Journal of Toxicology and Environmental Health
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Hepatic lipid peroxidation, sulfhydryl status, and toxicity of the blue‐green algal toxin microcystin‐LR in mice S. J. Hermansky , S. J. Stohs , R. S. Markin & W. J. Murray To cite this article: S. J. Hermansky , S. J. Stohs , R. S. Markin & W. J. Murray (1990) Hepatic lipid peroxidation, sulfhydryl status, and toxicity of the blue‐green algal toxin microcystin‐LR in mice, Journal of Toxicology and Environmental Health, 31:1, 71-91, DOI: 10.1080/15287399009531438 To link to this article: http://dx.doi.org/10.1080/15287399009531438
Published online: 20 Oct 2009.
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Date: 16 November 2015, At: 01:48
HEPATIC LIPID PEROXIDATION, SULFHYDRYL STATUS, AND TOXICITY OF THE BLUE-GREEN ALGAL TOXIN MICROCYSTIN-LR IN MICE
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S. J. Hermansky, S. J. Stohs Department of Pharmaceutical Sciences, University of Nebraska Medical Center and Creighton University, Omaha, Nebraska R. S. Markin Department of Pathology, University of Nebraska Medical Center, Omaha, Nebraska W. J. Murray Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, Nebraska
Microcystin-LR (MCLR), a cyclic heptapeptide produced by the blue-green algae Microcystis aeruginosa, produces death in female mice treated with 100 μg MCLR/kg. Kupffercell hyperplasia was observed histologically after treatment with 50 or 100μgMCLR/kg. No other changes or lethality were observed with the 50 μg MCLR/kg, while 100% lethality occurred in less than 2 h in mice treated with 100 μg/kg. In these animals liver weights increased by 45% and hepatic hemoglobin content increased 106% at 60 min posttreatment. Liver histology showed loss of hepatic architecture and necrosis 30 min after treatment, and congestion with blood became evident at 45 min after treatment. Serum enzymes were significantly increased 45 min posttreatment. Hepatic nonprotein sulfhydryl content decreased 19% when calculated on the basis of cytosolic protein and 39% when based upon the total protein content, respectively. The sulfhydryl content of the liver cytoskeletal fraction decreased 26% by 30 min after treatment. Decreased enzyme-mediated and increased non-enzyme-mediated lipid peroxidation were observed in hepatic microsomes following both in vivo and in vitro exposure of hepatic microsomes to MCLR. The toxicity of MCLR may be related to alterations in the sulfhydryl content of the cytoskeletal protein. Furthermore, MCLR may either directly or indirectly affect microsomes, suggesting alterations in structure and function of smooth endoplasmic reticulum.
INTRODUCTION Microcystin-LR (MCLR, microcystin-a, cyanoginosin-LR) is a toxin produced by the cyanobacterium Microcystis aeruginosa (Carmichael, 1988). Requests for reprints should be sent to S. J. Stohs, Department of Pharmaceutical Sciences, Creighton University, Omaha, NB 68178. 71 Journal of Toxicology and Environmental Health, 31:71-91, 1990 Copyright © 1990 by Hemisphere Publishing Corporation
Downloaded by [University of Toronto Libraries] at 01:48 16 November 2015
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This blue-green algae often forms blooms in waters where the nutrient concentration is elevated (Runnegar et al., 1988). The toxin may result in deaths of livestock and allergic or gastrointestinal problems in humans following contact with water blooms (Meyer, 1987; Carmichael, 1988). Microcystin-LR is a cyclic heptapeptide that is one of several closely related compounds produced by M. aeruginosa as well as other cyanobacteria (Aune and Berg, 1986; Krishnamurthy et al., 1986; Botes et al., 1982). To date, the mechanism of toxicity of this compound has not been identified (Dabholkar and Carmichael, 1986; Jackson et al., 1984; Theiss et al., 1988). Mice or rats treated ip or ¡v with a lethal dose of MCLR die within 1-3 h (Carmichael, 1988). The LD50 for rats and mice has been reported to be approximately 50 fig/kg (Theiss et al., 1988). The immediate cause of death in these animals is believed to be hemorrhagic shock secondary to the loss of blood from the vasculature into the damaged liver (Carmichael, 1988; Theiss et al., 1988). Falconer et al. (1981) reported an increase in serum lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) and an increase in the blood content of the liver following a massive (5 X LD100) ip dose of microcystin toxin. Unfortunately, the chemical structure of microcystin employed in these studies and the existence of several chemical forms of this toxin were not discovered until after the completion