http://vet.sagepub.com/Veterinary Pathology Online

http://vet.sagepub.com/content/26/3/246The online version of this article can be found at:

 DOI: 10.1177/030098588902600309

1989 26: 246Vet PatholS. B. Hooser, V. R. Beasley, R. A. Lovell, W. W. Carmichael and W. M. Haschek

Mice to Rats andMicrocystis aeruginosa,Toxicity of Microcystin LR, a Cyclic Heptapeptide Hepatotoxin from

  

Published by:

http://www.sagepublications.com

On behalf of: 

Pathologists.American College of Veterinary Pathologists, European College of Veterinary Pathologists, & the Japanese College of Veterinary

can be found at:Veterinary Pathology OnlineAdditional services and information for     

  http://vet.sagepub.com/cgi/alertsEmail Alerts:

 

http://vet.sagepub.com/subscriptionsSubscriptions:  

http://www.sagepub.com/journalsReprints.navReprints:  

http://www.sagepub.com/journalsPermissions.navPermissions:  

by guest on July 10, 2011vet.sagepub.comDownloaded from

Vet. Pathol. 26246-252 (1989)

Toxicity of Microcystin LR, a Cyclic Heptapeptide He pato toxin from Microcyst is aeruginosa,

to Rats and Mice

S. B. HOOSER, V. R. BEASLEY, R. A. LOVELL, W. W. CARMICHAEL, AND W. M. HASCHEK

Departments of Veterinary Pathobiology and Biosciences, University of Illinois, Urbana, IL; and Department of Biological Sciences, Wright State University, Dayton, OH

Abstract. Rats (Sprague-Dawley) and mice (Balb/c) were given microcystin LR intraperitoneally and were killed at intervals up to 24 hr (rats) or 90 min (mice) and necropsied. The lowest consistently lethal dose was 160 pg/kg in rats and 100 pg/kg in mice. Rats that were clinically unaffected had no lesion. All clinically affected rats in all dose groups died (from 20 to 32 hr after toxin) and had similar hepatic lesions. Livers were enlarged and dark red beginning 40 to 60 min after toxin. Mild disassociation and rounding of centrilobular hepatocytes developed within 20 min. By 60 rnin after toxin, degeneration and necrosis of hepatocytes involved most of the lobules except for small periportal zones. Weights of livers and kidneys were significantly increased. Eosin- ophilic fibrillar material filled renal glomerular capillaries as early as 9 hr after toxin. At 18 to 24 hr there was moderate vacuolation of proximal tubular epithelium with mild tubular dilatation. Beginning at 1 hr, intact hepatocytes and hepatic debris were present in pulmonary vessels. Analysis of serum revealed an increase in alanine aminotransferase 40 min after toxin; at 6 to 12 hr there were significant increases in alkaline phosphatase, total bilirubin, blood urea nitrogen, and creatinine. Mice survived only 60 to 90 rnin after toxin. Hepatic lesions were similar to those in rats, but renal and pulmonary lesions were not seen.

Toxins produced by the blue-green algae, Microcystis aeruginosa, have produced hepatotoxicity in sheep and cattle and have been implicated in human illnesses associated with proliferation of algae in reservoirs. Hepatotoxicity has been experimentally reproduced in pigs (Hooser, unpublished data), sheep, mice, rats, guinea pigs, rabbits, chickens, ducks, and calves by dosing with whole cells or toxin extract^.^,^.^.^-' l , l 6 - I 8

The pathogenesis and mechanism of action are not known. It has been hypothesized that hepatic damage may be due to a direct hepatotoxic effect,’~~ to venous hypertension caused by massive pulmonary throm- bosis,l,17 or to stimulation of the sympathetic nervous system leading to vasoconstriction with subsequent he- patic pooling of blood and peripheral anoxia.13 Death may be due to hypovolemic shock associated with he- patic necrosis and hemorrhage or to pulmonary em- bolism with subsequent venous hypertension, reduced cardiac output, and circulatory ~ol lapse .~J~ Sequential serum chemistry and corresponding morphologic al- terations using purified toxin have not yet been re- ported in rats.

