Toxic response indicators of microcystin-LR in F344 rats following a single-dose treatment

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Toxicon 51 (2008) 1068–1080 www.elsevier.com/locate/toxicon

Toxic response indicators of microcystin-LR in F344 rats following a single-dose treatment$ Madhavi Billam1, Sandeep Mukhi2, Lili Tang, Weimin Gao, Jia-Sheng Wang Department of Environmental Toxicology, The Institute of Environmental and Human Health, Texas Tech University, Box 41163, Lubbock, TX 79409-1163, USA Received 30 September 2007; received in revised form 20 January 2008; accepted 28 January 2008 Available online 6 February 2008

Abstract Microcystin-LR (MCLR) is the most common hepatotoxic cyanotoxin produced primarily by Microcystis aeruginosa. In this study, young F344 rats were intraperitoneally injected with a single dose (25, 50, 100, and 150 mg/kg) of MCLR to explore possible toxic effect and toxic response indicators. Acute toxic symptoms, including body weight loss and death, were monitored for 7 days. Mortality reached 100% (9/9) in rats treated with a single MCLR dose of 150 mg/kg. Histopathological examination showed spot necrosis in the liver of animals treated at low doses, while massive hemorrhage and widespread necrotic foci occurred at higher doses, indicating extensive liver damage. Protein phosphatase 2A (PP2A) expression showed a dose-dependent decrease in the liver. Immunohistochemical localization indicated that nuclear PP2A was affected first, followed by cytoplasmic PP2A. In addition, there was a significant increase in sphingolipid levels at higher doses, indicating the involvement of a ceramide-mediated apoptotic pathway. Expression of apoptosis and cell cycle regulatory proteins like Bax, Bcl2, and Bad showed a dose-dependent decrease. This study demonstrated that treatment with a single dose of MCLR caused liver damage, increased sphingolipid levels, and decreased PP2A expression, which ultimately down-regulated the expression of Bcl2 family proteins. r 2008 Elsevier Ltd. All rights reserved. Keywords: Microcystin-LR; Acute toxicity; F344 rats; PP2A; Toxic response indicators

1. Introduction $

Ethical statement: This study used young male Fisher 344 rats (90–110 g) obtained from the Harlan Lab Animals Inc. (Indianapolis, IN, USA). Animal maintenance, husbandry and treatment with MCLR was reviewed and approved by institutional Animal Care and Use Committee at Texas Tech University. Corresponding author. Tel.: +1 806 885 0320; fax: +1 806 885 4577. E-mail address: [email protected] (J.-S. Wang). 1 Current address: Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD 21231, USA. 2 Current address: Department of Embryology, Carnegie Institution of Washington, Baltimore, MD 21218, USA. 0041-0101/$ - see front matter r 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2008.01.014

Microcystins (MCs) are naturally occurring hepatotoxic cyanotoxins produced primarily by Microcystis spp. (Carmichael, 1981) and distributed in fresh water bodies around the world (Ueno et al., 1998). Exposure to MCs, especially microcystin-LR (MCLR), in ponds and water reservoirs has often resulted in acute and lethal toxicities to domestic animals (Carmichael, 1992). In humans, exposure to MCs resulted in hepatotoxicosis, gastroenteritis, allergic/irritation reactions, and also death (Pouria

