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Modulation of selected cell signaling genes in mouse liver by fumonisin B1
Chemico-Biological Interactions 139 (2002) 317– 331
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Modulation of selected cell signaling genes in mouse liver by fumonisin B1 Neetesh Bhandari, Raghubir P., Sharma * Department of Physiology and Pharmacology, College of Veterinary Medicine, The Uni6ersity of Georgia, Athens, GA 30602 -7389, USA Received 24 November 2001; received in revised form 10 January 2002; accepted 16 January 2002
Abstract Fumonisin B1 (FB1) is a naturally occurring mycotoxin produced primarily by Fusarium 6erticillioides and related fungi, common contaminants of corn throughout the world. FB1 is a carcinogen and causative agent of several lethal animal diseases, including equine leukoencephalomalacia and porcine pulmonary edema. Liver is the primary target organ in mice. In vivo and vitro, cells exposed to FB1 undergo a mixture of necrotic and apoptotic cell death. Our previous studies showed gender differences in hepatotoxicity caused after 5 day FB1 treatment. Gene alterations in cytokine network and apoptosis signaling molecules were also observed after an acute single dose of FB1. To further investigate the gene alterations after a subchronic FB1 exposure and its correlation to observed gender differences, male and female BALB/c mice (five per group) were injected subcutaneously with either saline or 2.25 mg/kg per day of FB1 for 5 days. FB1 caused increased expression of tumor necrosis factor a (TNFa), interleukin (IL)-1a, IL-1b, IL-1 receptor antagonist (IL-1Ra), IL-6, IL-10, IL-12 p40, IL-18 and interferon g (IFNg) in male liver, with a similar increase in females except for IL-1b and IL-18. Control females showed higher basal levels of IL-1a, IL-1Ra, IL-10, IL-12 p40 and IFNg compared with males. Expression of TNF receptor 55 and TNF receptor associated death domain (TRADD) was increased, with no changes in Fas signaling molecules, Fas, Fas ligand (FasL), Fas associated death domain (FADD) and Fas-associated protein factor (FAF). Expression of oncogenic transcription factors, c-Myc, B-Myc, Max and Mad, and apoptotic genes, namely Bcl-2, Bax and Bad, was increased after FB1 treatment. FB1 caused an activation of cytokine network in liver, particularly the TNFa signaling pathway, suggesting its involvement in hepatotoxic mechanisms. Induction of IL-1Ra and oncogenes is a likely mechanism for the cancer promoting properties of FB1, * Corresponding author. Tel.: + 1-706-542-2788; fax: + 1-706-542-3015. E-mail address: [email protected] (Sharma). 0009-2797/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 0 9 - 2 7 9 7 ( 0 2 ) 0 0 0 0 8 - X
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through a mechanism involving apoptotic necrosis, oncotic necrosis and consequent regeneration. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Fumonisin; Mouse; Liver; Gene expression; Cytokines; Gender; Signaling and apoptosis
1. Introduction Fumonisins are mycotoxins produced by Fusarium 6erticillioides (= F. moniliforme) and other Fusarium species, which commonly contaminate corn [1]. Of more than 14 fumonisins known, fumonisin B1 (FB1) is the most prevalent. Consumption of corn-based feed contaminated with high levels of fumonisins or pure FB1 has been shown to cause the farm animal diseases, equine leukoencephalomalacia and porcine pulmonary edema [2]. FB1 is carcinogenic in rats and mice [3] and has been implicated as a contributing factor in human esophageal cancer [4] and possibly primary liver cancer [5]. In laboratory animals FB1 has been shown to be hepatotoxic and nephrotoxic [2]. The mechanism of FB1 toxicity is complex; it is species-, gender-, and tissue-specific. For example, the primary target organ of FB1 toxicity is brain in horse, lungs in pig, kidney in rat and liver in the mouse [2]. In a 2-year feeding study conducted by the National Toxicology Program, the carcinogenic lesions were found in male F344/N rat kidney and female B6C3F1 mouse liver [3]. At present no carcinogenicity data is available on BALB/c mice. In a 5-day FB1 treatment study, female mice showed greater hepatotoxicity compared with males [6]. Biochemical alterations induced by FB1 include inhibition of ceramide synthase (sphinganine and sphingosine-N-acyl transferase) leading to accumulation of sphingoid bases, sphingoid base metabolites, depletion of more complex sphingolipids and inhibition of ceramide biosynthesis [2]. FB1 increased the expression of tumor necrosis factor-a (TNFa) in mouse liver [7,8]. In addition, the in vivo hematological effects of FB1 were