In the present study we characterize the acute tox- icity of the purified heptapeptide hepatotoxin, micro- cystin LR (cyanoginosin LR) produced by the labo- ratory strain 7820 of Microcystis aeruginosa. We have examined clinical signs and pathologic changes, and

have compared our findings to results obtained by oth- ers using cells or toxin extracts. Using the purified hepatotoxin we evaluated and compared the toxic ef- fects: (a) in male and female rats; (b) in male rats at minimal lethal doses and at doses greatly in excess of the minimum lethal dose; and (c) in mice and rats. In addition, we characterized the sequential morphologic and clinical pathologic changes occurring in male rats at a minimally lethal dose of the hepatotoxin.

Materials and Methods Animals

Male and female 175-200 g Sprague-Dawley rats were obtained from Harlan Sprague-Dawley, Inc. (Indianapolis, IN) and allowed to acclimate for 2 weeks or longer before use. Adult female Balb/c mice, raised in our own colony, were also used. Animals had free access to a commercial laboratory chow and water and were maintained on a 12-hr light: dark cycle.

Rats and mice were randomly assigned to the various dose groups immediately before dosing and then given free access to food and water until termination of the study.

Toxin

Microcystin LR (MCLR- approximately 9 5% pure) from Microcystis aeruginosa, laboratory strain 7820, was produced and purified in our (W. W. Carmichael) laboratory by meth-

246

by guest on July 10, 2011vet.sagepub.comDownloaded from

Microcystin LR Toxicity 247

anol/butanol cell extraction, centrifugation, sephadex filtra- tion, and purification by high performance liquid chroma- tography.12 MCLR was dissolved in 0.9% NaCl prior to use.

Pathologic examination

Animals not dying spontaneously were anesthetized with ether and killed by exsanguination. They were then weighed and necropsied. In the sequential studies, livers, kidneys (with adrenals), and spleens were weighed. Lungs were fixed by intratracheal instillation of 10% neutral buffered formalin. Sections of liver, lungs, kidneys, spleen, and thymus from all animals, as well as brain, heart, adrenal glands, small and large intestine, pancreas, urinary bladder, skeletal muscle, and eyes of rats from the acute toxicity study, were fixed by immersion in 10% neutral buffered formalin. Tissues were then routinely processed, embedded in paraffin, cut at 4-6 pm, stained with hematoxylin and eosin (HE), and examined by light microscopy. Selected kidney sections in the sequen- tial morphologic study were stained for fibrin deposition with phosphotungstic acid hematoxylin, Martius scarlet blue tri- chrome, Martius yellow scarlet red analine blue, or Carstair’s stains, or for calcium deposition with Von Kossa’s stain or Alizarin red stain.

Acute toxicity in rats

Male rats (two to eight per group [Table 11) were given an intraperitoneal (ip) injection of MCLR at the following doses: 20, 40, 80, 120, 160, 180, 200, 240, or 400 pg/kg. Volumes administered ranged from 0.1 to 1 .O ml. Animals were then observed continuously for clinical signs of toxicasis for 12 hr and periodically thereafter. Similarly, female rats (three per group) were given an ip injection of MCLR at 40, 80, 120, or 160 pg/kg. Surviving animals were killed 5 days after treatment.

In addition, male rats (three per group) were given a single ip injection of MCLR at the following doses: 0 (2 ml of the vehicle, 0.9% NaCl), 400, 800, or 1,200 pg/kg. Surviving animals were killed 24 hr after treatment.

Sequential acute toxicity study in rats

Male rats (two to seven per group) were given an ip injec- tion of a lethal dose of MCLR (160 pg/kg) and killed with ether at the following times: 5, 10, 20, 30, 40, 50, or 60 min, and 3, 6, 9, 12, 18, or 24 hr. No animals died spontaneously. Control rats (three per group) were injected ip with 0.5 ml of 0.9% NaCl and killed at 60 min and at 12 and 24 hr. Immediately prior to necropsy, blood samples were collected by cardiac puncture, immediately placed in serum tubes or tubes containing ethylenediaminetetraacetic acid (EDTA) as an anticoagulant, and put on ice. Clotted serum samples were centrifuged within 1 hr of collection, the clots were removed, and the samples were frozen until analyzed. Serum chemistry parameters evaluated included: creatinine, blood urea nitro- gen, total protein, albumin, calcium, phosphorus, sodium, potassium, chloride, glucose, alkaline phosphatase, alanine aminotransferase (ALT = serum glutamic-pyruvic trans- aminase), gamma glutamyl transferase, total bilirubin, al- bumin to globulin ratio, and sodium to potassium ratio.