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et al., 1998). Epidemiological studies in high-risk populations of China observed a strong correlation between liver cancer incidence and exposure to MCs (Yu, 1995; Ueno et al., 1996). The primary target organ of MCLR is liver, as a hepatocyte-specific organic anion transporting polypeptide (OATP) is required to carry MCLR into hepatocytes (Fischer et al., 2005). Acute hepatic toxicosis is attributed to rapid uptake of the toxin by carrier-mediated transport (Pace et al., 1991), inhibition of protein phosphatases (PPs) (Yoshizawa et al., 1990), subsequent breakdown and aggregation of actin filaments (Hooser et al., 1991), microtubules, and intermediate filaments (Wickstrom et al., 1995) resulting in cytoskeletal damage and morphological changes in hepatocytes. Exposure to MCLR results in apoptosis, which has been well documented in vitro and in vivo (Eriksson et al., 1990; Chen et al., 2005). Upon uptake of MCLR by OATP transporters, MCLR inhibits PP1 and 2A activity in the liver (Imanishi and Harada, 2004), and generates reactive oxygen species (ROS) (Nong et al., 2007; Weng et al., 2007). Inhibition of PP2A activates multiple mitogenactivated protein kinases including ERK1/2, JNK, and p38, all contributing to apoptosis (Komatsu et al., 2007). Studies suggest that mitochondrial permeability is also involved in MCLR-mediated apoptosis (Ding et al., 2000), and could be triggered by the ROS (Mikhailov et al., 2003). Furthermore, cysteine proteinases (calpain) are speculated to play an important role in MCLR-mediated apoptosis. For instance, in rat hepatocytes MCLR-induced apoptosis was caspase independent but was triggered by membrane permeability transition followed by calpain activation and release of cytochrome-c (Ding et al., 2002). Recently, Clark et al. (2007) have shown up-regulation of calpain subunits capn2 and capns1 in mice exposed to 40 mg/kg MCLR for 28 days, indicating a potential role of calpains in MCLR toxicity. Release of cytochrome-c results in caspase-dependent and/or caspase-independent apoptosis (Hampton et al., 1998). Upon exposure to MCLR, cytochrome-c does not bind to Apaf-1 (Li et al., 1997), but acts through activation of Ca2+-dependent calpains that cleave cytoskeletal proteins and pro-apoptotic Bax (Vanags et al., 1996; Wood and Newcomb, 1999). Finally, researchers have speculated on the role of ceramides in MCLR-mediated apoptosis (Gehringer, 2004); however, no studies have been reported. Collectively, alterations in the expression of genes/

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proteins involved in MCLR-induced apoptosis and tumor promotion are not well explored. Hence, studies designed to explore the molecular targets in MCLR-induced apoptosis pathways will provide data for understanding mechanisms involved in acute MCLR toxicosis. The objectives of this study were to investigate the mechanism of action involved in MCLR-induced toxicity/apoptosis following acute exposure. In addition to exploring the effect of MCLR on nuclear and cytosolic PP2A, the ceramide-mediated apoptotic pathway and the role of Bcl2 family proteins following acute MCLR treatment were also investigated. 2. Materials and methods 2.1. Animals Young male Fisher 344 rats (90–110 g) were obtained from Harlan Lab Animals Inc. (Indianapolis, IN, USA) a week before experiments were performed and housed individually in metabolic cages under controlled conditions of temperature (2271 1C), light (12 h light–dark cycle), relative humidity (50%75%), and ventilation frequency (18 times/h). NIH open formula diet (NIH-07 Rat and Mouse Feed; Zeigler Bros., Inc.; Gamers, PA) and deionized (DI) water were supplied ad libitum. Animal maintenance, husbandry, and treatment with MCLR were reviewed and approved by the institutional Animal Care and Use Committee at Texas Tech University. 2.2. Treatment Rats were randomly divided into five groups (nine animals per group) and housed in metabolic cages. Selection of doses was based on studies previously conducted in our laboratory (LD50 ¼ 250 mg/kg; unpublished) and from previously published MCLR data (Miura et al., 1991; Fawell et al., 1999). One group was given only solvent vehicle (DI water) and used as the control; the other four groups were treated with MCLR (Axorra LLC, San Diego, CA, USA) intra-peritoneally (i.p.) at 25, 50, 100, or 150 mg/kg body weights. Animals were carefully observed after dosing. Body weights and symptoms of toxicity were recorded. Three animals per group were euthanized by CO2 inhalation and necropsied at 1, 3, and 7 days after MCLR treatment. Blood was collected by a hypodermic syringe and all major

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organs were excised and divided into four sections. One section was fixed in 10% buffered formalin for histopathology and immunohistochemistry, and the remaining three sections were frozen in liquid nitrogen for western blot and enzyme activity studies. 2.3. Serum biochemical analysis Serum biochemical analysis was performed at the Clinical Pathology Lab, Texas Veterinary Medical Diagnostics Laboratory System (College Station, TX), using a Hitachi 911 automated serum biochemistry analyzer (Roche Laboratories, Indianapolis, IN). The parameters included total serum protein, albumin, calcium, phosphorous, glucose, blood urea nitrogen (BUN), creatinine, total bilirubin, alkaline phosphatase (ALP), creatine kinase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), globulin, albumin/globulin ratio, gamma glutamyl-transferase (GGT), amylase, and cholesterol. 2.4. Liver histopathology Liver sections were fixed in 10% formalin, embedded in paraffin wax, and sectioned by a microtome (4 mm). These sections were processed routinely by staining with hematoxylin and eosin (H&E) and observed under a microscope for pathological conditions.

