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Keywords:

  • Bacillus cereus;
  • Cereulide;
  • Microsteatosis;
  • Regeneration of liver;
  • Prognosis

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

Cereulide is the causative toxin of the emetic type of food-borne illness caused by Bacillus cereus. This toxin was previously shown to be associated with fulminant liver failure in a human case. Mice were injected i.p. with synthetic cereulide and the development of histopathological changes was examined. Hepatocytes showed mitochondrial swelling with loss of cristae, and dose-dependent increase of small fatty droplets. These microsteatotic hepatocytes were distributed mainly in the pericentral area. At higher cereulide doses, massive degeneration of hepatocytes occurred. The serum values of hepatic enzymes were highest on days 2–3 after the inoculation of cereulide, and rapidly decreased thereafter. General recovery from the pathological changes and regeneration of hepatocytes was observed after 4 weeks.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

Bacillus cereus is known to cause two different types of food poisoning, a diarrheal-type syndrome and an emetic-type syndrome. More than 90% of food poisonings caused by B. cereus are of the latter type. In addition, fatal cases of infections caused by contaminated foods have been reported [1]. Based on the vacuole formation activity in HEp-2 cells, we purified the emetic toxin from the B. cereus cultures, and determined its chemical structure and named it cereulide [2,3]. Cereulide is a cyclic dodecadepsipeptide, and a putative potassium ionophore. It causes mitochondrial swelling and consequently cell death [4,5]. Chemically synthesized cereulide caused emesis in animals through the 5-HT3 receptor [5].

A case of fulminant liver failure after ingestion of food contaminated with the B. cereus emetic toxin (cereulide) was reported in 1997 [6]. Autopsy of the patient's liver revealed diffuse microvesicular steatosis and midzonal necrosis. The purified toxin showed effects in rats similar to those in the human case, such as inhibition of rat liver mitochondrial fatty acid oxidation. However, the effect of possible contamination of other toxins in the purified preparation could not be excluded, since B. cereus secretes various toxins [1]. In this study, we examined the histopathological effects of chemically synthesized cereulide in BALB/c mice. Another purpose of this study was to examine sequelae of the effects of cereulide in the liver.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

2.1Administration of synthetic cereulide to mice

Cereulide was chemically synthesized as previously described [7] and dissolved in 10% ethanol for use. Male BALB/c mice were purchased from SLC (Hamamatsu, Japan) and used in the present experiments at 8–10 weeks of age. Experiments were performed according to the ethical guidelines of the Institute for Laboratory Animal Research, Nagoya University School of Medicine.

Mice were intraperitoneally injected with 200 μl of the solution of synthetic cereulide at doses of 5, 10, 15 or 20 μg per mouse. Mice were then killed and the sera and organs were obtained 24, 48, 72 or 96 h after the injection. Moreover, the organs and sera of the mice which received 10 μg of the toxin were obtained 4 weeks after the toxin challenge.

2.2Histological studies

Fixation and preparation of paraffin sections for light microscopy and electron microscopy examinations were performed with conventional methods as previously described [8]. For light microscopy, tissue samples were fixed, embedded in paraffin, and stained with hematoxylin-eosin (HE). For electron microscopy, fresh specimens were cut into 1-mm cubes, fixed with 2.5% glutaraldehyde buffered with 0.1 mol l−1 phosphate (pH 7.2), and post-fixed with 1% osmium tetroxide. After processing and embedding in epoxy resin, ultrathin sections stained with uranyl acetate and lead citrate were examined with an electron microscope (H-7100; Hitachi, Tokyo, Japan).

2.3Assay for enzymatic activities

Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) were measured with TEST-WAKO kits (Wako Pure Chemical Industries, Ltd., Osaka, Japan) according to the manufacturer's instructions.

3Results

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

3.1Histopathological changes caused by cereulide

Apparent pathological changes were observed only in liver after the injection of cereulide and no visible changes were observed in other organs (data not shown). These lesions were developed within 6 h after toxin injection and persisted for several days, depending on the toxin dosage. As Fig. 1 shows, the severity of the lesions roughly depended on the given dose. At 5 μg of cereulide hepatocytes were regularly arranged with slightly vacuolated cytoplasm. In the liver of mice which were given higher doses of cereulide, more hepatocytes were degenerated. Fig. 2a,c,d exhibit typical lesions on day 3 after injection of 10 μg of cereulide. Mice receiving other doses also showed similar lesions in the liver. The liver of the mice in Fig. 2a,c had congestion around central veins, small foci of well demarcated confluent necrosis and swollen hepatocytes. These transformed hepatocytes had vacuolization in their cytoplasm. Central veins were markedly dilated and perivenular sinusoids were also slightly dilated. As Fig. 2d shows, necrosis was accompanied by inflammatory reaction, neutrophils were infiltrated around the necrosis. Proliferation of Kupffer cells in sinusoids was also observed. In the group of mice which received 15 μg of cereulide, marked swelling of hepatocytes with vacuolation and massive microsteatosis was observed and injection of 20 μg of the toxin, a lethal dose, caused far more severe lesions in the liver (unpublished observations). The distribution of the hepatocytes with fatty degeneration was correlated with Rappaport's zone II, and was similar to that of the human case.

image

Figure 1. Dose-dependent morphological changes in liver. Liver samples were removed and fixed 48 h post inoculation with cereulide (HE stain, ×380). BALB/c mice were injected with solvent (a), 5 μg of cereulide (b), 10 μg of cereulide (c), 15 μg of cereulide (d), and 20 μg of cereulide (e); V, central vein.