partially reversed by anti-TNFa antibodies [9], and mouse strain lacking p75 or p55 TNFa receptor (TNFR) showed reduced hepatotoxicity after FB1 treatment [7,8]. FB1 treatment caused an increased expression of TNFa in liver with relatively less effect in kidney [6]. Inhibition of apoptosis protein, an inhibitor of TNFa pathway also prevented FB1 toxicity in CV-1 cells [10]. In a recent study, cleavage of caspase 8, was involved in FB1-induced apoptosis, further supporting the role of TNFa signal transduction pathway in FB1 toxicity [11]. In vivo studies in mice and rats have demonstrated that FB1 induces apoptosis in liver and kidney [2,12]. FB1 treatment caused apoptosis in several different monkey and human cells [13,14]. Since apoptosis can be involved both in cancer development and disease process, the ability of FB1 to induce apoptosis appears to be important with respect to its toxicological effects. TNFa causes apoptosis in cells and its induction is one of the earlier events in hepatic inflammation, which triggers a cascade of other cytokines [15]. In a recent study we showed the time related induction of various cytokines and apoptosis signaling genes at 4 and 8 h after single acute FB1 treatment [16]. The
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present study was undertaken to investigate these gene alterations after a 5-day sub-chronic FB1 treatment and also to correlate any of these alterations to the observed gender differences. In our previous study we showed that TNFa interleukin (IL)-1b, IL-6, IL-10, IL-12 p40 and interferon g (IFNg), measured by reverse transcriptase-polymerase chain reaction (RT-PCR) in the liver of male mice, were induced after FB1 treatment [6]. Female mice were more sensitive to FB1 toxicity and showed higher basal levels of TNFa, IL-1b, IL-12 p40 and IFNg compared with males. Suggesting that higher basal levels of certain cytokines may predispose female mouse liver to FB1 toxicity. To further evaluate the activation of cytokine network in liver using a quantitative method, expression of various cytokines was measured by ribonuclease protection assay (RPA) in mouse liver after FB1 treatment of animals. FB1 toxicity in liver has been described to cause both oncotic necrosis as well as apoptotic necrosis [17]. The two major death receptors involved in liver pathology are that of TNFa and Fas ligand (FasL) [18]. The FB1-induced c-Myc expression has also been suggested in cancer promoting effects of FB1 [19], therefore, expression of Myc/Max/Mad network oncogenes was measured after FB1 treatment. Other intracellular molecules involved in regulation of apoptosis, like caspases (caspase 3, caspase 6 and caspase 8) and Bcl-2 family pro-apoptotic (Bax and Bad) and anti-apoptotic (Bcl-2) molecules were also measured. FB1 treatment in mice caused induction of both apoptotic and anti-apoptotic genes in the liver. The induction of cytokine network and the Myc/Max/Mad complex in mice liver could play an important role in the observed FB1 toxicity.
2. Materials and methods
2.1. Animal and housing Adult male and female BALB/c mice, 7 weeks old (17–20 g body weight), were obtained from Harlan Laboratories (Indianapolis, IN). The animals were acclimated in the University of Georgia Animal Resources facility for 1 week at the targeted temperature of 23 °C and 50% relative humidity, with a 12-h light: 12-h dark cycle. Pelted feed (fumonisin-free, Teklad 22/5 rodent diet from Harlan– Teklad, Madison, WI) and fresh water were provided ad libitum. Food and water consumptions were recorded daily. The protocols for animal use followed the Public Health Service Policy on Humane Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee.
2.2. Treatment and sampling Mice (five per group per sex) were treated daily with subcutaneous injections (in a volume of 1 ml/100 g body wt.) of endotoxin-free FB1 (99.6% purity; Promec, Tygerberg, South Africa) in physiological buffered saline for 5 days; the dose per mouse being 0 (saline-treated controls) or 2.25 mg FB1/kg per day. The protocol for treatment was based on our previous studies in which FB1 produced consistent liver
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injury characterized by apoptosis in both male and female mice, and females showing greater hepatotoxixicty [6,12]. One day after the last injection, animals were euthanized with halothane, and pieces of liver tissue were obtained, quickly frozen in aliquots on dry ice and stored at − 85 °C until further analysis. The mice were sacrificed in the order alternating between treatment group and sex. All the animals were sacrificed within a 90 min interval starting at 09:00 h.