Table 1. Mortality of male and female rats following in- traperitoneal microcystin LR injection.

Number Dead/Number Treated

Males Females Dose (&kd

20 40 80

120 160 180 200 240 400 800

1.200

0/3 - 0/3 013 0/3 1 /3 2/3 2/3 718 3/3 3/3 - 313 - 1 /2 - 3/3 - 3/3 - 3/3 -

Sequential morphologic alterations in mice

Female mice were given an ip injection of 100 pg/kg of MCLR and killed (two per group) at 15, 30, 60, and 90 min post-dosing. Three mice that died at 90 to 120 min post- dosing were necropsied immediately. Control mice (given 0.9% NaC1) were killed at 120 min post-dosing.

Statistical analysis

Serum chemistry data were analyzed using a one-way anal- ysis of variance (SAS Institute, SAS Circle, Cary, NC) with Tukey’s comparison of means to evaluate differences be- tween individual times. Since controls were not done at each individual time point, all control data were pooled to com- pare to the observations from treated rats. Results were con- sidered significant when P < 0.05.

Results

Acute toxicity in rats

Male rats dosed at 20, 40, or 80 pg/kg and female rats dosed at 40 pg/kg did not have any clinical signs of toxicity and had no gross or microscopic lesions. Variable numbers of animals dosed between 40 pg mi- crocystin LR (MCLR)/kg (females) or 80 pg MCLR/ kg (males) and 160 pg MCLR/kg were affected (Table 1). All clinically unaffected animals had no lesions. All clinically affected animals in all dose groups died and had similar gross and histologic lesions irrespective of sex or dose. Clinical signs in affected animals consisted, of lethargy and ruffling of fur which began several hr after toxin administration and increased in severity until the time of death. Time from injection of the hepatotoxin to death ranged from 20 to 32 hr in both males and females injected with less than 240 pg/kg of the toxin. All rats given very high doses of MCLR (400-1,200 pg/kg) died 6 to 8 hr following treatment (Table 1).

Gross lesions were seen only in the liver. Livers from all rats which died were dark red and enlarged, espe-

by guest on July 10, 2011vet.sagepub.comDownloaded from

248 Hooser et al.

E m .-

2

Liver Weights Kidney Weights

T 1.0 r

5.5 I-- I I T

3.0 I I I I I I I I 200 400 600 800 1000 1200 1400

Time in Minutes

Fig. 1. Mean liver weights (as O/o body weight) k SEM in rats (n = 2 to 7) injected intraperitoneally with 160 wg mi- crocystin LR/kg. Time 0 point is mean control value.

cially from those receiving the higher doses. Severe centrilobular and midzonal necrosis, with occasional degenerate and necrotic hepatocytes in central veins, were seen with equal severity in all affected rats of both sexes.

Glomerular capillaries frequently contained eosin- ophilic, finely fibrillar to homogeneous material. Renal cortical proximal tubular epithelial cells were moder- ately vacuolated, and scattered tubules were mildly dilated and contained moderate amounts of homoge- neous, basophilic to eosinophilic material. With very high doses there were multifocal areas of tubular epi- thelial necrosis with small amounts of luminal necrotic debris. Many pulmonary capillaries and larger vessels were dilated and contained eosinophilic globular de- bris or intact cells which appeared to be hepatocytes. Other tissues were unaffected.