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image

Figure 2. (a) Liver sample 48 h post inoculation with 10 μg of cereulide (HE stain, ×95); V, central vein. (b) Electron microscopy of an injured hepatocyte (×7600, bar=1 μm). Arrow indicates an abnormal mitochondria with a loss of cristae; L, small fat droplets. (c) Liver sample 48 h post inoculation with 10 μg of cereulide (HE stain, ×95); N, focus of necrosis. (d) Higher magnification of panel c (HE stain, ×380). (e) Liver sample 4 weeks post inoculation with 10 μg of cereulide (HE stain, ×95). (f) Higher magnification of panel e (HE stain, ×380).

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3.2Electron microscopic observations

Fig. 2b shows electron microscopic changes of hepatocytes after the injection of cereulide. Hepatocytes showed abnormality of the mitochondria, such as swelling and loss of cristae. Degenerated hepatocytes were mainly distributed around the pericentral area and increased in number dose-dependently. Dose-dependent increases of small fatty droplets in the degenerated hepatocytes were also observed.

3.3Regeneration of liver

It is clinically important to know if sequelae remain after the acute liver dysfunction caused by cereulide. As Fig. 2e shows, general recovery from the pathological changes in the liver was observed 4 weeks after the injection of cereulide. Hepatocytes were arranged almost regularly and pericentral congestion was not observed. In Fig. 2f at higher magnification, cytoplasm of hepatocytes scarcely contained small fat vesicles. Minor histological lesions which still remained in the liver were as follows. Clumps of lymphocytes and Kupffer cells were seen in perivascular and hepatocyte plates (cords) and low-grade patchy loss of hepatocytes and infiltration of lymphocytes were still observed.

3.4Liver function

Liver function was evaluated using the values of serum AST, ALT and LDH in mice injected with 10 μg of the toxin. As shown in Fig. 3, values of serum LDH, AST and ALT reached their highest levels at 24 or 48 h after toxin injection, and then rapidly decreased. After 4 days, liver function returned to within the normal range.

image

Figure 3. Kinetic responses of serum AST, ALT and LDH after cereulide inoculation. The values of serum AST, ALT and LDH were measured in mice which received i.p. injection of 10 μg of cereulide. (a) Serum AST. (b) Serum ALT. (c) Serum LDH. Each value represents the mean of the levels in three mice±S.E.M. 0* indicates the value just before inoculation with cereulide. IU, international unit.

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4Discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

The B. cereus emetic toxin cereulide is a cyclic depsipeptide and structurally resembles valinomycin. Cereulide, as well as valinomycin, disrupts mitochondrial membrane potential and deenergizes mitochondria, and thereby impairs mitochondrial fatty acid metabolism [6,9]. Vacuoles in the cytoplasm of hepatocytes of mice treated with cereulide are correlated with histological changes such as swollen mitochondria and accumulated fatty droplets. The effect of cereulide on mitochondria was mainly responsible for acute liver failure in our animal model, as previously suggested in a human case [6]. The kinetics of the responses of mice to cereulide indicates rapid development of liver injury. The duration of the lesions varied with the dose administered. The maximum pathological effect in the mouse liver was observed 1–2 days after the injection of cereulide. The values of serum enzymes released from the liver correlated with the histological changes. At 25 μg per mouse, cereulide killed mice within several hours, perhaps because of acute liver failure.

It is clinically important to know whether the liver injury caused by cereulide is reversible, or whether some sequelae remain. As the present study clearly exhibited, the tissue of the liver, as well as serum enzyme values 4 weeks after cereulide challenge, became almost normal. We therefore conclude that intensive supportive therapy during the acute phase to reduce the load on the liver, which is severely injured by B. cereus food poisoning, will improve the prognosis of patients. It should be noted, however, that the potential to recover from liver injury depends on age, nutrient condition and even animal species.

The histological examination revealed that observable pathological changes occurred only in liver. This does not necessarily indicate that the effect of cereulide is confined to the liver, because cereulide induced emesis in animals at much lower doses than did valinomycin, via stimulation of the vagus afferent [5].

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

We are grateful to Kenji Kamiya for helpful suggestions. This study was supported by a Grant-in-Aid (No. 08457086) for Scientific Research from the Ministry of Education, Science and Culture, Japan.

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References
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