of this work. As a result, the specific toxin or toxins involved in this research are not known. A good correlation has been shown to exist between morphological damage and LDH leakage following exposure to MCLR (Runnegar and Falconer, 1986). However, no detailed dose- and time-dependent studies have compared serum LDH as well as other enzymes with morphological and biochemical changes in livers of MCLR-treated mice. Thus, mice were treated with 0,12.5, 25, 50, or 100 /xg MCLR/kg, and liver histology, serum LDH, serum aspartate aminotransferase (AST, SCOT), and serum alanine aminotransferase (ALT, SGPT), liver and kidney hemoglobin content, and lethality were determined with time after treatment. The sulfhydryl content of the microsomal, cytosolic (both protein and nonprotein bound), and mitochondrial fractions and the lipid peroxidation of the liver and kidney microsomal fractions also were examined. Additionally, hepatic microsomal fractions isolated from untreated mice were incubated with varying concentrations of MCLR and assayed with time for the development of both enzymatically and nonenzymatically mediated in vitro lipid peroxidation. MATERIALS AND METHODS
Microcystin-LR (MCLR) was provided by Dr. Wayne W. Carmichael, who isolated the toxin from cultured Microcystis aeruginosa strain 7820. The MCLR had greater than 95% purity as verified by HPLC with a trace of the less active demethylated product being present. The MCLR was stored
Downloaded by [University of Toronto Libraries] at 01:48 16 November 2015
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73
at -80°C as a lyophilized powder. At the time of use, it was dissolved in 20% methanol in distilled water, because MCLR does not directly dissolve in water. The methanol was subsequently removed with nitrogen gas (Dr. W. Carmichael, personal communication), and the resulting solution of toxin was diluted to a concentration that allowed a volume of 0.15-0.25 ml to be administered per mouse at all doses. The MCLR was administered ip in all studies. Female NIH non-Swiss, outbred mice (Amitech, Omaha, Neb.) weighing 20-24 g were used in all studies. Animals were housed under conditions of controlled temperature (24°Q and lighting (12-h light/dark cycles), and were allowed free access to Purina Rodent Chow (Ralston Purina Co., St. Louis, Mo.) and tap water. All mice were allowed to acclimate for 3-5 d before the initiation of experiments. A minimum of four mice was killed at each time point by cervical dislocation, and the appropriate tissues were removed and placed in ice-cold phosphate buffer (pH 7.4). All subsequent operations were performed at 0-4°C. Blood was collected from the orbital sinus using a heparinized microcapillary tube (Fisher Scientific, Pittsburgh, Pa.). A minimum of 80 p\ blood was drawn from each mouse just prior to cervical dislocation. Serum was separated via centrifugaron for 4 min in a Fisher microcentrifuge, and the serum was frozen at -80°C. Enzyme assays were performed on the serum within 1 wk of collection. Preliminary studies by us and others (Ward et al., 1985) have shown no loss of enzyme activity under these conditions. Assays for determining LDH, AST, and ALT were performed as described by Moss et al. (1986) using 10 ¿il serum. Chemicals were of standard laboratory grade and were purchased from Sigma Chemical Company (St. Louis, Mo.). Hemoglobin content of livers and kidneys was assessed using a Sigma kit 525. Livers and kidneys were rinsed in ice-cold saline, blotted, and weighed prior to homogenizing. Tissues were homogenized in 9 volumes of distilled water using a Brinkmann Polytron Tissue Homogenizer (Brinkmann Instruments, Westburg, N.Y.). The homogenates were centrifuged at 100,000 x g for 45 min and the supernatant fractions were assayed for hemoglobin content. Specimens of liver were fixed in formalin, paraffin-embedded, and histologie sections were cut. The sections were stained with hematoxylin and eosin for evaluation. The sections were examined and photographed under a light microscope. Microsomes were prepared according to the method of Stohs et al. (1986). Mitochondria were isolated according to the method of Reitman et al. (1988). Plasma membranes were prepared using the Percoll densitygradient method of Inui et al. (1981). The cytoskeletal protein fraction of liver was isolated by a modification of the method of DiMonte et al. (1984). Briefly, tissues were homogenized in 4 volumes of phosphate buffer (0.10 M, pH 7.4), and 0.25 ml of
Downloaded by [University of Toronto Libraries] at 01:48 16 November 2015
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S. J. HERMANSKY ET AL.
