The data indicate the mean ± SDM (n = 5). p < 0.05 was considered statistically significant.

and ATP6 (Figure 3D) were not significantly changed in the 1, 5, and 10 μg/L groups compared to the control groups, although their mRNA expressions increased among these genes. However, their protein expressions decreased when exposed to higher MC-LR concentrations (20 and 40 μg/L). Expression Alterations of the Regulation Genes for mtDNA Gene Replication in the Liver Cells. To explore the possible mechanism for mitochondrial damage with MC-LR exposure, the mRNA and protein expressions of these genes associated with mtDNA replication were quantified. We chose the regulation genes of mtDNA replication, which were components of the mtDNA replication fork and the nucleoid structure, including POLG, mtSSB, TWINKLE, and TFAM, to detect their expression levels by qRT-PCR and western blot analysis. The results showed that the mRNA expressions of POLG and mtSSB were significantly elevated in the 1 μg/L group compared to the control (Figure 4A). The mRNA expressions remained upregulated in the 5, 10, 20, and 40 μg/L groups compared to the control but showed a decrease when compared to the 1 μg/L group (Figure 4A). However, the protein levels did not change accordingly (panels C and D of Figure 4). The protein levels of POLG and TWINKLE were not significantly changed in the 1 and 5 μg/L groups compared to that in the control group (panels C and E of Figure 4), although their mRNA expressions increased in these groups (panels A and B of Figure 4). Notably, the protein expressions of POLG and TWINKLE genes remained at the higher level in the 10 and 20 μg/L groups and then decreased in the 40 μg/L group (panels C and E of Figure 4). In contrast, the protein levels of mtSSB and TFAM decreased in the MC-LR exposure groups (panels D and F of Figure 4).

downward trend of the mtDNA content was observed among the low-concentration MC-LR groups (1, 5, and 10 μg/L) compared to the control group (Figure 2C), but the difference was not statistically significant. However, the amount of mtDNA in the liver cells was significantly increased in the 20 μg/L group (p = 0.036 for 20 μg/L group versus control group). However, the mtDNA content turned down again when the mice were exposed to a higher concentration of MCLR (40 μg/L). Protein Expressions of MUTYH and OGG1 with MC-LR Exposures in the Liver Cells. Panels B and C of Figure 1 illustrate the changes of the MUTYH and OGG1 protein expression in the liver cells from MC-LR-exposed mice with the concentrations of 1, 5, 10, 20, and 40 μg/L in the drinking water for 12 months. In comparison to the control group, the protein levels of MUTYH (Figure 1B) and OGG1 (Figure 1C) showed an upward trend in the low-dose groups (1 and 5 μg/ L), and then the expressions were maintained in the 10 and 20 μg/L groups. However, their protein levels decreased when exposed to a higher MC-LR concentration (40 μg/L). MC-LR Exposure Altered the Expressions of the Mitochondrial Genes in the Liver Cells. To investigate the mechanism of the mitochondrial function alterations in these mice exposed to MC-LR, we chose mitochondrial genes that were important components of ETC and OXPHOS as well as mitochondrial rRNA, including COX3, CYTB, ATP6, and RNR2, to determine their expression levels by qRT-PCR and western blot analysis. The transcriptional changes of mitochondrial genes are shown in Figure 3A. In comparison to the control, the mRNA levels of COX3, CYTB, ATP6, and RNR2 genes showed an upward trend in the low-dose groups (1, 5, and 10 μg/L MC-LR), and then the expressions of these genes were either maintained (COX3 and ATP6) or decreased (CYTB and RNR2) in the 20 and 40 μg/L groups. In contrast, the protein levels of COX3 (Figure 3B), CTYB (Figure 3C),

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DISCUSSION The cumulative evidence showed that MC-LR-induced genotoxicity was mediated by the formation of ROS, which 4669