by a fluorometer. The method used was adapted with modifications from the published procedure (Fontal et al., 1999). Briefly, 35 ml of sample (cell or tissue homogenates) was mixed and incubated at 37 1C for 10 min with 5 ml of NiCl2 (40 mM), 5 ml of 5 mg/ml bovine serum albumin, and 35 ml of phosphatase assay buffer (pH 7.4). To this mixture, 120 ml of 100 mM DiFMUP was added, mixed, incubated at 37 1C, and assayed after 30 min. The fluorescence was measured using a fluorescence microplate reader (Fmax, Molecular Devices, Sunnyvale, CA, USA) at 355 nm (excitation) and 460 nm (emission). 2.7. Sphingolipid analysis Sphingolipid metabolites, sphinganine (Sa) and sphingosine (So), in serum samples were analyzed by HPLC according to a recently published method (Cai et al., 2007). Briefly, 50 ml of serum was mixed with 20 ml of internal standard (carbon-20) and 130 ml of 50 mM phosphate buffer (pH 9.0). The mixture was extracted with 800 ml of dichloromethane (CH2Cl2) and centrifuged at 3000 rpm for phase separation. Organic phase (600 ml) was fractionated and dried in vacuo at room temperature. The dried pellet was mixed with 275 ml of methanol and 25 ml of O-pthaladehyde for derivatization, and analyzed by HPLC with fluorescence detection at excitation/emission of 340/455 nm. 2.8. Western blot

2.5. Tissue lysate preparation Tissue lysate was prepared using T-PER (Tissue protein extraction reagent; Pierce Biotechnology Inc., Rockford, IL, USA). Briefly, for 0.5 mg of liver tissue, 1 ml T-PER reagent was added and the mixture homogenized. Samples were centrifuged at 14,000 rpm for 5 min. Supernatant tissue lysate was stored at 20 1C or used immediately. For the PP inhibition assay, these lysates were prepared at a concentration of 0.5 mg tissue/ml T-PER reagent. 2.6. Measurement of protein phosphatase activity The principle of the assay is that PP can specifically remove phosphate groups from a fluorescent substrate, 6, 8-difluoro-4-methylumbelliferyl phosphate (DiFMUP, Molecular Probes Eugene, OR, USA) to produce 6, 8-difluoro-7hydroxy-4-methylcoumarin, which can be measured

Western blot was done using a standard protocol. Briefly, tissue lysates were prepared and their protein content determined. Lysates were reduced by heating (95 1C) for 5 min in the presence of 2-mercaptoethanol. The reduced samples were electrophoresed on Bio-rad precast gradient gel (4–20%) and transferred to a PVDF membrane using a Trans-blot SD semidry electrophoretic transfer cell (Bio-Rad Laboratories, Richmond, CA, USA). The membrane was blocked by 3% non-fat dry milk in 0.1% Tween-20, and then allowed to bind to primary antibody overnight on an orbital shaker. Primary monoclonal antibody for PP2A (anti-PP2A C subunit, clone 1D6) was obtained from Upstate (Upstate Biotechnology, Lake Placid, NY, USA), and Bax, Bcl2, and Bad from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The membrane was then placed in secondary antibody (anti-mouse HRP), washed, treated with

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2.9. Immunohistochemistry Immunostaining for subcellular distribution of PP2A was performed using a commercially available kit (Vectastains Elite ABC kit, Vector Labs, Burlingame, CA, USA). Briefly, tissue sections (4–6 mm thick) on the slides were deparaffinized in xylene and subsequently hydrated gradually by graded alcohol washes. Antigen was retrieved by placing the hydrated sections in antigen retrieval solution. After washing, the slides were incubated with 3% hydrogen peroxide and further washed with PBS. Slides were then incubated in normal horse serum, followed by blocking with avidin and biotin. Next, slides were incubated with primary antibody for PP2A (10 mg/ml) overnight at 4 1C. After washing with PBS, sections were incubated with biotin-conjugated secondary antibody at 1 mg/ml, followed by incubation with avidin–biotin peroxidase enzyme reagent and peroxidase substrate (DAB). Slides were then washed, dehydrated, and mounted. Digital images of DAB-stained sections were imported to Image-Pros Express (Media Cybernetics Inc., Bethesda, MD, USA) software for further analysis. A grid was placed on each image and random spots in the nuclear/cytoplasmic region were selected. The optical density of 30 spots per image was obtained, and the average optical density of each sample was calculated. For validation, representative sections were processed with only normal horse serum (used as blocking agent), and with mouse IgG only as a negative control. 2.10. Data analysis Data were expressed as mean7SE for each treatment group. One-way ANOVA was used for normally distributed variables, followed by a posthoc test (Tukeys HSD or Dunnets) as required. For parameters that were not normally distributed, Kruskal–Wallis test or Wilcoxon rank sum test was used to compare the differences among treatment groups and time points. Significance was set at