the homogenate was digested in 4.75 ml of a buffer containing 1% Triton X-100 (as described by Mirabelli et al., 1988) for 30 min at 4°C. Non-Triton X-100-soluble protein was separated by centrifuging at 4000 x g for 15 min at 4°C, washed twice in the digestion buffer, and dissolved in 8 M urea/1% sodium lauryl sulfate (SLS). Total sulfhydryl content of all tissue fractions was determined by the method of Jocelyn (1987). The sulfhydryl concentration of the nonprotein fractions was assayed following precipitation of protein using trichloroacetic acid (2% final concentration). All tissue fractions were suspended in 1% SLS in 0.10 M phosphate buffer, pH 7.4, prior to addition of 5,5'dithiobis(2-nitrobenzoic acid) (DTNB, 100 nM final concentration). Protein sulfhydryl content was calculated by subtracting nonprotetin sulfhydryl content from the total sulfhydryl content of each cellular fraction. All samples were read at 412 and 520 nm in a spectrophotometer (Perkin Elmer Lambda 6), and sulfhydryl content was determined using the molar absorptivity constant of 1.415 x 104 M'1 cm" 1 (Riddles et al., 1979) using glutathione as the standard. The protein sulfhydryl content of all cellular fractions was calculated per milligram protein. Lipid peroxidation was determined colorimetrically by measuring the amount of thiobarbituric acid-reactive substances (TBARS) formed by hepatic microsomes (Miles et al., 1979). Malondialdehyde was used as the standard. Following ip administration of 100 /¿g/kg MCLR, microsomal fractions were obtained from individual mice 0,10,20, and 30 min following an ip injection of MCLR and analyzed for the development of in vitro lipid peroxidation. Enzyme-mediated lipid peroxidation was initiated by the addition of NADPH (200 (iM, final concentration) to the incubation media and incubating at 37°C for 10 min. In some experiments, sodium ascorbate (0.5 mM, final concentration) was substituted for the NADPH to assess the development of non-enzyme-mediated lipid peroxidation (Engineer and Sridhar, 1989). The reaction was terminated by the addition of trichloroacetic acid (3%, final concentration) followed by the addition of 2.5 ml of a 0.67% solution of 2-thiobarbituric acid, and the reaction mixture was heated at 90°C for 15-20 min. The reaction mixtures were cooled to room temperature and centrifuged to remove the precipitated protein, and the absorbance of the supernatant determined at 535 nm. Malondialdehyde equivalent concentrations were calculated using a molar absorptivity coefficient of 1.56 x 105 M" 1 cm" 1 (Sinnuber and Lu, 1958). For studies on the in vitro effect of MCLR on microsomal lipid peroxidation, hepatic microsomes from untreated mice were pooled at a final concentration of 1.5 mg protein/ml (Engineer and Sridhar, 1989). Concentrations of MCLR were calculated based upon a 100 fig/kg dose to intact mice assuming a volume of distribution of 2 times the total body weight (0.05 /¿g/ml in the liver) and 100% localization of the dose in the liver (2 ¿tg/ml in the liver) (Brooks and Codd, 1987). Thus, hepatic microsomes
HEPATIC TOXICITY OF MYCROCYSTIN-LR
75
were preincubated with concentrations of MCLR of 0, 0.05, 0.10, 0.50, 1.00, or 2.00 /ig/ml for 10, 20, or 30 min. The microsomal suspensions were then assayed for the development of TBARS as described above. Significant differences between mean values were analyzed by Student's f-test. A p value of g MCLR/kg produced no changes in the percent weight of the heart, lungs, or kidneys over the 60-min survival time, as shown in Table 1. However, the percent weight of the liver increased 17%, 41%, and 44% at 30, 45, and 60 min, respectively, after administration of the toxin. Liver and kidney hemoglobin content of the animals treated with 100 Hg MCLR/kg were assayed (Table 2). No increase in hepatic hemoglobin content occurred with a dose of 50 /Leg MCLR/kg at any time point (data not shown) or at 15 or 30 min after treatment with 100 ¡ig MCLR/kg. However, significant increases in hepatic hemoglobin content were observed 45 and 60 min after treatment with 100 /*g/kg, with a 49% increase in hemoglobin content being observed at the 60-min time point. Hemoglobin content of the kidneys increased by 79, 106, and 117% at 30, 45, and 60 min after treatment with 100 ng MCLR/kg, respectively (Table 2).
