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© 1999 Oxford University Press

Article

Enhancement of glutathione S-transferase placental-form positive liver cell foci development by microcystin-LR in aflatoxin B1-initiated rats

Masaru Sekijima1,2, Tomoaki Tsutsumi1, Toshinori Yoshida3, Takanori Harada3, Fumio Tashiro4, Gang Chen5, Shun-Zhang Yu5 and Yoshio Ueno1,6

1 Department of Toxicology and Microbial Chemistry, Faculty of Pharmaceutical Sciences, Science University of Tokyo, Ichigaya, Tokyo 162,
2 Kashima Laboratory, Mitsubishi Chemical Safety Institute, Ibaraki 314-02,
3 Toxicology Division, Institute of Environmental Toxicology, Ibaraki 303,
4 Faculty of Industry and Technology, Science University of Tokyo, Chiba, Japan and
5 Institute of Preventive Medicine, Shanghai Medical University, Shanghai, China


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
The objective of this study was to elucidate whether microcystin-LR (MC-LR), a hepatotoxic blue-green algal toxin in drinking water, is carcinogenic or possesses the ability to modulate aflatoxin B1 (AFB1)-induced hepatocarcinogenicity. In a medium-term liver bioassay, male Fischer 344 rats were given a single i.p. injection of diethylnitrosamine (DEN, 200 mg/kg) followed by an i.p. injection of MC-LR for 6 weeks after 2 weeks of DEN treatment. To study the synergism between AFB1 and MC-LR, DEN-treated rats were given an i.p. injection of AFB1 (0.5 mg/kg) dissolved in dimethyl sulfoxide (DMSO) followed by MC-LR at 2 weeks after the treatment. In a separate experiment, the rats were first given AFB1 (0.5 mg/kg) and 2 weeks later an i.p. injection of 1 or 10 µg/kg of MC-LR twice a week for 6 weeks. Most rats were subjected to a two-thirds partial hepatectomy (PH) at week 3 and were killed under anesthesia at week 8. Liver sections were analyzed for glutathione S-transferase placental form (GST-P) expression, and subjected to histopathological examination for phenotypic alteration of hepatocellular foci. In rats that did not receive DEN, MC-LR did not cause a significant increase in the numbers of GST-P-positive foci, whereas AFB1 induced a slight increase in GST-P-positive foci development. In rats given DEN, MC-LR enhanced the expression of GST-P-positive foci, as did AFB1 but no synergism was observed. Histopathological analysis revealed that the area of eosinophilic foci, a biomarker for preneoplastic liver lesion, markedly increased because of MC-LR. In rats given AFB1 as an initiator, treatment with MC-LR resulted in a synergistic increase in the development of GST-P-positive foci. These results suggest that the hepatocarcinogenicities of MC-LR and AFB1 can be predicted in experimental animals with a medium-term bioassay. Furthermore, tumor promoting activity of MC-LR was demonstrated in rats treated with AFB1.

Abbreviations: ABC, avidin–biotin–peroxidase complex; AFB1 , aflatoxin B1; DEN, diethylnitrosamine; DMSO, dimethyl sulfoxide; ELISA, enzyme-linked immunosorbent assay; GGT, {gamma}-glutamyl transpeptidase; GST-P, glutathione S-transferase placental form; H&E, hematoxylin and eosin; MC-LR, microcystin-LR; PH, partial hepatectomy; PLC, primary liver cancer.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Epidemiological studies have demonstrated that environmental carcinogens in dietary compounds markedly influence the genesis of primary liver cancer (PLC), along with hepatitis B and C viruses (1,2). In recent years, carcinogenic mycotoxins have been isolated from various species of fungi, and aflatoxin B1 (AFB1), which is produced by Aspergillus flavus, has been recognized as a risk factor for PLC (3). In addition to carcinogenic mycotoxins, non-genotoxic fungal products, such as trichothecenes and fumonisins, have been isolated from the metabolites of several plant-pathogenic Fusarium species, and these mycotoxins contaminate cereals and foodstuffs worldwide (4–7). A medium-term rat liver bioassay demonstrated that fumonisins possess an ability to promote liver cancer in N-acetylaminofluorene-initiated rats (8). We previously demonstrated that nivalenol and fumonisins enhanced the expression of glutathione S-transferase placental form (GST-P)-positive liver foci in AFB1-initiated rats (9,10), suggesting that co-exposure to the potent initiator AFB1 and to these fungal promoters through diet causes an enhancement of PLC.