2.3. RNA isolation and RNase protection assay (RPA) RNA was isolated from frozen livers (each 70–80 mg) with TRI® reagent (Molecular Research Center, Cincinnati, OH), using a protocol described earlier [7]. Frozen tissues were grounded in liquid nitrogen in a mortar and then TRI reagent added to the mortar and tissues further homogenized. RPA was performed on samples using RiboQuant™ RPA starter kit (Pharmingen, San Diego, CA) as described in our previous study [16]. In brief, three Pharmingen template sets, one for cytokines (mCK-2b), the second for TNFa and Fas signaling molecules (mAPO-3), and a third mouse template set (containing TNFa/Myc/Max/Mad/Bcl2/Bax/Bad/Caspases) were used. The synthesis of high-specific-activity, [a32P]UTPlabeled, T7 RNA polymerase-directed, anti-sense RNA probes was done using the in vitro transcription kit and manufacturer’s directions (Pharmingen, San Diego, CA). About 50 mg of sample RNA was hybridized with the probe over night and digested with RNase A and RNase T1. The RNase-protected probes were purified and resolved on a denaturing polyacrylamide gel. The relative gene expression was quantified via densitometric imaging using UN-SCAN-IT software (Silk Scientific Inc., Orem, UT) and normalized against ribosomal protein L32 and glyceraldehydes-3phosphate dehydrogenase (GAPDH) house keeping genes.
2.4. Statistics Data from these studies were analyzed by one-way analysis of variance (ANOVA) followed by the Duncan’s Multiple Range test. The SAS computer program (SAS Institute, Cary, NC) was employed for all calculations. The level of PB 0.05 was considered statistically significant for all comparisons.
3. Results Multiple dose treatment of either male or female mice with FB1 for 5 days produced no obvious gross clinical or behavioral effects. Gene alterations in mice liver after FB1 treatment were measured by RPA. Fig. 1 shows the representative gels obtained from the three RPA templates. The data reported have been normalized to ribosomal protein L32 (accession cK02060). Identical results were obtained with GAPDH as the normalizing gene (accession c M32599, data not shown). Genes from the three template sets have been grouped into different figures based on their relevance and the range of their relative expression values.
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Gene alterations in cytokine network were observed in both males and females after FB1 treatment. FB1 treatment in males caused increased expression of TNFa, IL-1a, IL-1b, IL-1Ra, IL-6, IL-10, IL-18 and IFNg in liver (Fig. 2). In males maximum increase was noted in TNFa (33-fold) followed by IL-1Ra (18-fold), IFNg (12-fold), and IL-6 (11-fold). Table 1 displays calculated fold-increase in expression of cytokine genes, which were significantly altered after FB1 treatment either in males or females. FB1 treatment of female mice caused increased expression of TNFa, IL-1a, IL-1Ra, IL-6, IL-10 and IFNg (Fig. 2). In females, maximum increase was noted in IL-6 followed by TNFa and IFNg. In general, control females showed higher basal levels of most cytokines compared with control males, of which expression of IL-1a, IL-1Ra, IL-10, and IFNg was significantly greater in females than males (Fig. 2). Expression of IL-12 p40 was increased in males but unaltered in females after FB1 treatment (Fig. 3). Females also showed higher basal levels of IL-12 p40 compared with males. IL-12 p35 (Fig. 3) and macrophage migration inhibitory factor (MIF, data not shown) expression was unaltered in both males and females after FB1 treatment. Despite significant differences in basal levels of IL-1a, IL-1Ra, IL-10 and IFNg between male and female, their absolute amounts following FB1 treatment were nearly identical.
Fig. 1. Representative gel from an RNase protection assay. Three template sets were used (A) for cytokines (mCK-2b), (B) for TNFa and Fas signaling molecules (mAPO-3), and (C) for apoptosis signaling molecules (a custom template). A non-hybridized probe set (P) was run as a size marker. The gene names are listed on the left side of each probe set. Treatment groups are indicated as (1) male, saline-treated, (2) male, FB1-treated, (3) female, saline-treated and (4) female, FB1-treated. These bands were obtained from exposure at different times. The efficiency of exposure is linear for several orders of magnitude.
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Fig. 2. Modulation of cytokine expression in male (A) and female (B) mice liver after FB1 treatment. RPA was performed with liver RNA from individual animals. The relative mRNA expression is indicated and normalized against L32. Results are expressed as mean 9 standard error (S.E.; n =5) *, Indicates significantly different from the control group at P B0.05 and a, indicates significantly different from the corresponding male group at PB0.05.