a ¼ 0.05. All analyses were performed using 15.0 SPSS software (2006, SPSS, Chicago, IL, USA). 3. Results 3.1. General acute toxic effects General acute toxic effects were monitored in rats treated with a single dose of MCLR for up to 7 days. A significant decrease in body weight was observed in animals treated with a single dose of MCLR at 100 and 150 mg/kg (Fig. 1). Deaths were recorded for 9/9 (100%) rats receiving a single dose (150 mg/kg) within 7 days post-treatment. Significant dose-dependent alterations in serum biochemical parameters were observed in animals 1 day after treatment with a single dose of MCLR (Table 1). As compared to control animals, levels of ALP, AST, and ALT were significantly elevated in animals treated with a single MCLR dose of 50 mg/kg or higher. Levels of creatinine, GGT, BUN, and bilirubin were significantly elevated in animals treated with 100 mg/kg MCLR or higher. In contrast, levels of amylase, glucose, and cholesterol significantly decreased in a dose-dependent manner (Table 1). No significant changes in serum biochemical parameters were observed at 3 and 7 days post-treatment (data not presented), which suggests a recovery from acute hepatic toxic effect. 3.2. Histopathology of liver tissues Histopathologic evaluation for H&E-stained liver sections is shown in Fig. 2. At 1 day after treatment 45 Weight gain/loss (gm)

chemiluminescent reagents (ECL), and then exposed to X-ray film. Quantification was done using a TM Chemimager 4400 low light imaging system (Alpha Innotech Corporation, San Leandro, CA) TM and densitometric analysis using Alpha Ease Software (Alpha Innotech Corporation, San Leandro, CA, USA).

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control 25 ug/kg 50 ug/kg 100 ug/kg 150 ug/kg

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Fig. 1. Effect of acute doses of MCLR on body weight at 1, 3, and 7 days post-exposure. On 1 and 3 days post-exposure, there was a significant dose-dependent loss in weight at both the highest doses, while no effect was observed 7 days post-exposure.

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Table 1 Serum biochemical analysis of animals treated with different doses of MCLR for 1 day represented as mean7SD Control

25 mg/kg

50 mg/kg

Parameters with significant decrease Albumin (g/dl) 4.270.1 Amylase (U/I) 2382.07104.0 Cholesterol (mg/dl) 84.778.1 Glucose (mg/dl) 138.7732.3

4.270.2 2299.07208.2 101.3714.6 149.0747.8

4.070.1 1576.37189.0** 94.073.0 103.3710.0

Parameters with significant increase ALP (U/I) 488.7719.7 ALT (U/I) 79.077.9 AST (U/I) 229.7793.5 BUN (mg/dl) 20.371.4 Creatinine (mg/dl) 0.270.1 GGT (U/I) 3.070.0 Total bilirubin (mg/dl) 0.170.0

515.3733.8 81.7715.0 283.3790.3 18.471.2 0.170.0 3.070.0 0.170.0

650.0760.4** 1238.77166.3** 1177.77242.7** 20.973.1 0.270.0 3.070.0 0.170.0

Parameters with no changes A/G CK (U/I) Globulins (mg/dl) Serum calcium (mg/dl) Serum phosphorous (mg/dl) Total protein (g/dl)

2.470.2 1882.07598.4 1.870.2 11.270.3 12.070.8 6.070.3

2.570.1 3158.07811.2 1.770.1 11.971.0 14.271.8 5.970.3

100 mg/kg

4.270.0 772.57210.0** 95.5736.1 78.5714.9 846.071.4** 14,398.574256.1** 20,170.577022.3** 51.878.6** 0.470.1* 5.070.0 2.271.3*

2.470.3 1762.07310.2 1.770.2 11.270.4 11.071.1 5.870.3

2.470.1 2692.072517.3 1.770.1 11.070.1 10.570.6 6.070.1

150 mg/kg

3.870.3* 628.57225.6** 44.070.0** 45.5723.3** 792.5767.2** 16,445.571788.3** 19,713.571539.4** 90.279.1** 0.570.1** 21.0711.3** 2.771.6* 2.570.1 1754.57914.3 1.570.1 10.570.6 12.870.5 5.370.4