Beside these fungal toxins, a family of hepatotoxic cyclic peptides, called microcystins (MCs) (Figure 1Go), is found in various species of blue-green algae such as Microcystis aeruginosa (11–13). Epidemiological studies have shown that the mortality rate of PLC, the highest among various tumors in China, was closely associated with the sources of drinking water in areas at high risk for PLC (14–16). Based on experimental findings that microcystin-LR (MC-LR), one of the naturally occurring microcystin derivatives, possesses an ability to inhibit protein phosphatases, and consequently exhibits tumor-promoting activity in rats (17), we previously analyzed MC levels in more than 1000 samples of drinking water collected from various water sources in Haimen and Fusui (areas at high risk for PLC), using a highly sensitive enzyme-linked immunosorbent assay (ELISA) recently developed in our laboratory (18,19). The results showed that the levels of MCs were higher in pond-ditch and river water than shallow- and deep-well water, which were in accordance with the mortality rates for PLC in the population. Furthermore, the daily uptake of MCs was estimated to be 0.19 ng per person (20).



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  		Fig. 1. Microcystins. *Adda refers to a side chain moiety attached to the cyclic structure, and is essential for the toxicity of this family. Chemically, it is 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid.

 
In this paper, we attempted to clarify whether MCs possess an ability to modulate the hepatocarcinogenic potential of AFB1 in two medium-term rat liver bioassays: one used diethylnitrosamine (DEN), a two-thirds partial hepatectomy (PH) bioassay, and the other used a AFB1–PH bioassay. GST-P-positive liver foci and altered hepatocyte lesions were employed as an endpoint marker.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Chemicals
DEN was obtained from Wako Pure Chemical (Osaka, Japan), and AFB1 was obtained from Sigma (St Louis, MO). MC-LR (>95% purity in HPLC), isolated from algal bloom sampled from Suwa Lake (Japan) was dissolved in saline and kept at –20°C until use. The antibody for GST-P was kindly provided by the late Dr Sato, Hirosaki University, Japan.

Animals and treatments
Evaluation of carcinogenic activity was essentially carried out by the medium-term rat liver bioassay system proposed by Ito et al. (21,22) by estimating GST-P-positive liver foci. In addition, the development of phenotypic changes in hepatocellular foci was estimated histochemistry (23,24). The procedure for experiment 1 is presented in Figure 2Go. Briefly, a total of 169 male F344 rats (Charles River Japan, Kanagawa, Japan), aged 6 weeks at the commencement of the experiments, were maintained on a basal diet (SIPPK/BK, Shanghai, China), given tap water ad libitum, and housed in plastic cages on wood chips for bedding, in an air-conditioned room at 24 ± 2°C and 55 ± 5% relative humidity with a 12 h light/dark cycle. For the bioassay of hepatocarcinogenicity, the rats were initially given an i.p. injection of 200 mg/kg body weight of DEN dissolved in saline and then, 2 weeks later, were given an i.p. injection of 1 (group 1) or 10 (group 2) µg/kg of MC-LR twice a week for 6 weeks. To study the combined effect of MC-LR and AFB1, DEN-treated rats received either a single i.p. injection of 0.5 mg/kg body wt of AFB1 dissolved in dimethyl sulfoxide (DMSO) (group 4) or MC-LR as above (groups 5 and 6). Other groups not given DEN received MC-LR without (group 8) or with AFB1 (group 9).



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  		Fig. 2. Experimental protocol for medium-term hepatocarcinogenicity assay (experiment 1).