Signaling molecules involved in TNFa apoptotic pathway were increased in both males and females after FB1 treatment. Expression of TNFR 55 increased 40% over control after FB1 treatment (Fig. 4). No alterations were observed in expression of receptor interacting protein (RIP) with FB1 (Fig. 4). FB1 treatment increased TNF receptor associated death domain (TRADD) gene expression 70% over control (Fig. 5). Observed increases in expression of apoptosis and signaling genes, which were significantly altered after FB1 treatment, are shown in Table 2. FB1 treatment caused no alteration in Fas signaling molecules, namely Fas-associated protein factor (FAF, Fig. 4), Fas and Fas-associated death domain (FADD, Fig. 5). Expression of FasL and Fas-associated phosphatase (FAP) could not be detected in any of the liver samples (Fig. 1). No alterations were observed in the expression of TNF-related apoptosis-inducing ligand (TRAIL) after FB1 treatment (Fig. 5). Gene expression of c-Myc, B-Myc, Max and Mad, involved in the Myc/Max/ Mad network were all activated after FB1 treatment, without any gender differences
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Table 1 Expression of cytokines after FB1 treatment in mice liver Gene
GenBank accession number
Fold increase over respective controls
Significance
Male
Female
TNFa IL-1a IL-1b IL-1Ra
M11731 X01450 M15131 M57525
33.3* 5.4* 3.7* 17.8*
4.6* 2.3* 1.6 2.9*
IL-6 IL-10 IL-12 p40 IL-18 IFNg
J03783 M37897 M86671 D49949 K00083
11.0* 4.0* 3.7* 1.6* 11.8*
5.0* 2.5* 1.5 1.0 3.8*
Pro-inflammatory (hepatotoxic) Pro-inflammatory Pro-inflammatory (hepatotoxic) Anti-inflammatory (cancer promoter) Pro-inflammatory Anti-inflammatory Induce IFNg Induce IFNg Activate Kupffer cells (hepatotoxic)
*, Indicates FB1 treatment caused significant increase in gene expression compared with control group at PB0.05. See Figs. 2 and 3 for relative high expression of many cytokines in females than in males.
(Fig. 6). Among these four genes the induction of c-Myc was highest, followed by B-Myc, Mad and Max. FB1 treatment caused induction of pro-apoptotic Bcl-2 family genes, Bax (Fig. 4) and Bad (Fig. 6). Expression of anti-apoptotic gene Bcl-2 was induced only in females after FB1 treatment (Fig. 6). In males, the Bcl-2 expression did increase two-fold after FB1 treatment, but was not significant because of variation within animals.
Fig. 3. Alterations in IL-12 p35 and IL-12 p40 cytokine expression in male (A) and female (B) mice liver after FB1 treatment. RPA was performed with liver RNA from individual animals. The relative mRNA expression is indicated and normalized against L32. Results are expressed as mean 9 S.E. (n = 5) *, Indicates significantly different from the control group at PB 0.05 and a, indicates significantly different from the corresponding male group at PB0.05.
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Fig. 4. Alterations in the expression of TNFR55, RIP, FAF, caspase 6 and Bax in male (A) and female (B) liver after FB1 treatment. RPA was performed with liver RNA from individual animals. The relative mRNA expression is indicated and normalized against L32. Results are expressed as mean 9 S.E. (n = 5) *, Indicates significantly different from the control group at P B0.05.
FB1 treatment caused increased expression of caspase 3 in both males and females (Fig. 5). The expression of caspase 6 (Fig. 4) and caspase 8 (Fig. 5) was unaltered after FB1 treatment. This assay was capable of detecting less than 50% increase in gene expression (for TNFR 55). Only very few genes with fold increase greater than 1.5 were shown unaltered.
4. Discussion FB1-induced hepatotoxicity in male and female BALB/c mice is accompanied by induction of several pro-inflammatory cytokines in liver. Expression of TNFa signaling molecules was also induced after FB1 treatment with no involvement of Fas death receptor activated pathway. Both pro-apoptotic and anti-apoptotic classes of intracellular signaling molecules were induced after FB1 treatment.