Asterisk (*) indicates significant differences from control (po0.05) and (**) indicates po0.01.

with 25 mg/kg MCLR, rat liver tissue sections generally showed normal morphology with scattered spot necrosis and occasional cellular karyomitosis (Fig. 2B). For rats receiving a single MCLR dose of 50 mg/kg, scattered necrotic foci with leukocyte infiltration was observed around central veins in the liver. Eosinophilic bodies and pyknotic nucleated cells were also observed. In rats treated with 100 mg/kg MCLR, a great number of hepatocytes became necrotic and large necrotic foci were observed (Fig. 2C). Necrosis usually occurred in centrilobular and midzones. Polymorphonuclear leukocytes infiltrated into the foci. In rats receiving a single dose of 150 mg/kg MCLR, widespread necrosis, intra-hepatic hemorrhage, and infiltration of inflammatory cells were observed (Fig. 2D). Liver cell plates showed disarray and some apoptotic bodies. At 3 days post-treatment, acidophilic apoptotic bodies, vacuolated cells, and a few karyomitotic cells were noted in rats receiving a single dose of 25 and 50 mg/kg MCLR. Hypertrophic and pyknotic cells with infiltrated inflammatory cells were observed in rats receiving a single dose of 100 or 150 mg/kg MCLR. At 7 days post-treatment, a great number of hypertrophic cells were observed with massive hypertrophic foci and eosinophilic plasma found in animals receiving

a single dose of 100 mg/kg MCLR (slides not shown). 3.3. Serum and liver PP activity Serum PP activity in animals treated with a single dose of 100 or 150 mg/kg MCLR was significantly elevated at 1 day post-treatment as compared to control (po0.05); the elevation lasted up to 7 days post-treatment in the 100 mg/kg treatment group. No significant difference in the PP activity was observed in the liver cytosol between treated and control animals at any time point. 3.4. Sphingolipid levels in serum Alteration in the levels of sphingolipid metabolites in serum samples of rats treated with different doses of MCLR was observed. Serum levels of Sa and So in MCLR-treated rats were significantly higher than controls (p40.05) at both the 100 and 150 mg/kg dose groups 1 day after treatment (Fig. 3A and B). No significant differences in Sa and So levels were observed between treated groups and controls at 3 and 7 days post-treatment (Fig. 3A and B).

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Fig. 2. Histopathology of the rat liver after 1-day exposure to acute MCLR. (A) Control liver with normal vacuolation; (B) at 24 h postexposure, inflammatory infiltration at 25 mg/kg treatment; (C) massive centrilobular and midzonal necrosis at 100 mg/kg treatment; (D) massive necrosis, leukocyte infiltration and intra-hepatic hemorrhage at 150 mg/kg treatment.

3.5. Protein expressions of PP2A, Bax, Bcl 2, and Bad Alteration of protein expression patterns was detected by western blot analysis of liver tissues collected 1 day after MCLR treatment. The expression of PP2A showed a significant decrease in animals treated with a single dose of 100 mg/kg (po0.05) (Fig. 4A). No significant difference was observed in animals at the other doses. A significant decrease in the expression of Bax was observed in rats treated with the highest dose (150 mg/kg) (po0.05) (Fig. 4B). However, there was no significant decrease in the expression of Bax protein in other treated groups. A dose-dependent decrease in Bcl 2 expression was observed in liver tissues from animals treated with single doses of MCLR. As

compared to control animals, the decrease in Bcl 2 expression was significant in all dose groups (po0.05), with the exception of the lowest dose (25 mg/kg) (Fig. 5A). Similar patterns were observed in Bad expression (po0.05) (Fig. 5B) except for the lowest dose (25 mg/kg) group. 3.6. PP2A distribution pattern Nuclear and cytoplasmic distributions of PP2A were evaluated by immunohistochemistry in liver sections collected 1 day after the single dose treatment. As compared to control sections, nuclear PP2A staining intensity was decreased; cytoplasmic PP2A was slightly increased in rats receiving a single dose of 25 mg/kg (Fig. 6D and E). In rats treated with 50 mg/kg, nuclear PP2A was completely absent,

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Sa levels (nM)