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25.0
20.0 - -
15.0 -•
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10.0 -
5.0 CD
05
Í
1.20 +
ALDH
C
• SGPT (ALT) 0.B0 --
• SGOT (AST)
0.40 --
0.00
10
20
30
40
50
60
70
Time After Treatment (min) FIGURE 1. Serum LDH, AST, and ALT activities in female mice treated with 100 ¡ig MCLR/kg ip in water. Control animals received the vehicle. Control values are indicated by the point where the traces intersect the V axis. 'Statistically significant at p < .05. Each value is the mean ± SD of four mice. TABLE 1. Effect of Microcystin-LR (100 uglkg) on Organ Weights Expressed as Percent Body Weight Time posttreatment
Liver
Control 15 min 30 min 45 min 60 min 2h
5.53 5.83 6.46 7.78 7.95 NSfc
Heart ± ± ± ± ±
0.17 0.44 0.533 0.41 a 0.32a
0.48 0.48 0.53 0.49 0.48 NS
± ± ± ± ±
Kidneys
Lungs 0.04 0.04 0.03 0.03 0.08
0.92 0.85 0.84 0.81 0.82 NS
± + ± ± ±
0.14 0.08 0.07 0.10 0.07
1.42 1.35 1.38 1.30 1.44 NS
± ± ± ± ±
0.11 0.05 0.09 0.04 0.06
Note. Female mice were treated with 100 ¡ig MCLR/kg ip in water. Control animals received the vehicle. Each value is the mean ± SD of four mice. Statistically significant at p < .05. ''NS indicates that experimental animals did not survive.
HEPATIC TOXICITY OF MYCROCYSTIN-LR
77
TABLE 2. Effect of Microcystin-LR (100 fig/kg) on Hemoglobin Content
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mg Hemoglobin/g tissue Time posttreatment
Liver
Control 15 min 30 min 45 min 60 min 2h
11.39 11.67 9.42 23.50 16.98 NS6
Kidney ± ± ± ± ±
2.62 1.91 1.74 6.01a 3.30a
3.56 4.10 6.39 7.32 7.74 NS
± ± ± ± ±
0.59 0.12 1.04a 0.81a 0.70a
Note. Female mice were treated with 100 ng MCLR/kg ip in water. Control animals received the vehicle. Each value is the mean ± SD of four mice. Statistically significant at p < .05. b NS indicates that experimental animals did not survive.
Histologie evaluations of the livers were performed at all doses of MCLR, and served as a basis of comparison for the biochemical measurements. No morphological changes were observed in any of the livers from mice treated with 12.5 or 25 /xg MCLR/kg. Kupffer-cell hyperplasia was observed at all time points in livers of mice that were treated with 50 or 100 /¿g MCLR/kg. A representative control liver and a liver from a mouse 60 min after treatment with 50 /*g/kg are presented in Figures 2 and 3, respectively. No other morphological changes were observed at the 50 /¿g/kg dose of MCLR. Hepatic histological changes other than Kupffer-cell hyperplasia following a dose of 100 /¿g/kg were first observed in the form of isolated areas of necrotic tissue at 30 min after MCLR administration (Figure 4). However, hepatic congestion with blood did not become apparent until 45 min after treatment, and the liver is markedly congested with blood at 60 min after treatment (Figure 5). Hepatic necrosis became more pronounced and widespread at 45 and 60 min posttreatment (Figure 5). Histological evaluations of all other organs showed no changes as compared to controls (data not shown). Following a 100 /¿g/kg dose of MCLR, no differences existed between control and treated groups in the total and protein-bound sulfhydryl content of the liver and kidney by 30 min after treatment (data not shown). In addition, no differences were observed in the protein-bound sulfhydryl content of the microsomal and mitochondrial fractions of the liver and kidneys of plasma membrane fraction of the liver after administering 100 /ig MCLR/kg (data not shown). However, the nonprotein sulfhydryl content of the liver decreased 30% at 30 min after treatment as compared to controls when calculations were based upon the total protein content of the liver (Figure 6). When calculations were based upon the protein content of the cytosolic fraction, the nonprotein sulfhydryl content of
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FIGURE 2. Section of a control liver from a female mouse. Specimens were fixed in formalin, paraffin-embedded, and stained with hematoxylin and eosin. PV, Portal vein; B, bile duct; A, artery; CV, central vein. Original magnification, x200.
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FIGURE 3. Section of a liver from a female mouse 60 min after treatment with 50 ¿ig MCLR/kg. Specimens were fixed in formalin, paraffin-embedded, and stained with hematoxylin and eosin. CV, Central vein. Original magnification, X200.
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CO
o
'''«#• -_. " Â . _^
' ' iw.