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The induction of cytokine network in liver is important because liver has a complete innate immune system consisting of at least Kupffer cells and hepatic natural killer (NK) cells [20]. Kupffer cells are an essential immunological component of host defense, which can induce proinflammatory cytokine like TNFa and IL-1. IL-18, another cytokine produced by Kupffer cells can induce IFNg production by Th1 cells in presence of IL-12 [21]. TNFa, IL-12 and IL-18 can also activate NK cells to produce IFNg. This IFNg through a positive feedback can further activate the Kupffer cells to produce more IL-12 and TNFa [22]. IL-12 is a disulphide linked heterodimeric cytokine consisting of p35 and p40 kDa subunits. Out of the two subunits, p40 is mostly regulated whereas p35 is expressed in an almost constitutive fashion [23]. This could be the reason why we see changes only in IL-12 p40 expression after FB1 exposure and not IL-12 p35 expression. The induced cytokines after FB1 treatment may play an important role in the severity of the observed liver toxicity in mice. In our previous study, females were shown to be more sensitive to FB1 toxicity, showing greater increase in circulating alanine aminotransferase and more apoptotic cells in liver after FB1 treatment [6].
Fig. 5. Alterations in Fas, FADD, TRADD, TRAIL, Caspase 8 and Caspase 3 expression in male (A) and female (B) mice liver after FB1 treatment. RPA was performed with liver RNA from individual animals. The relative mRNA expression is indicated and normalized against L32. Results are expressed as mean 9 S.E. (n= 5) *, Indicates significantly different from the control group at PB 0.05.
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Table 2 Expression of apoptosis and signaling genes after FB1 treatment in mice liver Gene
GenBank Fold increase over accession number respective controls
Significance
Male
Female
1.4* 1.7* 5.4*
1.4* 1.8* 4.7*
TNFR 55 TRADD B-Myc
L26349 AA013699 W18410
c-Myc
X00195
51.4*
29.5*
Max
M63903
2.0*
1.6*
Mad Bcl-2 Bax Bad Caspase 3
L38926 M16506 L22472 L37296 U49929
4.1* 2.1 5.1* 1.8* 2.6*
3.9* 4.6* 3.2* 1.9* 1.5*
TNFa signaling receptor TNFa signaling adapter protein Inhibit growth (hormonally regulated) Activate transcription, proliferation and apoptosis (cancer promoter) Neutral, heterodimerizes with c-Myc or Mad Repress transcription Anti-apoptotic Pro-apoptotic Pro-apoptotic Effector caspase (execute apoptosis)
*, Indicates FB1 treatment caused significant increase in gene expression compared with control group at PB0.05.
In the current study, females also showed higher basal expression levels of IL-1a, IL-1Ra, IL-10, IL-12 p40, and IFNg cytokines in liver compared with males, suggesting the possible role of these increased cytokines in making the females more sensitive to FB1 induced toxicity. Various cytokines are involved in different experimental mouse models of hepatotoxicity. Neutralizing antibodies to TNFa or IL-1 partially prevented liver damage in mice, initiated by hepatotoxic doses of acetaminophen [24]. TNFa, IFNg and IL-12 were involved in liver toxicity known as Shwartzman reaction caused by repeated lipopolysaccharide challenge [25]. IFNg over-expressed mice also develop chronic active hepatitis [26]. TNFa and IL-6 are involved in alcoholic liver disease [18]. TNFa and IFNg have also been related to concanavalin A-induced hepatitis [27]. IL-1Ra was the primary gene that showed altered expression in mouse hepatocellular carcinoma relative to normal liver [28]. IL-1Ra is a natural antagonist of IL-1, where its expression has been clearly correlated with an increase in the proliferating cell nuclear antigen index in hepatocellular adenomas in mice [28]. In FB1 toxicity its increased expression could support a micro-environment for promoting tumors. IL-1Ra expression was increased 18-fold in males and three-fold in females after FB1 treatment. Females also had higher basal levels of IL-1Ra (7.5-fold) as compared with males. TNFR 55 and Fas are two important death receptors whose activation by TNFa and FasL, respectively, has been shown in various liver diseases. FB1 toxicity caused increased expression of TNFR 55 and TRADD involved in TNFa signal transduction pathway, with no alteration in molecules involved in Fas pathway. Expression of TNF type 1 receptor associated protein 2 was also induced in