Sequential acute toxicity study in rats

Clinical signs were similar to those seen in the acute studies. Livers from hepatotoxin-treated (1 60 Mglkg) rats killed as early as 40 to 60 min after treatment were dark red and enlarged. Liver weight (as % of body weight) of treated rats was significantly (P = 0.01) great- er than that of controls over the 24-hr time period (Fig. 1). No other gross lesions were noted. Kidney weight (as O/o of body weight) progressively increased beginning at 3 hr post-dosing (Fig. 2). Spleen weight of toxin- treated animals did not differ from that of controls.

Hepatic lesions were first seen at 20 min and con- sisted of mild disassociation and rounding of centri- lobular hepatocytes. At 60 min there was severe he- patocyte disassociation, degeneration, and necrosis with severe hepatocyte loss and hemorrhage (Fig. 3)

g 0.9

z I $ 0.7 m

3 1

0.5 I I I I

200 400 600 800 1000 1200 1400

Time in Minutes

Fig. 2. Mean kidney weights (as 9’0 body weight) f SEM in rats (n = 2 to 7) injected intraperitoneally with 160 l g microcystin LR/kg. Time 0 point is mean control value.

throughout the entire liver lobule, except for areas ap- proximately six to seven cells wide surrounding portal regions. Central veins were frequently replaced by dis- associated hepatocytes, cellular debris, and erythro- cytes (Fig. 4). Intact central veins frequently contained hepatocytes or hepatic debris (Fig. 5). The lesions in- creased in severity until at 9 to 24 hr post-dosing there was a progressive reduction in the number of cells with the remaining cellular debris intermingled with red blood cells (Fig. 6).

Beginning 60 min after dosing, scattered renal cor- tical tubules contained progressively increasing amounts of homogeneous, basophilic material. Begin- ning at 9 hr post-dosing, glomerular capillaries con- tained small amounts of eosinophilic, fibrillar material which did not stain for fibrin with phosphotungstic acid hematoxylin, Martius scarlet blue trichrome, Martius yellow scarlet red analine blue, or Carstair’s stains. Scattered basophilic stippling that did not stain for calcium with Von Kossa’s or Alizarin red stains was present within the eosinophilic material. By 18 hr post-dosing, cortical proximal tubular epithelial cells frequently contained one to several variably sized, clear, intracytoplasmic vacuoles.

Beginning 1 hr post-dosing, many pulmonary cap- illaries and arterioles contained eosinophilic granular debris or intact rounded cells (Figs. 7, 8). These cells had abundant eosinophilic cytoplasm, occasional bi- nucleate and frequently pyknotic nuclei, and were much larger than erythrocytes or leukocytes. In all rats killed between 12 and 24 hr post-dosing, the cells in the pulmonary capillaries became increasingly degenerate with nuclear karyolysis and intracytoplasmic vacuola- tion. This correlated with the increased hepatocyte de-

by guest on July 10, 2011vet.sagepub.comDownloaded from

Microcystin LR Toxicity 249

Fig. 3. Centrilobular hepatocyte degeneration, disassociation, and rounding with small areas of hemorrhage, 1 hr post- dosing; liver from rat injected intraperitoneally with 160 pg microcystin LWkg. HE.

Fig. 4. Increased loss of centrilobular architecture with destruction of central vein (arrowhead) and increased hepatocyte degeneration and necrosis, 3 hr post-dosing; liver from rat injected intraperitoneally with 160 pg microcystin LWkg. HE.

Fig. 5. Hepatocytes in central vein (arrowheads), 1 hr post-dosing; liver from rat injected intraperitoneally with 160 pg microcystin LWkg. HE.

Fig. 6. Most hepatocytes, except for a rim periportally, have been replaced by necrotic debris and erythrocytes, 24 hr post-dosing. Portal vein (P); liver from rat injected intraperitoneally with 160 pg microcystin LWkg. HE.

generation and necrosis in the liver observed at the same time points. The morphology as well as the nu- clear pyknosis and cytoplasmic vacuolation of these cells was identical to that of affected hepatocytes.