FIGURE 4. Section of a liver from a female mouse 30 min after treatment with 100
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Hepatic lipid peroxidation, sulfhydryl status, and toxicity of the blue‐green algal toxin microcystin‐LR in mice S. J. Hermansky , S. J. Stohs , R. S. Markin & W. J. Murray To cite this article: S. J. Hermansky , S. J. Stohs , R. S. Markin & W. J. Murray (1990) Hepatic lipid peroxidation, sulfhydryl status, and toxicity of the blue‐green algal toxin microcystin‐LR in mice, Journal of Toxicology and Environmental Health, 31:1, 71-91, DOI: 10.1080/15287399009531438 To link to this article: http://dx.doi.org/10.1080/15287399009531438
Published online: 20 Oct 2009.
Submit your article to this journal
Article views: 4
View related articles
Citing articles: 16 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=uteh20 Download by: [University of Toronto Libraries]
Date: 16 November 2015, At: 01:48
HEPATIC LIPID PEROXIDATION, SULFHYDRYL STATUS, AND TOXICITY OF THE BLUE-GREEN ALGAL TOXIN MICROCYSTIN-LR IN MICE
Downloaded by [University of Toronto Libraries] at 01:48 16 November 2015
S. J. Hermansky, S. J. Stohs Department of Pharmaceutical Sciences, University of Nebraska Medical Center and Creighton University, Omaha, Nebraska R. S. Markin Department of Pathology, University of Nebraska Medical Center, Omaha, Nebraska W. J. Murray Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, Nebraska
Microcystin-LR (MCLR), a cyclic heptapeptide produced by the blue-green algae Microcystis aeruginosa, produces death in female mice treated with 100 μg MCLR/kg. Kupffercell hyperplasia was observed histologically after treatment with 50 or 100μgMCLR/kg. No other changes or lethality were observed with the 50 μg MCLR/kg, while 100% lethality occurred in less than 2 h in mice treated with 100 μg/kg. In these animals liver weights increased by 45% and hepatic hemoglobin content increased 106% at 60 min posttreatment. Liver histology showed loss of hepatic architecture and necrosis 30 min after treatment, and congestion with blood became evident at 45 min after treatment. Serum enzymes were significantly increased 45 min posttreatment. Hepatic nonprotein sulfhydryl content decreased 19% when calculated on the basis of cytosolic protein and 39% when based upon the total protein content, respectively. The sulfhydryl content of the liver cytoskeletal fraction decreased 26% by 30 min after treatment. Decreased enzyme-mediated and increased non-enzyme-mediated lipid peroxidation were observed in hepatic microsomes following both in vivo and in vitro exposure of hepatic microsomes to MCLR. The toxicity of MCLR may be related to alterations in the sulfhydryl content of the cytoskeletal protein. Furthermore, MCLR may either directly or indirectly affect microsomes, suggesting alterations in structure and function of smooth endoplasmic reticulum.
INTRODUCTION Microcystin-LR (MCLR, microcystin-a, cyanoginosin-LR) is a toxin produced by the cyanobacterium Microcystis aeruginosa (Carmichael, 1988). Requests for reprints should be sent to S. J. Stohs, Department of Pharmaceutical Sciences, Creighton University, Omaha, NB 68178. 71 Journal of Toxicology and Environmental Health, 31:71-91, 1990 Copyright © 1990 by Hemisphere Publishing Corporation
Downloaded by [University of Toronto Libraries] at 01:48 16 November 2015
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This blue-green algae often forms blooms in waters where the nutrient concentration is elevated (Runnegar et al., 1988). The toxin may result in deaths of livestock and allergic or gastrointestinal problems in humans following contact with water blooms (Meyer, 1987; Carmichael, 1988). Microcystin-LR is a cyclic heptapeptide that is one of several closely related compounds produced by M. aeruginosa as well as other cyanobacteria (Aune and Berg, 1986; Krishnamurthy et al., 1986; Botes et al., 1982). To date, the mechanism of toxicity of this compound has not been identified (Dabholkar and Carmichael, 1986; Jackson et al., 1984; Theiss et al., 1988). Mice or rats treated ip or ¡v with a lethal dose of MCLR die within 1-3 h (Carmichael, 1988). The LD50 for rats and mice has been reported to be approximately 50 fig/kg (Theiss et al., 1988). The immediate cause of death in these animals is believed to be hemorrhagic shock secondary to the loss of blood from the vasculature into the damaged liver (Carmichael, 1988; Theiss et al., 1988). Falconer et al. (1981) reported an increase in serum lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) and an increase in the blood content