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FB1-sensitive CV-1 cells but repressed in FB1-resistant COS-7 cells demonstrating the involvement of TNFa-induced death pathway in FB1-induced apoptosis [29]. TNFR 55 is heavily expressed in hepatocytes and Kupffer cells. The expression of TNFR 55 was strongly enhanced in hepatitis B, hepatitis C, autoimmune hepatitis and especially alcohol-induced hepatitis [18]. Signaling via the TNFR 55 is extremely complex, leading to both cell death and cell survival signals [30]. The apoptotic pathway after TNF binding to TNFR 55 involves receptor oligomerization, leading to recruitment of the adapter protein TRADD. TRADD recruits with its self another adapter protein FADD of the Fas signaling pathway, which can activate the caspase 8. An alternative apoptotic pathway by TNFR 55 also involves binding of TRADD with RIP [30]. The RIP expression was unaltered after FB1 treatment. Another death receptor ligand, TRAIL, which could play a role in the liver toxicity [18] showed no change with FB1. Fas/FasL receptor interactions also play an important role in induction of apoptosis in liver. The hepatocytes in the liver appear to be especially susceptible to Fas mediated death, because injection of anti-Fas induces massive injury to liver and not elsewhere [31]. Fas signaling pathway was not involved in the FB1
Fig. 6. Alterations in c-Myc, B-Myc, Max, Mad, Bad and Bcl-2 expression in male (A) and female (B) mice liver after FB1 treatment. RPA was performed with liver RNA from individual animals. The relative mRNA expression is indicated and normalized against L32. Results are expressed as mean 9 S.E. (n=5) *, Indicates significantly different from the control group at PB 0.05.
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hepatotoxicity, because expression of Fas signaling molecules (Fas, FADD and FAF) remained unaltered after FB1 treatment. Hepatocytes constitutively express Fas and may upregulate expression of this receptor in a variety of liver diseases like viral hepatitis or alcohol induced liver disease [32]. Fas expression has been shown to be induced and upregulated by inflammatory cytokines such as IL-1 [33]. At the same time Fas expression was also reduced in particularly aggressive forms of hepatocellular carcinoma [34]. FB1 treatment caused an induction of Myc/Max/Mad network in both male and female mice. The Myc, Max and Mad proteins form a network centered on Max, which forms transactivating complexes when associated with c-Myc but repressive complexes when bound to Mad [35]. The increase in c-Myc expression after FB1 treatment was much greater than the increase in Mad, suggestive of c-Myc dominated response. Induced expression of c-Myc after FB1 exposure has also been reported earlier [19]. The ability of overexpressed c-Myc to facilitate proliferation and inhibit terminal differentiation can thus be involved in the cancer promoting effects of FB1. Overexpression of cyclin D1 protein has also been suggested as a mechanism for FB1-induced hepatocarcinoma [36]. There is conflicting data in the litrature on how c-Myc regulates cyclin D1 expression, some suggesting down-regulation and others up-regulation of cyclin D1 by c-Myc [37]. Bcl-2 family of genes represents another class of proteins, which are involved in the modulation phase of apoptosis, shifting the balance either to cell survival or cell death. Overexpression of Bad, Bax and Bcl-XS enhance apoptosis process whereas Bcl-2 and Bcl-XL expression inhibits apoptosis [38]. In this study, FB1 treatment caused induction of Bax and Bad in both sexes, whereas Bcl-2 expression was increased only in females after FB1 treatment. The Bcl-2 induction in females could be a protective feedback outcome, in response to the greater toxicity observed in females compared with males. A western blot analysis of mouse liver showed expression of Bcl-XL and Bcl-XS proteins with no detectable levels of Bcl-2 [39], thus suggesting that expression of Bcl-2 might be of lesser significance in mouse liver than Bcl-XL. Bcl-2 was also shown not to be an effective inhibitor of FB1-induced apoptosis in CV-1 cells [11]. The caspases are the final executioners of apoptosis. They are expressed as inactive proenzymes in the cytosol and are sequentially activated by proteolysis during apoptosis [40]. Initiator caspases (caspases 8, 9 and 10) activate downstream effector caspases (caspases 3, 6 and 7). Expression of caspase 3 was increased after FB1 treatment in both males and females, whereas caspase 6 and caspase 8 remained unaltered. Expression of caspase 8, being an initiator caspase in TNFa signaling pathway, was increased after 8 h of acute FB1 treatment [16]. FB1 treatment in CV-1 cells also caused cleavage of caspase 8 [11]. In the present study the liver samples were analyzed 24 h after last treatment of FB1 and caspase 8 may be induced at earlier times and then its expression reduced by I-Flice, an endogenous dominant-negative form of caspase 8 [41]. Many genes like caspases and Bcl-2 are activated by proteolytic cleavage. Their activity can be regulated both at the mRNA level as well as at the protein level. More information regarding their protein alterations is required to better understand their involvement in FB1-induced toxicity.