 
In experiment 2 (Figure 3Go), the rats were initially given the same dose of AFB1, and 2 weeks later, were given i.p. injections of 1 (group 11) or 10 (group 12) µg/kg of MC-LR twice a week for 6 weeks. The control group (group 13) received the vehicle alone and was fed the basal diet. The rats not given AFB1 also received 1 (group 14) or 10 (group 15) µg/kg of MC-LR as above. Most rats were subjected to PH, which is a prerequisite for effective initiation by carcinogens (23). The number of rats employed in each group is listed in the respective tables. In these investigations, the experimental dose of MC-LR was quoted from a previous report (17) and based on human uptake of MC-LR (0.19 ng/day per person), the present doses were calculated to be 50–500 times higher than the proposed advisory level (20). Body weights were recorded every week. All animals were killed at week 8 under ether anesthesia, and the livers were immediately excised and sectioned to a thickness of 2–3 mm with a razor blade. Three slices, one from the caudate lobe and two from the anterior lobe, were fixed in an ice-cold acetone solution for immunohistochemical examination of GST-P expression. Additional slices were fixed in 10% phosphate-buffered formalin for further staining with hematoxylin and eosin (H&E).



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  		Fig. 3. Experimental protocol for medium-term hepatocarcinogenicity assay (experiment 2).

 
Phenotypic alteration of hepatocellular foci
The avidin–biotin–peroxidase complex (ABC) method described by Hsu et al. (25) was used to demonstrate GST-P positive liver cells. Tissue preparation, GST-P immunohistochemistry and quantitative analysis of GST-P-positive foci were performed as previously described (9,10). After deparaffinization, liver sections were treated sequentially with normal goat serum, rabbit anti-GST-P (1:8000), biotin-labeled goat anti-rabbit IgG (1:400) and ABC. The sites of peroxidase binding were demonstrated by the diaminobenzidine method. Sections were then counter-stained with hematoxylin for microscopic examination. As a negative control for the specificity of anti-GST-P antibody binding, pre-immune rabbit serum was used instead of antiserum. The numbers and the areas of GST-P-positive foci >0.1 mm in diameter and total areas of the liver sections examined were measured using a color video image processor (Luzex III).

Altered hepatocellular foci in H&E-stained sections were classified into eosinophilic, clear, basophilic and mixed types according to the criteria reported (23,24). The results were assessed by comparing the values of foci between DEN plus test chemicals and DEN alone, and DEN-AFB1 groups. Additive effects were considered to have occurred when net values in the groups administered together were almost the same as the sum total of the individual net values, and synergism was defined when net values in the combined treatment groups were superior to the sum totals. Statistical analysis of the observed values, numbers and areas of GST-P-positive foci, were carried out using a Student's t-test and a Welch's t-test combined with the t-test for variability (26).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
No marked changes in final body weight gains or liver weights were observed among DEN-, AFB1-initiated or non-initiated rats (Table IGo) in experiment 1. To evaluate the hepatocarcinogenicity of MC-LR and the combined effect of MC-LR and AFB1, the effects of a single i.p. injection of AFB1 and repeated i.p. injection of MC-LR for 6 weeks on the numbers and areas of GST-P-positive foci per unit area of liver section were analyzed in rats that were given DEN or saline, as summarized in Table IIGo. In rats initiated with DEN, the numbers and areas of GST-P-positive foci increased (group 7). These values did not significantly increase after repeated i.p. injections of 1 µg/kg of MC-LR (group 1), but they increased significantly to 4.94/cm2 and 0.92 mm2/cm2 with a high dose (10 µg/kg) of MC-LR (group 2). A single i.p. injection of 0.5 mg/kg of AFB1 to DEN-treated rats resulted in marked elevation in the focus numbers (8.34/cm2) and areas (1.69 mm2/cm2) (group 4), reconfirming the hepatocarcinogenic potential of AFB1, as previously reported (9,10).