of the liver following a massive (5 X LD100) ip dose of microcystin toxin. Unfortunately, the chemical structure of microcystin employed in these studies and the existence of several chemical forms of this toxin were not discovered until after the completion of this work. As a result, the specific toxin or toxins involved in this research are not known. A good correlation has been shown to exist between morphological damage and LDH leakage following exposure to MCLR (Runnegar and Falconer, 1986). However, no detailed dose- and time-dependent studies have compared serum LDH as well as other enzymes with morphological and biochemical changes in livers of MCLR-treated mice. Thus, mice were treated with 0,12.5, 25, 50, or 100 /xg MCLR/kg, and liver histology, serum LDH, serum aspartate aminotransferase (AST, SCOT), and serum alanine aminotransferase (ALT, SGPT), liver and kidney hemoglobin content, and lethality were determined with time after treatment. The sulfhydryl content of the microsomal, cytosolic (both protein and nonprotein bound), and mitochondrial fractions and the lipid peroxidation of the liver and kidney microsomal fractions also were examined. Additionally, hepatic microsomal fractions isolated from untreated mice were incubated with varying concentrations of MCLR and assayed with time for the development of both enzymatically and nonenzymatically mediated in vitro lipid peroxidation. MATERIALS AND METHODS
Microcystin-LR (MCLR) was provided by Dr. Wayne W. Carmichael, who isolated the toxin from cultured Microcystis aeruginosa strain 7820. The MCLR had greater than 95% purity as verified by HPLC with a trace of the less active demethylated product being present. The MCLR was stored
Downloaded by [University of Toronto Libraries] at 01:48 16 November 2015
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at -80°C as a lyophilized powder. At the time of use, it was dissolved in 20% methanol in distilled water, because MCLR does not directly dissolve in water. The methanol was subsequently removed with nitrogen gas (Dr. W. Carmichael, personal communication), and the resulting solution of toxin was diluted to a concentration that allowed a volume of 0.15-0.25 ml to be administered per mouse at all doses. The MCLR was administered ip in all studies. Female NIH non-Swiss, outbred mice (Amitech, Omaha, Neb.) weighing 20-24 g were used in all studies. Animals were housed under conditions of controlled temperature (24°Q and lighting (12-h light/dark cycles), and were allowed free access to Purina Rodent Chow (Ralston Purina Co., St. Louis, Mo.) and tap water. All mice were allowed to acclimate for 3-5 d before the initiation of experiments. A minimum of four mice was killed at each time point by cervical dislocation, and the appropriate tissues were removed and placed in ice-cold phosphate buffer (pH 7.4). All subsequent operations were performed at 0-4°C. Blood was collected from the orbital sinus using a heparinized microcapillary tube (Fisher Scientific, Pittsburgh, Pa.). A minimum of 80 p\ blood was drawn from each mouse just prior to cervical dislocation. Serum was separated via centrifugaron for 4 min in a Fisher microcentrifuge, and the serum was frozen at -80°C. Enzyme assays were performed on the serum within 1 wk of collection. Preliminary studies by us and others (Ward et al., 1985) have shown no loss of enzyme activity under these conditions. Assays for determining LDH, AST, and ALT were performed as described by Moss et al. (1986) using 10 ¿il serum. Chemicals were of standard laboratory grade and were purchased from Sigma Chemical Company (St. Louis, Mo.). Hemoglobin content of livers and kidneys was assessed using a Sigma kit 525. Livers and kidneys were rinsed in ice-cold saline, blotted, and weighed prior to homogenizing. Tissues were homogenized in 9 volumes of distilled water using a Brinkmann Polytron Tissue Homogenizer (Brinkmann Instruments, Westburg, N.Y.). The homogenates were centrifuged at 100,000 x g for 45 min and the supernatant fractions were assayed for hemoglobin content. Specimens of liver were fixed in formalin, paraffin-embedded, and histologie sections were cut. The sections were stained with hematoxylin and eosin for evaluation. The sections were examined and photographed under a light microscope. Microsomes were prepared according to the method of Stohs et al. (1986). Mitochondria were isolated according to the method of Reitman et al. (1988). Plasma membranes were prepared using the Percoll densitygradient method of Inui et al. (1981). The cytoskeletal protein fraction of liver was isolated by a modification of the method of DiMonte et al. (1984). Briefly, tissues were homogenized in 4 volumes of phosphate buffer (0.10 M, pH 7.4), and 0.25 ml of