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A generalized activation of a variety of genes by FB1 may suggest an effect on transcription factors like nuclear factor-kB (NF-kB), activating protein-1, or nuclear factor of activated T cells, which are known to bind to regulatory elements of TNFa promoter, thus influencing its expression [42]. TNFa induction can activate the cytokine network in liver, thus leading to stimulation of multiple intracellular signaling pathways. However, an increased translocation of NF-kB to nuclei may not be relevant in FB1 hepatotoxicity, because such increase of NF-kB noted in mice carrying a human TNFa gene resulted in a reduced hepatotoxic response [43]. Since many of these genes are also known to play a protective role along with their cytotoxic effects, their induction could also be a possible outcome of liver regeneration. Cytokine network induction was also noted after short term (acute) FB1 treatment, thus suggesting these effects to be directly related to the liver damage and not to regeneration [16]. Although in the present study, c-Myc and Bcl-2 family genes were induced after FB1 treatment, their expression was unaltered in liver after an acute FB1 treatment [16]. This suggests the possible role of c-Myc and Bcl-2 family genes in liver regeneration, after FB1 toxicity. The results of the current study show an induction of cytokine network in liver after FB1 treatment. FB1-induced expression of TNFa, IL-1, IL-6 and IFN g play a role in the observed liver toxicity, whereas FB1-induced expression of IL-1Ra maybe involved in hepatocyte proliferation. TNFa death receptor pathway was activated by FB1 with no alterations in Fas signaling pathway. FB1 treatment caused increased expression of c-Myc, B-Myc, Max and Mad oncogenes, with a c-Myc-dominated response, suggesting c-Myc’s involvement in FB1-induced hepatocyte proliferation. Both pro-apoptotic and anti-apoptotic Bcl-2 family genes along with caspase 3 expression were increased after FB1 treatment. Further studies are required to understand the importance of individual altered gene in toxicity caused by FB1.
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Modulation of selected cell signaling genes in mouse liver by fumonisin B1 Neetesh Bhandari, Raghubir P., Sharma * Department of Physiology and Pharmacology, College of Veterinary Medicine, The Uni6ersity of Georgia, Athens, GA 30602 -7389, USA Received 24 November 2001; received in revised form 10 January 2002; accepted 16 January 2002
Abstract Fumonisin B1 (FB1) is a naturally occurring mycotoxin produced primarily by Fusarium 6erticillioides and related fungi, common contaminants of corn throughout the world. FB1 is a carcinogen and causative agent of several lethal animal diseases, including equine leukoencephalomalacia and porcine pulmonary edema. Liver is the primary target organ in mice. In vivo and vitro, cells exposed to FB1 undergo a mixture of necrotic and apoptotic cell death. Our previous studies showed gender differences in hepatotoxicity caused after 5 day FB1 treatment. Gene alterations in cytokine network and apoptosis signaling molecules were also observed after an acute single dose of FB1. To further investigate the gene alterations after a subchronic FB1 exposure and its correlation to observed gender differences, male and female BALB/c mice (five per group) were injected subcutaneously with either saline or 2.25 mg/kg per day of FB1 for 5 days. FB1 caused increased expression of tumor necrosis factor a (TNFa), interleukin (IL)-1a, IL-1b, IL-1 receptor antagonist (IL-1Ra), IL-6, IL-10, IL-12 p40, IL-18 and interferon g (IFNg) in male liver, with a similar increase in females except for IL-1b and IL-18. Control females showed higher basal levels of IL-1a, IL-1Ra, IL-10, IL-12 p40 and IFNg compared with males. Expression of TNF receptor 55 and TNF receptor associated death domain (TRADD) was increased, with no changes in Fas signaling molecules, Fas, Fas ligand (FasL), Fas associated death domain (FADD) and Fas-associated protein factor (FAF). Expression of oncogenic transcription factors, c-Myc, B-Myc, Max and Mad, and apoptotic genes, namely Bcl-2, Bax and Bad, was increased after FB1 treatment. FB1 caused an activation of cytokine network in liver, particularly the TNFa signaling pathway, suggesting its involvement in hepatotoxic mechanisms. Induction of IL-1Ra and oncogenes is a likely mechanism for the cancer promoting properties of FB1, * Corresponding author. Tel.: + 1-706-542-2788; fax: + 1-706-542-3015. E-mail address: [email protected] (Sharma). 0009-2797/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 0 9 - 2 7 9 7 ( 0 2 ) 0 0 0 0 8 - X