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  		Table I. Final body and liver weights (experiment 1)
 

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  		Table II. Evaluation of microcystin-LR in the rat liver medium-term test (experiment 1)
 
In rats given a single i.p. injection of AFB1 followed by repeated i.p. injections of MC-LR (group 5), further increases in the focus numbers (10.72/cm2) and the areas (2.26 mm2/cm2) were observed with a low dose (1 µg/kg) of MC-LR (group 5); however, 10 µg/kg MC-LR showed no increase in these values (group 6). To clarify whether the increased values observed after the subsequent injection of MC-LR (1 µg/kg) were additive or synergistic, the background level of GST-P-positive foci in DEN-treated rats (group 5) was subtracted, and the net changes were calculated. The increase in GST-P-positive foci (8.26/cm2 and 1.87 mm2/cm2) of AFB1-1 µg/kg MC-LR (group 5) was not superior to the sum total (5.88/cm2 and 1.30 mm2/cm2) of individual values of AFB1 either alone or with 1 µg/kg of MC-LR (group 1). Thus, the GST-P-positive foci in DEN-treated and AFB1-treated rats were not synergistically enhanced by the subsequent treatment of MC-LR under the present conditions. However, as shown in Table IIIGo, the quantitative analysis of altered hepatocellular foci in the H&E stained section demonstrated that all types, including the eosinophilic/clear cell type, an endpoint marker for neoplastic lesions (22,23), were significantly higher in DEN–AFB1–MC (groups 5 and 6) than DEN–AFB1 (group 4) or DEN–MC (groups 1, 2). In experiment 2, the rats were first treated with AFB1 then followed by MC-LR for evaluation of the interaction between AFB1 and MC-LR. As summarized in Table IVGo, no significant changes in the final body or liver weights were observed among the groups. Immunochemical staining of GST-P-positive foci revealed (Table VGo) that, in rats not given AFB1, no significant expression of GST-P-positive foci was observed in the control (group 16) or MC-treated rats (groups 14, 15). Whereas, in the rats initiated with 0.5 mg/kg AFB1, injections of MC-LR twice weekly for 6 weeks increased the numbers and areas of GST-P-positive foci (groups 11, 12). To clarify whether the increased values observed with the subsequent injection of MC-LR (1 µg/kg) were additive or synergistic, the background level of GST-P-positive foci in rats not treated with AFB1 (group 14) was subtracted, and the net changes were calculated. The increase in GST-P-positive foci (3.17/cm2 and 2.17 mm2/cm2) of AFB1-1 µg/kg MC-LR (group 16) was superior to the sum total (1.90/cm2 and 0.83 mm2/cm2) of individual values of AFB1 either alone or with 1 µg/kg of MC-LR (group 11). Thus, the GST-P-positive foci in AFB1-treated rats were synergistically enhanced by the subsequent treatment with MC-LR under the present conditions.


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  		Table III. Quantitative analysis of altered hepatocellular foci (experiment 1)
 

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  		Table IV. Final body and organ weights (experiment 2)
 

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  		Table V. Quantitative values for GST-P-positive foci in experiment 2
 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
To detect carcinogens, especially hepatocarcinogens, histochemical markers such as {gamma}-glutamyl transpeptidase (GGT) and GST-P are widely employed as end point markers. Extensive research on 227 chemicals using GST-P-positive foci in rats initiated with DEN revealed a close relationship between the results of medium-term assays and the findings from long-term carcinogenic studies (21,27). Adopting this bioassay system, we previously demonstrated the tumor-promoting activity of nivalenol and fumonisins, food contaminants derived from Fusarium spp. (9,10). Among various carcinogenic mycotoxins, AFB1 is well-known as a potent hepatocarcinogen affecting human populations worldwide, especially in sub-Sahara Africa, southern Asia and China, where PLC is prevalent (3). Recently, employing ELISA, we analyzed AFB1-albumin adducts in sera collected from Haimen and Fusui, areas at high risk for PLC in China. The findings revealed that the population was continually exposed to AFB1, suggesting that AFB1 plays an important role in the initiation of hepatocarcinogenesis (16,28). Another environmental risk factor is derived from drinking water, as suggested by several epidemiological surveys carried out in China by the collaborators of the present study (15,16). Actually, our recent study on drinking water in these high risk areas showed an association between the mortality rates of PLC and the levels of MCs in the drinking water (20).