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probably a compensatory mechanism to maintain the normal level of mtRNA transcripts.27 However, the increase of the mtDNA content in the 20 μg/L group had not remained in the 40 μg/L group. We considered that mice exposed to the concentration of 40 μg/L MC-LR would impair the mtDNA synthesis and cause the mitochondrial function failure in the liver cells. MC-LR Damaged the Compensatory Mechanism and Then Induced a Change in the Expression of Mitochondrial Genes. A crucial question was whether the observed changes in the integrity and relative content of mtDNA as well as the decreased repair capacity of DNA oxidative damage in the cells exposed to MC-LR would substantially impair mitochondrial and cellular functions. The human mtDNA genome contains 37 genes coding for 13 subunits of the respiratory complexes I, III, IV, and V, 22 mitochondrial tRNAs, and 2 rRNAs.17 We selected three genes of the encoding subunits of the respiratory complexes, ATP6, COX3, and CYTB, and one of the rRNAs to address whether the integrity and content of mtDNA would affect the expression of mtDNA genes. We observed the transcription expressions of ATP6, COX3, CYTB, and RNR2 genes and detected the expression levels of ATP6, COX3, and CYTB proteins simultaneously. The results showed a trend in the upregulation of the transcription expression of the observed genes with increasing concentrations of MC-LR. We considered that the raised transcriptional level could be a compensatory mechanism for the increased mtDNA oxidative lesion, which could produce the abnormal mtRNA. If the compensation mechanism worked adequately, the mitochondrial function in cells could be maintained. In our current work, the transcriptional RNA levels of the four mtDNA genes kept rising in the 1, 5, and 10 μg/L MC-LR exposure groups but the protein expressions of the three genes encoding proteins, ATP6, COX3, and CYTB, showed no significant alteration among these concentrations. However, the protein levels of the three genes showed a decrease in the groups exposed to MC-LR at 20 and 40 μg/L, indicating that the higher MC-LR concentrations disrupted the regulation mechanism of compensatory proteins.14,28 MC-LR Exposure Affected the Regulation of mtDNA Replication. On the basis of the gene-dosage mechanism, the transcriptional expressions and protein products of mitochondrial genes were tightly concerned with the mtDNA content in the cells.29 The increased mtDNA content in the cells with oxidative stress could be considered as a moderate effect to maintain a stable level of mtRNA. However, the mtRNA level could also depend upon the integrity of mtDNA and the expressions of the nuclear genes that were involved in the regulation of mtDNA replication.28,30 To date, POLG, mtSSB, and TWINKLE, which make up the mtDNA replication complex, as well as TFAM, an essential component of the mitochondrial nucleoid structure, have been shown to be involved in mtDNA replication.15,31 POLG is the only mtDNA polymerase involved in mtDNA replication and damage repair. mtSSB, associated with mitochondrial biogenesis, can bind to and stabilize single-stranded DNA (ssDNA) and prevent the formation of secondary ssDNA structures, which could stop POLG. TWINKLE, known to co-localize with mtSSB in mitochondrial nucleoid, can bind both ssDNA and dsDNA and catalyze DNA unwinding. We observed the transcription expressions of POLG, mtSSB, and TWINKLE in the liver cells of the different MC-LR groups

leads to DNA damage and condensed chromatin in humans and animals.7,23 However, few studies have examined the alteration of the mtDNA content. Our present work provided a natural exposure mode of MC-LR and addressed the mechanism of the MC-LR effect on the mtDNA content and mtDNA replication. MC-LR Long Exposure Induced the Change of the 8OHdG Level in the Genomic DNA of Liver Cells. MC-LR induces an increase of 8-OHdG in mouse liver cells. The increase may be influenced by an elevated ROS level and insufficient function of the BER system in cells. The MUTYH and OGG1 are the main genes of the BER system and participate in the lesion repair of 8-OHdG in DNA.11 The upregulation of MUTYH and OGG1 expressions can enhance the capacity of DNA repair in the cells.24 Thus, the content of 8-OHdG in the DNA genome reflected the combined results of oxidative stress and the impaired DNA repair in the cells. Our results showed that a different 8-OHdG level among the mice was exposed to MC-LR at different concentrations. As shown in Figure 1A, no significant increase in 8-OHdG was observed in the mice exposed to 1 and 5 μg/L MC-LR compared to the control mice, but the expression levels of MUTYH and OGG1 proteins were obviously upregulated in both of the exposed groups (panels B−E of Figure 1). These data indicated that the oxidative stress caused by MC-LR exposure in the liver cells could induce the expressions of MUTYH and OGG1 to repair the increased DNA lesion. However, a significantly elevated level of 8-OHdG appeared in the 10 μg/L group. We believed that the mice exposed to MC-LR at the concentration of 10 μg/ L experienced increased oxidative stress compared to the mice exposed to 1 or 5 μg/L MC-LR. We also noted the protein expressions of MUTYH and OGG1 did not show an upregulation at this concentration (panels B and C of Figure 1). Higher 8-OHdG of liver DNA in the 10 μg/L group can be explained by the disturbance and/or exhaustion of the BER system.25 Interestingly, the level of 8-OHdG in the liver cells unexpectedly decreased with the increase in MC-LR concentrations at 20 and 40 μg/L groups. The histopathological results showed focal fatty degeneration, and apoptosis appeared in the liver tissues in the 20 and 40 μg/L groups (see Figure S2 of the Supporting Information). The reduced 8-OHdG level could be due to mitochondrial function failure of the liver cells. MC-LR Induced mtDNA Damage and the Change of the Mitochondrial Content. Our results showed that MCLR could cause DNA damage and the increase of the 8-OHdG level. mtDNA was more sensitive to oxidative damage than nDNA because it was in close proximity to the free-radicalproducing ETC. Moreover, MC-LR could cause the decline of the respiratory function of mitochondria.14,27 We investigated the alterations of mtDNA and mitochondrial function with MC-LR exposure and observed the relationships between the MC-LR concentration and the change of mtDNA integrity and mtDNA contents in the liver cells of the exposed mice. We observed that a decline in the efficiency of long-range PCR for nDNA and mtDNA was associated with the MC-LR concentration. Meanwhile, the content of mtDNA in the liver cells showed a negative correlation with the exposed MC-LR at relatively low concentrations (1, 5, and 10 μg/L), while an obvious increase in the mtDNA content appeared in the 20 μg/ L group. Hou et al.26 reported that the medium level of oxidative stress activated mtDNA synthesis, while the oxidative stress in height impaired the mtDNA synthesis. The increase in the mtDNA content observed in the 20 μg/L group was 4670