Statistically significant increases were seen in the serum activities of the enzymes alanine aminotrans- ferase (ALT) (P = 0.003) and alkaline phosphatase (ALP) (P = 0.001), and in blood urea nitrogen (BUN) (P = 0.001) and creatinine (P = 0.002) from treated

animals as compared to controls. Beginning at 40 min, there was an increase in ALT which continued until the end of the study (Figs. 9, 10). ALP (Fig. 11) and total bilirubin (Fig. 12) were elevated beginning 360 min post-dosing. The BUN increased from 22 to 70 mg/dl over the 1,440-min (24-hr) period. Blood glu- cose concentration decreased by 67% over time, and phosphorous varied inconsistently over the 1,440-min period (data not presented). Total protein, albumin,

by guest on July 10, 2011vet.sagepub.comDownloaded from

250 Hooser et al.

A LT

loo0l 800

0 5 10 15 20 25 30 35 40 \ 45 50 55 60

Time in Minutes

Fig. 9. Alanine aminotransferase k SEM in rats (n = 2 to 7) after intraperitoneal injection with 160 pg microcystin LWkg over the first 60 min. Time 0 point is mean control value.

several central veins. In the kidney, mild dilation of cortical tubules was first seen 60 min post-dosing and became more severe by 90 min. Many tubules con- tained eosinophilic granular to fibrillar material. Sim- ilar material was occasionally noted in Bowman’s space and in glomerular capillaries. No treatment-related le- sions were seen in the lungs.

Discussion

The liver is the major target organ of the microcys- The marked hepatocyte disassociation and

necrosis which occurred in rats and mice in our ex- periments are similar to that reported in mice and sheep given whole cells or toxin extracts. An exception

de~cribed.~ No differences were noted in the response of male rats as compared to females. A steep dose- response curve was found with no effect below 80 pg/

kg and virtually 100% lethality above 160 pg/kg. After a single intraperitoneal (ip) exposure, rats injected with

Fig. 7. Pulmonary capillaries containing intact hepato- cytes and erythrocytes. One hepatocyte is binucleate (arrow-

neally with 160 pg microcystin LWkg. HE. Fig. 8. Pulmonary vessel containing several hepatocytes

and erythrocytes. One hepatocyte is binucleate (arrowhead), 6 hr post-dosing; lung from rat injected intraperitoneally with 160 pg microcystin LR/kg. HE.

head), 3 hr post-dosing; lung from rat injected intraperito- is One study in mice in which periportal necrosis was

by guest on July 10, 2011vet.sagepub.comDownloaded from

Microcystin LR Toxicity 25 1

Alkaline Phosphatase

1400 i T

Time in Minutes

Fig. 11. Serum alkaline phosphatase f SEM in rats (n =

2 to 7) after intraperitoneal injection with 160 pg microcystin LWkg. Time 0 point is control value.

80-160 p g microcystin LR (MCLR)/kg were either clinically affected and died with massive liver destruc- tion and hemorrhage, or were clinically unaffected and had no gross or microscopic lesions. In both rats and mice, microscopic lesions were present in the liver as early as 20 to 30 min post-dosing. At 60 rnin the his- tologic lesions in rats were comparable to those in mice. However, while mice died at 60 to 90 rnin post- dosing, rats survived for 20 to 32 hr. Even with doses greater than 240 pdkg of MCLR, the survival time in rats was still 6 to 8 hr. The reason for this species difference is intriguing but undetermined at the present time.

A good correlation was found between the initial hepatocyte lesions seen at 30 rnin and the initial in- crease in alanine aminotransferase (ALT) at 40 rnin post-dosing. The increases in alkaline phosphatase (ALP) and total bilirubin, while more often correlated

Total Bilirubin

0 200 400 P 600 800 1000 1200 1400

Time in Minutes

Fig. 12. Total bilirubin f SEM in rats (n = 2 to 7) after intrapentoneal injection with 160 pg microcystin LWkg. Time 0 point is mean control value.