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Fig. 3. Effect of MCLR exposure on sphinganine (Sa) (Panel A) and sphingosine (So) (Panel B) levels in the serum of rats. After 1-day exposure, both Sa and So levels in serum were significantly higher than control at the 100 and 150 mg/kg doses when compared by ANOVA. No effect was observed at any of the doses after 3 and 7 days post-exposure. Asterisk (*) indicates significance (po0.05).

while cytoplasmic PP2A significantly increased (Fig. 6B, D, E). Both nuclear and cytoplasmic PP2A decreased significantly (po0.05) in rats receiving a single dose of 100 mg/kg MCLR (Fig. 6C–E). However, the expression of PP2A was very strong in both nuclear and cytoplasmic regions in animals receiving a single dose of 150 mg/kg MCLR. 4. Discussion MCLR is a ubiquitously distributed hepatotoxic cyanotoxin (Yu, 1995; Ueno et al., 1996). Although many reports in the literature have described its toxic effects, toxic response indicators, especially molecular targets of MCLR, are mostly speculated. In this study, we investigated acute effects of MCLR on toxicity symptoms, serum biochemistry, histopathological changes in the liver, PP activity, expression of pro- and anti-apoptotic protein levels, as well as changes in PP2A distribution patterns. After receiving a single injection of different doses of MCLR, apparent dose-dependent body weight loss was observed and serum biochemical parameters were significantly altered. Serum levels of ALP, AST, ALT, GGT, BUN, and creatinine,

which represent liver function, increased significantly, while levels of amylase, glucose, and cholesterol significantly decreased (po0.05); this suggests damage primarily to the liver (Renner and Dallenbach, 1992). Comparable results were reported in both fed and fasted rats treated with 100 mg/kg MCLR, where levels of ALT and sorbitol dehydrogenase were increased significantly over controls (Miura et al., 1991). In another study, rats exposed to 100 mg/kg MCLR for 15–90 min had a significant increase in BUN, creatinine, ALT, and AST, while serum glucose levels declined (Miura et al., 1989). Liver toxicity observed in this study is consistent with previous single dose reports. Balb/c mice treated with 45 mg/kg MCLR had hepatocellular hypertrophy, loss of cytosolic vacuolation, apoptosis, hepatocytomegaly, and karyomegaly (Guzman and Solter, 2002). An i.p. injection of 180 mg/kg MCLR for 10–60 min in rats led to hepatocyte dissociation, massive necrosis, endothelial damage, and finally the release of hepatocytes and debri into circulation (Hooser et al., 1990). In another study, SD rats i.p. injected with 500 mg/kg MCLR showed loss of cell–cell contact, shrinkage and rounding of hepatocytes, and loss of microvilli. This was

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Fig. 4. Expression of PP2A and Bax protein in liver of rats exposed to acute doses of MCLR. Panel A represents a significant decrease in PP2A protein expression at 100 mg/kg, while a significant decrease in Bax was observed at the highest dose (150 mg/kg) (panel B). Asterisk (*) indicates significant difference from control (ANOVA and Tukey’s HSD, po0.05).

Fig. 5. Alteration in the expression of Bcl2 and Bad proteins in liver upon exposure to a single dose of MCLR. Significant decrease in Bcl2 protein was observed at the three highest doses (panel A), while there was a significant decrease in Bad expression at all doses tested (panel B). Asterisk (*) indicates significant difference from control (ANOVA and Tukey’s HSD, po0.05).

followed by breakup of hepatocytes into apoptotic bodies, which indicated that hepatic damage by MCLR was due to rapid induction and progression of apoptosis in hepatocytes (Hooser, 2000). Similar to these results, we observed scattered necrosis and karyomitosis at low doses, transitioning into necrotic foci, leukocyte infiltration and pyknotic nucleated cells at 50 mg/kg, large necrotic foci at 100 mg/kg, and finally massive necrosis and intrahepatic hemorrhage at 150 mg/kg. A recent study has also found hepatocellular hypertrophy in mice treated daily with 40 mg/kg MCLR for 4 days (Clark et al., 2007). Studies conducted on different vertebrate and invertebrate models show that MCLR inhibits PP1 and 2A enzymes (Eriksson et al., 1990) and it has 40

times more affinity for PP2A than PP1 (Suganuma et al., 1992). In this study, we observed that serum PP activity was significantly (po0.05) increased 1 day after treatment with 100 or 150 mg/kg MCLR, while no changes were found at lower doses. This may be due to rapid elimination of MCLR from the circulation as observed in mice treated with 35 mg/kg radiolabelled MCLR, where the toxin was completely eliminated from plasma within an hour after exposure (Robinson et al., 1991). Similarly, no significant change in liver tissue lysate PP activity was observed. This is presumably because MCLR in liver forms adducts with PP, which could be subsequently eliminated (Jayaraj and Lakshmana Rao, 2006). Because our study samples were collected 1 day post-treatment or later, it appears