DOI: 10.1021/es5059132 Environ. Sci. Technol. 2015, 49, 4665−4672

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total RNA (Protocol S3), antibodies (Protocol S4), change of body and liver weights (Figure S1), and hematoxylin and eosin (H&E) staining of liver tissues (Figure S2). This material is available free of charge via the Internet at http://pubs.acs.org.

and determined the protein levels of these genes simultaneously. Our results showed an increased transcription expression in the 1, 5, and 10 μg/L groups compared to the control group. However, the protein expressions of POLG, mtSSB, and TWINKLE showed no significant alteration among these concentrations, which was similar to the mtDNA content in the liver cells of the three groups. These results indicated that MC-LR exposure in the concentrations of 1, 5, and 10 μg/ L had not disturbed the balance regulation of the mtDNA content in these groups. The protein levels of POLG and TWINKLE as well as the mtDNA content showed an increase in the 20 μg/L group, indicating that the cumulative lesion of mtDNA in this group could mediate the activation of the compensatory mechanism for mtDNA replication. In addition, mtSSB was a known regulator for mtDNA transcription and replication and was considered to be a candidate for the regulation of the mtDNA content.12,32 However, our results showed that a declined protein level of mtSSB was associated with the exposed concentration of MCLR, which was consistent with the change in the integrity of nDNA but not the mtDNA content. Previous studies indicated that the expression patterns of mtSSB were strikingly similar to those of TFAM in both brain and kidney tissues.33 Nuclearencoding TFAM showed a powerful effect on the transcription initiation of mtDNA and could have a regulatory role on the mtDNA content.32,34 A similar change pattern of the mtSSB and TFAM protein expression was also observed in these groups of MC-LR exposure to different concentrations. However, no direct evidence showed that the TFAM expression was involved in the mtDNA replication in the current study. Monttier et al.15 hypothesized that the mtDNA content was regulated by its threshold. When the copy numbers of mtDNA in the cells were reduced or increased to a lower or higher threshold, the mechanism of mtDNA replication or degradation would be triggered to stabilize the mtDNA level. This hypothesis could be used to explain the significant increase in the mtDNA content in the 20 μg/L group compared to that in the 10 μg/L group. Epidemiologic studies suggest that chronic exposure to low MC concentrations in drinking water is associated with a high risk for cancer, especially for hepatocellular carcinoma.35,36 The pathogenic mutations in mtDNA would contribute to the promotion of cancer by prevention of apoptosis.37 The limitation of the current works is that we had not observed the alteration of apoptosis and cell-cycle regulation and the altered mitochondrial function, which would be associated with the mtDNA impairment. Both of the assays could help with the understanding of the mitochondrial mechanism for the MCLR-induced hepatocellular carcinoma. Moreover, the current data that we presented of the MC-LR-induced impairment of mtDNA were limited with the sample size in this study. Expanded works should be administrated to address the molecular mechanism of the expression regulation of these genes associated with mtDNA replication and the phenotypic characteristics related with the impairments of mtDNA integrity and mtDNA content.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +86-25-83686495. Fax: +86-25-83686451. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21107048 and 81270152), the Open Foundation of the State Key Laboratory of Pharmaceutical Biotechnology (KF-GN-201201), and the Innovative Engineering Training of Jiangsu Province (CXLX12-0059).

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ASSOCIATED CONTENT

S Supporting Information *

Details on the materials, methods, and results, including the primer sequences (Table S1), process for the 8-OHdG assay (Protocol S1), long-range PCRs (Protocol S2), extraction of 4671

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Article

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DOI: 10.1021/es5059132 Environ. Sci. Technol. 2015, 49, 4665−4672