Table 2. Liver and kidney weights (as Yo body weight [BW]) of mice injected intraperitoneally with 100 pg micro- cystin LR/kg.*

Liver (Yo BW) Kidney (Yo BW) Time after Dosing (rnin)

Saline controls (1 20) 5.37 0.76 15 5.37 0.75 30 5.63 0.75 60 9.01 0.82 90 10.48 0.97 90-120t 9.69 1 .oo

* Mean of two mice per group. t Spontaneous death.

with biliary obstruction, can also be related to necrosis with release of these enzymes into the circulation. This contrasts with the effects of M. aeruginosa toxin ex- tracts when added to suspensions of isolated hepato- cytes. Although isolated hepatocytes had morphologic changes (plasma membrane blebbing) within 5 to 10 min, intracellular enzymes are not released for 60 to 100 min.3J"16 Possible explanations are that hepato- cytes are altered during isolation, some contributing factor is present in vivo which is not present in vitro, or that the number of hepatocytes damaged in vivo is large enough to allow an increase in ALT to be detected earlier than in vitro. The decrease in serum glucose may be due to the inability of the damaged liver to convert glycogen to glucose or to synthesize glucose from precursor molecules.

Endothelial damage in the liver was evident from the observation of massive hemorrhage and loss of central veins and sinusoidal architecture. Whether the initial effect was on the endothelial cells or on hepa- tocytes, with subsequent endothelial damage, is un- clear at this time. However, breakdown in hepatic en- dothelial integrity, in conjunction with massive hepatocyte disassociation, appears to allow intact he- patocytes and necrotic cellular debris into the venous circulation, subsequently into the pulmonary artery, and eventually into the lungs. Much later in the course of the toxicosis this debris is thought to be deposited as eosinophilic fibrillar material in the renal glomerular capillaries. In rats, necrotic debris and intact cells were seen in the pulmonary vasculature as early as 1 hr post- dosing. Recent ultrastructural studies have confirmed that these cells are hepatocytes (unpublished data). It must be stressed that severe hepatic lesions preceded the finding of intact cells and necrotic cellular debris in the pulmonary vasculature. Thus, the hypothesis that pulmonary thrombosis causes an increase in ve- nous pressure and is responsible for hepatic damage should be discounted. In our studies, hepatocyte mi- croemboli, although present in many pulmonary ves- sels, did not appear to cause significant pulmonary or

by guest on July 10, 2011vet.sagepub.comDownloaded from

252 Hooser et al.

extrapulmonary damage. We did not see such intact cells in the pulmonary circulation of mice. The release of intact cells and cell fragments from the liver into the venous circulation has not been seen with other hepatotoxins and thus appears to be unique to MCLR. Pulmonary thrombosis and embolism have been de- scribed previously and reported as being acellular. 1,17

Renal tubular lesions in mice and rats were sugges- tive of glomerular damage with leakage of protein into cortical tubules. Renal lesions were more extensive in rats than mice, again perhaps due to the short time to death in mice. In rats, the presence of large amounts of eosinophilic material within glomerular capillaries at 18 and 24 hr coincided with increased amounts of eosinophilic to basophilic material within the lumens of renal cortical tubules. This could also be from is- chemia due to hypovolemic shock with damage to glo- merular capillaries. Renal proximal tubular necrosis was evident in rats at very high doses of MCLR. This may be due to a direct effect of the toxin on the tubular epithelium or, more likely, to a secondary ischemic effect from shock induced by the massive hepatic necrosis and hemorrhage. It is possible that this is exacerbated by partial obstruction of glomerular and peritubular capillaries with the eosinophilic fibrillar material observed. Recent ultrastructural studies (un- published) indicate that this material is hepatic debris. The time at which renal lesions were seen roughly cor- related with the elevation of blood urea nitrogen and creatinine.

While the exact mechanism of action of the toxin produced by Microcystis aeruginosa remains to be de- termined, death in affected animals is due to massive hepatic destruction and hemorrhage resulting in severe circulatory shock and loss of liver function.

Acknowledgements

These studies were supported in part by United States Army Medical Research and Development Command con- tract DAMD 17-85-C-5241. The authors thank Ms. Leslie Waite and Ms. Joanie Dye for technical assistance.