To elucidate whether MC-LR possesses an ability to modulate the development of PLC in AFB1-initiated humans, we conducted experiments using rats as an animal model. Since no marked alterations in body weight gain or hepatic weights were observed in rats given DEN and/or AFB1, these chemicals have no significant toxic effects under the present experimental conditions (Figure 2Go; Table IGo). First, we examined the effect of a single i.p. injection of AFB1 in rats not given MCs. The data showing a slight increase in the development of GST-P-positive foci (Table IIGo) suggested the hepatocarcinogenic potential of this mycotoxin. This result reconfirms previous findings (9,22,29). A significant elevation of GST-P-positive foci in rats treated with DEN followed by a repeated i.p. injection of a high dose (10 µg/kg) of MC-LR predicted the hepatocarcinogenic potential of this phycotoxin (Table IIGo). An important finding in this experiment was derived from the rats receiving both AFB1 and MC-LR. Upon subtracting the background values obtained from DEN-treated rats, we could not demonstrate a synergistic effect for MC-LR on the AFB1-induced increment of GST-P-positive foci, even though rats were given a high dose of MC-LR. In contrast, the histopathological findings of increases in the area of all types of foci, and consequently in the eosinophilic/clear cell type in the rats given both natural toxins (Table IIIGo), suggested that MC-LR enhanced neoplastic lesions in AFB1-treated rats, as observed with phenobarbital (24). As a whole, the promoting effect of MC-LR on the hepatocarcinogenicity was predicted in AFB1-initiated rats. However, no significant difference in GST-P expression or neoplastic lesion was observed between the two doses of MC-LR. It was presumed that a high dose (10 µg/kg) of MC-LR had cytotoxic effects on the normal and/or altered hepatocytes. In this experiment, MC-LR was given to rats i.p., since this route is widely used (17). However, in human cases, fresh water as well as drinking water are the major sources of exposure to MCs. Actually, 0.19 ng MC/day was estimated to be the human exposure dose in Haimen, a high risk area for PLC in China (20). Based on epidemiological investigation, 0.01 µg/l is proposed as an advisory level of MC in drinking water for long-term exposure (20). Moreover, an estimated dose of 100 or more was observed in the MC-LR levels in areas at high risk for PLC in China (20). Since MCs contaminate environmental water worldwide (30), their toxicological significance in fresh and drinking water has to be clarified with special reference to cancer induction.

With regard to the mechanism of the hepatotoxic effects of MCs, numerous experiments have demonstrated their selective accumulation in hepatic tissues, inhibition of protein phosphatases, and that hyperphosphorylation of cytoskeletal microfilaments and regulatory factors give rise to a modulation of signal transduction (31,32). Recently, employing an anti-MC-monoclonal antibody, immunohistochemical analyses revealed localization of MC-LR, in the nuclei, along with cytoplasmic hepatocytes (33,34). Some populations of apoptotic hepatocytes are immunohistochemically positive for MC-LR (34). These findings suggest a modulation of nuclear function by selective binding of MC-LR with nuclear phosphatases, which may influence cellular growth and differentiation. If MC-response elements in nuclear DNA of AFB1-initiated hepatic cells possess a high affinity for MCs, the combined effects of these natural toxins in the development of tumor promotion can be expected.

In consideration of our previous findings in areas at high risk for PLC in China where human sera were positive for AFB1-albumin adducts, a biomarker for AFB1 exposure, and the level of MC-LR in drinking water was associated with the mortality rate for PLC, the present findings support the theory that the hepatocarcinogenic mycotoxin AFB1 in dietary foods may act together with the cancer-promoting phycotoxin, MC, in drinking water to promote the development of PLC.


   Notes
 
6 To whom correspondence should be addressed Email: youeno{at}ps.kagu.sut.ac.jp Back


   Acknowledgments
 
The present study was partly aided by research grants from the Ministry of Education, Science, Sports and Culture (Japan), the Asahi Glass Foundation (Tokyo) and the Mitsubishi Foundation (Tokyo).


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received August 6, 1998; revised September 24, 1998; accepted September 29, 1998.


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T. Chen, Q. Wang, J. Cui, W. Yang, Q. Shi, Z. Hua, J. Ji, and P. Shen
Induction of Apoptosis in Mouse Liver by Microcystin-LR: A Combined Transcriptomic, Proteomic, And Simulation Strategy
Mol. Cell. Proteomics, July 1, 2005; 4(7): 958 - 974.
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