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Fig. 6. Immunohistochemical localization of PP2A in the liver of rats exposed to acute doses of MCLR (A) control; (B) 50 mg/kg—nuclear PP2A expression has declined, while that of cytoplasm is greater than control; (C) 100 mg/kg—both nuclear and cytoplasmic PP2A expression are completely inhibited. Immunohistochemical quantification of nuclear (D) and cytosolic PP2A expression (E) in sections of liver. Asterisk (*) indicates significant difference from control (ANOVA and Tukey’s HSD, po0.05).

that PP inhibition is the earliest toxic response to MCLR. The inhibition may mobilize the release of more PPs from liver to circulation, as shown by a dose-dependent decrease in PP2A protein in the liver. There are two types of PP2A, nuclear PP2A and cytoplasmic PP2A. Currently it is still not clear which one is the target of MCLR or if both are targets. There have been some contradicting results published (Hooser et al., 1991; Yoshida et al., 1998). An uptake and subcellular localization study of 10 mg/ml dihydro-MCLR in hepatocytes and in perfused rat livers showed that 66–77% of MCLR localizes in the cytoplasmic fraction, of which 50–60% was bound to cytoplasmic protein, while

13–18% was found in the plasma membrane/ nuclear fraction (Hooser et al., 1991). Yoshida et al. (1998) reported that MCLR in mice was localized in both nucleus and cytoplasm. In this study, although the lower dose of MCLR did not change total PP2A protein levels, a significant decrease in nuclear PP2A and an increase in cytoplasmic PP2A were observed. Both nuclear and cytoplasmic PP2A were reduced significantly at 100 mg/kg MCLR, indicating that dose determines the localization of PP2A. The expression of PP2A was very strong in both nuclear and cytoplasmic regions at the 150 mg/kg dose, which could reflect a survival mechanism in stressed cells. On the other hand, the high PP2A levels at 150 mg/kg observed in

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this study could be biased as the liver was completely damaged at this dose; our analysis involved only intact or viable cells. A previous study also suggested nuclear localization of MCLR as the primary event in mice exposed to a single i.p. dose (45 mg/kg), and an immunopositive reaction with anti-MC antibody was observed in both cytoplasm and nuclei in mice exposed to lethal doses of MCLR (57.6–101.3 mg/kg) (Yoshida et al., 1998). However, the nuclear localization of MCLR was weak and the intensity was equal to or less than that detected in the cytoplasm of mice exposed to repeated doses of MCLR (Guzman and Solter, 2002). It appears that MCLR binds to nuclear PP2A at low doses, and binds to both nuclear and cytoplasmic PP2A at high doses. Therefore, nuclear PP2A is the primary target of MCLR after acute exposure, followed by cytoplasmic PP2A. PP2A or PP1 inhibition by MCLR results in hyperphosphorylation of cytosolic and cytoskeletal proteins (Eriksson et al., 1990; Falconer et al., 1992) and increased phosphorylation stimulates polymerization of microtubule proteins (Yamamoto et al., 1988; Brugg and Matus, 1991) and alters cytoskeletal organization and function (Geisler and Weber, 1988; Chou et al., 1990; Cadrin et al., 1992). Subsequent breakdown of the cytoskeleton may lead to cell death. Hence, inhibition of PP2A by MCLR could be a pathway responsible for the induction of apoptosis after acute exposure. In this study, MCLR up-regulates the sphingolipid metabolites Sa and So after 1-day exposure, suggesting the involvement of ceramide biosynthesis. Ceramide, a naturally occurring membrane sphingolipid, plays an important role in sphingomyelin biosynthesis that regulates cell membrane porosity, signaling, apoptosis, and cell growth arrest (Cinque et al., 2003). It is reported that upregulation of ceramide opens mitochondrial membrane pores and releases cytochrome-c, resulting in apoptosis (Gulbins, 2003). So is pro-apoptotic by activating caspases (Sweeney et al., 1998), JNK and MAP kinases (Jarvis et al., 1998) and inhibiting PKCs (Hannun and Bell, 1989), c-Src/v-Src protein kinases (Igarashi et al., 1989), and CaMKII (Jefferson and Schulman, 1988). The increase in Sa found after 1-day exposure indicates up-regulation of ceramide, while the increase in So indicates activation of the ceramide-mediated signaling pathway (Hannun, 1994; Kolesnick and Fuks, 1995). These observations suggest that MCLR-induced cytochrome-c-dependent apoptosis (McDermott