References

1 Adams WH, Stoner RD, Adams DG, Slatkin DN, Sie- gelman H W: Pathophysiologic effects of a toxic peptide from Microcystis aeruginosa. Toxicon 23:44 1-447, 1985

2 Ashworth CT, Mason MF: Observations on the patho- logical changes produced by a toxic substance present in blue-green algae (Microcystis aeruginosa). Am J Pathol

3 Aune T, Berg KJ: Use of freshly prepared rat hepato- cytes to study toxicity of blooms of the blue-green alga Microcystis aeruginosa and Oscillatoria agardhiri. J Tox- icol Environ Health 19:325-336, 1986

22~369-383, 1946

4 Elleman TC, Falconer IR, Jackson AR, Runnegar MT: Isolation, characterization, and pathology of the toxin from a Microcystis aeruginosa (=Anacystis cyanea) bloom. Aust J Biol Sci 31:209-218, 1978

5 Falconer IR, Beresford A, Runnegar MT: Evidence of liver damage by toxin from a bloom of the blue-green alga, Microcystis aeruginosa. Med J Aust 1:5 11-5 14, 1983

6 Falconer IR, Buckley TC, Runnegar MT: Biological half-life organ distribution and excretion of 124-labelled toxic peptide from the blue-green alga, Microcystis aeru- ginosa. Aust J Biol Sci 39:17-22, 1986

7 Falconer IR, Jackson AR, Langley J, Runnegar MT: Liver pathology in mice in poisoning by the blue-green alga Microcystis aeruginosa. Aust J Biol Sci 34: 179-1 87, 1981

8 Galey FD, Beasley VR, Carmichael WW, Kleppe G, Hooser SB, Haschek WM: Blue-green algae (Microcys- tis aeruginosa) hepatoxicosis in dairy cows. Am J Vet Res 48:1415-1420, 1987

9 Jackson AR, McInnes A, Falconer IR, Runnegar MT: Toxicity for sheep of the blue-green alga, Microcystis aeruginosa. Toxicon Suppl3: 19 1-1 94, 1983

10 Jackson ARB, McInnes A, Falconer IR, Runnegar MTC: Clinical and pathological changes in sheep ex- perimentally poisoned by the blue-green alga, Microcystis aeruginosa. Vet Pathol21:102-113, 1984

11 Konst H, McKercher PD, Gorham PR, Robertson A, Howell J: Symptoms and pathology produced by toxic Microcystis aeruginosa NRC- 1 in laboratory and do- mestic animals. Can J Comp Med Vet Sci 29:221-228, 1965

12 Krishnamurthy T, Carmichael WW, Sarver EW: Toxic peptides from freshwater cyanobacteria (blue-green al- gae). I. Isolation, purification, and characterization of peptides from Microcystis aeruginosa and AnabaenaJlos- aquae. Toxicon 24:865-869, 1986

13 Oishi S, Watanabe MF: Acute toxicity of Microcystis aeruginosa and its cardiovascular effects. Environ Res

14 Runnegar MTC, Falconer IR: The in vivo and in vitro biological effects of the peptide hepatotoxin from the blue-green alga Microcystis aeruginosa. S Afr J Sci 78:

15 Runnegar MTC, Falconer IR: Effect of toxin from the cyanobacterium Microcystis aeruginosa on ultrastruc- tural morphology and actin polymerization in isolated hepatocytes. Toxicon 24: 109-1 15, 1986

16 Runnegar MT, Falconer IR, Silver J: Deformation of isolated rat hepatocytes by a peptide hepatotoxin from the blue-green alga Microcystis aeruginosa. Naunyn Schmiedebergs Arch Pharmacol317268-272, 198 1

17 Slatkin DN, Stoner RD, Adams WH, Kycia JH, Sie- gelman H W: Atypical pulmonary thrombosis caused by a toxic cyanobacterial peptide. Science 220: 1383-1385, 1983

18 Zin LL, Edwards W C: Toxicity of blue-green algae in livestock. Bov Practitioner 14: 15 1-153, 1979

40~518-524, 1986

363-366, 1982

Request reprints from Dr. V. R. Beasley, Department of Veterinary Biosciences, University of Illinois, 2001 South Lincoln, Urbana, IL 6 180 1 (USA).

by guest on July 10, 2011vet.sagepub.comDownloaded from