Downloaded by [University of Toronto Libraries] at 01:48 16 November 2015
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the homogenate was digested in 4.75 ml of a buffer containing 1% Triton X-100 (as described by Mirabelli et al., 1988) for 30 min at 4°C. Non-Triton X-100-soluble protein was separated by centrifuging at 4000 x g for 15 min at 4°C, washed twice in the digestion buffer, and dissolved in 8 M urea/1% sodium lauryl sulfate (SLS). Total sulfhydryl content of all tissue fractions was determined by the method of Jocelyn (1987). The sulfhydryl concentration of the nonprotein fractions was assayed following precipitation of protein using trichloroacetic acid (2% final concentration). All tissue fractions were suspended in 1% SLS in 0.10 M phosphate buffer, pH 7.4, prior to addition of 5,5'dithiobis(2-nitrobenzoic acid) (DTNB, 100 nM final concentration). Protein sulfhydryl content was calculated by subtracting nonprotetin sulfhydryl content from the total sulfhydryl content of each cellular fraction. All samples were read at 412 and 520 nm in a spectrophotometer (Perkin Elmer Lambda 6), and sulfhydryl content was determined using the molar absorptivity constant of 1.415 x 104 M'1 cm" 1 (Riddles et al., 1979) using glutathione as the standard. The protein sulfhydryl content of all cellular fractions was calculated per milligram protein. Lipid peroxidation was determined colorimetrically by measuring the amount of thiobarbituric acid-reactive substances (TBARS) formed by hepatic microsomes (Miles et al., 1979). Malondialdehyde was used as the standard. Following ip administration of 100 /¿g/kg MCLR, microsomal fractions were obtained from individual mice 0,10,20, and 30 min following an ip injection of MCLR and analyzed for the development of in vitro lipid peroxidation. Enzyme-mediated lipid peroxidation was initiated by the addition of NADPH (200 (iM, final concentration) to the incubation media and incubating at 37°C for 10 min. In some experiments, sodium ascorbate (0.5 mM, final concentration) was substituted for the NADPH to assess the development of non-enzyme-mediated lipid peroxidation (Engineer and Sridhar, 1989). The reaction was terminated by the addition of trichloroacetic acid (3%, final concentration) followed by the addition of 2.5 ml of a 0.67% solution of 2-thiobarbituric acid, and the reaction mixture was heated at 90°C for 15-20 min. The reaction mixtures were cooled to room temperature and centrifuged to remove the precipitated protein, and the absorbance of the supernatant determined at 535 nm. Malondialdehyde equivalent concentrations were calculated using a molar absorptivity coefficient of 1.56 x 105 M" 1 cm" 1 (Sinnuber and Lu, 1958). For studies on the in vitro effect of MCLR on microsomal lipid peroxidation, hepatic microsomes from untreated mice were pooled at a final concentration of 1.5 mg protein/ml (Engineer and Sridhar, 1989). Concentrations of MCLR were calculated based upon a 100 fig/kg dose to intact mice assuming a volume of distribution of 2 times the total body weight (0.05 /¿g/ml in the liver) and 100% localization of the dose in the liver (2 ¿tg/ml in the liver) (Brooks and Codd, 1987). Thus, hepatic microsomes
HEPATIC TOXICITY OF MYCROCYSTIN-LR
75
were preincubated with concentrations of MCLR of 0, 0.05, 0.10, 0.50, 1.00, or 2.00 /ig/ml for 10, 20, or 30 min. The microsomal suspensions were then assayed for the development of TBARS as described above. Significant differences between mean values were analyzed by Student's f-test. A p value of g MCLR/kg produced no changes in the percent weight of the heart, lungs, or kidneys over the 60-min survival time, as shown in Table 1. However, the percent weight of the liver increased 17%, 41%, and 44% at 30, 45, and 60 min, respectively, after administration of the toxin. Liver and kidney hemoglobin content of the animals treated with 100 Hg MCLR/kg were assayed (Table 2). No increase in hepatic hemoglobin content occurred with a dose of 50 /Leg MCLR/kg at any time point (data not shown) or at 15 or 30 min after treatment with 100 ¡ig MCLR/kg. However, significant increases in hepatic hemoglobin content were observed 45 and 60 min after treatment with 100 /*g/kg, with a 49% increase in hemoglobin content being observed at the 60-min time point. Hemoglobin content of the kidneys increased by 79, 106, and 117% at 30, 45, and 60 min after treatment with 100 ng MCLR/kg, respectively (Table 2).