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et al., 1998; Li et al., 2001; Ding et al., 2002) could be controlled through up-regulation of sphingolipids and ceramide, enhancing the opening of mitochondrial membrane pores and the release of cytochrome-c. Future study will focus on analysis of both sphingolipids and cytochrome-c. A significant decrease in the protein expression of Bcl2 was observed at doses X50 mg/kg and in Bad at doses from 25 to 150 mg/kg. The alterations in the expression of Bax, Bad, and Bcl2 observed in this study are consistent with previous studies (Hu et al., 2002). For instance, Bax gene expression was significantly decreased in MCLR-dosed rats, and MCLR-induced apoptosis was reported to be through a Bcl2-mediated pathway (Hu et al., 2002). Chen et al. (2005) also reported that female BALB/c mice exposed to 50–70 mg/kg MCLR for 24 h had a significant decrease in Bcl2 and Bad protein expression, and a slight increase in Bax protein expression. Bad and Bax are pro-apoptotic and act by dimerization with Bcl2 proteins, which inactivates Bcl2 function on mitochondrial membrane and the release of cytochrome-c (Yang et al., 1995; Zha et al., 1996). Alternatively, inhibition of PP2A by MCLR could also affect the dephosphorylation and synthesis of Bcl2 family proteins (Ruvolo et al., 1999). In a mouse model, MCLR-induced apoptosis in the liver is proposed to be regulated by two independent pathways: a Bid–Bax–Bcl-2-mediated apoptosis in mice exposed to a single dose of 50 mg/kg for 24 h, and a ROS-mediated apoptotic pathway in mice exposed to a single dose greater than 70 mg/kg (Chen et al., 2005). The ROSmediated apoptotic pathway may play an important role in regulating apoptosis (Nong et al., 2007; Weng et al., 2007), but additional studies are needed. In conclusion, treatment with single doses of MCLR reduced body weight gain, altered serum biochemical parameters, and caused liver damage in F344 rats. After 1-day post-treatment, serum and liver PP activities returned to normal, while PP2A expression in the liver was reduced in a dosedependent manner. Levels of sphingolipid metabolites were increased and Bcl2-regulated apoptosis pathways were involved. Based on the findings in this study and others, a possible mechanism for toxic response following single-dose exposure to MCLR was proposed (Fig. 7). Upon acute exposure to MCLR, sphingolipid levels were increased significantly, which stimulates ceramide-mediated

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Fig. 7. Possible mechanism for toxic response indicators of MCLR following a single dose. Upon acute exposure to MCLR, sphingolipid levels increase significantly, which stimulates ceramide-mediated signaling pathway, resulting in caspase-independent and cytochrome-c-mediated apoptosis pathway. Alternatively, MCLR exposure inhibits PP2A levels, which regulate both Bax and Bad levels and further enhance the Bcl2-regulated apoptotic pathway.

signaling pathway, resulting in caspase-independent and cytochrome-c-mediated apoptosis pathway, which down-regulated Bcl2 proteins. Alternatively, MCLR exposure inhibits PP2A levels, which regulate both Bax and Bad levels and further activates the Bcl2-regulated apoptotic pathway. This mechanism will be further explored in various in vitro and in vivo models, including concurrent measurements of both cytochrome-c and sphingolipids. Acknowledgments This study was partially supported by the research grant of CA98643 from NIH (to JS Wang) and the research contracts DAAD13-00-C-0056 and DAAD13-01-C-0053 from the Research, Development and Engineering Command, US Army (to JS Wang). We acknowledge Drs. Ernest Smith and Reynaldo Patin˜o for providing access to their research facilities, Dr. Qingsong Cai for sphingolipid analysis, Dr. Jianjia Su for histopathological evaluations, and Dr. Todd Anderson for reviewing the manuscript. References Brugg, B., Matus, A., 1991. Phosphorylation determines the binding of microtubule-associated protein 2 (MAP2) to microtubules in living cells. J. Cell Biol. 114, 735–743. Cadrin, M., McFarlane-Anderson, N., Aasheim, L.H., Kawahara, H., Franks, D.J., Marceau, N., French, S.W., 1992.

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