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15.0 -•
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10.0 -
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05
Í
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ALDH
C
• SGPT (ALT) 0.B0 --
• SGOT (AST)
0.40 --
0.00
10
20
30
40
50
60
70
Time After Treatment (min) FIGURE 1. Serum LDH, AST, and ALT activities in female mice treated with 100 ¡ig MCLR/kg ip in water. Control animals received the vehicle. Control values are indicated by the point where the traces intersect the V axis. 'Statistically significant at p < .05. Each value is the mean ± SD of four mice. TABLE 1. Effect of Microcystin-LR (100 uglkg) on Organ Weights Expressed as Percent Body Weight Time posttreatment
Liver
Control 15 min 30 min 45 min 60 min 2h
5.53 5.83 6.46 7.78 7.95 NSfc
Heart ± ± ± ± ±
0.17 0.44 0.533 0.41 a 0.32a
0.48 0.48 0.53 0.49 0.48 NS
± ± ± ± ±
Kidneys
Lungs 0.04 0.04 0.03 0.03 0.08
0.92 0.85 0.84 0.81 0.82 NS
± + ± ± ±
0.14 0.08 0.07 0.10 0.07
1.42 1.35 1.38 1.30 1.44 NS
± ± ± ± ±
0.11 0.05 0.09 0.04 0.06
Note. Female mice were treated with 100 ¡ig MCLR/kg ip in water. Control animals received the vehicle. Each value is the mean ± SD of four mice. Statistically significant at p < .05. ''NS indicates that experimental animals did not survive.
HEPATIC TOXICITY OF MYCROCYSTIN-LR
77
TABLE 2. Effect of Microcystin-LR (100 fig/kg) on Hemoglobin Content
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mg Hemoglobin/g tissue Time posttreatment
Liver
Control 15 min 30 min 45 min 60 min 2h
11.39 11.67 9.42 23.50 16.98 NS6
Kidney ± ± ± ± ±
2.62 1.91 1.74 6.01a 3.30a
3.56 4.10 6.39 7.32 7.74 NS
± ± ± ± ±
0.59 0.12 1.04a 0.81a 0.70a
Note. Female mice were treated with 100 ng MCLR/kg ip in water. Control animals received the vehicle. Each value is the mean ± SD of four mice. Statistically significant at p < .05. b NS indicates that experimental animals did not survive.
Histologie evaluations of the livers were performed at all doses of MCLR, and served as a basis of comparison for the biochemical measurements. No morphological changes were observed in any of the livers from mice treated with 12.5 or 25 /xg MCLR/kg. Kupffer-cell hyperplasia was observed at all time points in livers of mice that were treated with 50 or 100 /¿g MCLR/kg. A representative control liver and a liver from a mouse 60 min after treatment with 50 /*g/kg are presented in Figures 2 and 3, respectively. No other morphological changes were observed at the 50 /¿g/kg dose of MCLR. Hepatic histological changes other than Kupffer-cell hyperplasia following a dose of 100 /¿g/kg were first observed in the form of isolated areas of necrotic tissue at 30 min after MCLR administration (Figure 4). However, hepatic congestion with blood did not become apparent until 45 min after treatment, and the liver is markedly congested with blood at 60 min after treatment (Figure 5). Hepatic necrosis became more pronounced and widespread at 45 and 60 min posttreatment (Figure 5). Histological evaluations of all other organs showed no changes as compared to controls (data not shown). Following a 100 /¿g/kg dose of MCLR, no differences existed between control and treated groups in the total and protein-bound sulfhydryl content of the liver and kidney by 30 min after treatment (data not shown). In addition, no differences were observed in the protein-bound sulfhydryl content of the microsomal and mitochondrial fractions of the liver and kidneys of plasma membrane fraction of the liver after administering 100 /ig MCLR/kg (data not shown). However, the nonprotein sulfhydryl content of the liver decreased 30% at 30 min after treatment as compared to controls when calculations were based upon the total protein content of the liver (Figure 6). When calculations were based upon the protein content of the cytosolic fraction, the nonprotein sulfhydryl content of
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FIGURE 2. Section of a control liver from a female mouse. Specimens were fixed in formalin, paraffin-embedded, and stained with hematoxylin and eosin. PV, Portal vein; B, bile duct; A, artery; CV, central vein. Original magnification, x200.
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FIGURE 3. Section of a liver from a female mouse 60 min after treatment with 50 ¿ig MCLR/kg. Specimens were fixed in formalin, paraffin-embedded, and stained with hematoxylin and eosin. CV, Central vein. Original magnification, X200.
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FIGURE 4. Section of a liver from a female mouse 30 min after treatment with 100