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through a mechanism involving apoptotic necrosis, oncotic necrosis and consequent regeneration. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Fumonisin; Mouse; Liver; Gene expression; Cytokines; Gender; Signaling and apoptosis
1. Introduction Fumonisins are mycotoxins produced by Fusarium 6erticillioides (= F. moniliforme) and other Fusarium species, which commonly contaminate corn [1]. Of more than 14 fumonisins known, fumonisin B1 (FB1) is the most prevalent. Consumption of corn-based feed contaminated with high levels of fumonisins or pure FB1 has been shown to cause the farm animal diseases, equine leukoencephalomalacia and porcine pulmonary edema [2]. FB1 is carcinogenic in rats and mice [3] and has been implicated as a contributing factor in human esophageal cancer [4] and possibly primary liver cancer [5]. In laboratory animals FB1 has been shown to be hepatotoxic and nephrotoxic [2]. The mechanism of FB1 toxicity is complex; it is species-, gender-, and tissue-specific. For example, the primary target organ of FB1 toxicity is brain in horse, lungs in pig, kidney in rat and liver in the mouse [2]. In a 2-year feeding study conducted by the National Toxicology Program, the carcinogenic lesions were found in male F344/N rat kidney and female B6C3F1 mouse liver [3]. At present no carcinogenicity data is available on BALB/c mice. In a 5-day FB1 treatment study, female mice showed greater hepatotoxicity compared with males [6]. Biochemical alterations induced by FB1 include inhibition of ceramide synthase (sphinganine and sphingosine-N-acyl transferase) leading to accumulation of sphingoid bases, sphingoid base metabolites, depletion of more complex sphingolipids and inhibition of ceramide biosynthesis [2]. FB1 increased the expression of tumor necrosis factor-a (TNFa) in mouse liver [7,8]. In addition, the in vivo hematological effects of FB1 were partially reversed by anti-TNFa antibodies [9], and mouse strain lacking p75 or p55 TNFa receptor (TNFR) showed reduced hepatotoxicity after FB1 treatment [7,8]. FB1 treatment caused an increased expression of TNFa in liver with relatively less effect in kidney [6]. Inhibition of apoptosis protein, an inhibitor of TNFa pathway also prevented FB1 toxicity in CV-1 cells [10]. In a recent study, cleavage of caspase 8, was involved in FB1-induced apoptosis, further supporting the role of TNFa signal transduction pathway in FB1 toxicity [11]. In vivo studies in mice and rats have demonstrated that FB1 induces apoptosis in liver and kidney [2,12]. FB1 treatment caused apoptosis in several different monkey and human cells [13,14]. Since apoptosis can be involved both in cancer development and disease process, the ability of FB1 to induce apoptosis appears to be important with respect to its toxicological effects. TNFa causes apoptosis in cells and its induction is one of the earlier events in hepatic inflammation, which triggers a cascade of other cytokines [15]. In a recent study we showed the time related induction of various cytokines and apoptosis signaling genes at 4 and 8 h after single acute FB1 treatment [16]. The
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present study was undertaken to investigate these gene alterations after a 5-day sub-chronic FB1 treatment and also to correlate any of these alterations to the observed gender differences. In our previous study we showed that TNFa interleukin (IL)-1b, IL-6, IL-10, IL-12 p40 and interferon g (IFNg), measured by reverse transcriptase-polymerase chain reaction (RT-PCR) in the liver of male mice, were induced after FB1 treatment [6]. Female mice were more sensitive to FB1 toxicity and showed higher basal levels of TNFa, IL-1b, IL-12 p40 and IFNg compared with males. Suggesting that higher basal levels of certain cytokines may predispose female mouse liver to FB1 toxicity. To further evaluate the activation of cytokine network in liver using a quantitative method, expression of various cytokines was measured by ribonuclease protection assay (RPA) in mouse liver after FB1 treatment of animals. FB1 toxicity in liver has been described to cause both oncotic necrosis as well as apoptotic necrosis [17]. The two major death receptors involved in liver pathology are that of TNFa and Fas ligand (FasL) [18]. The FB1-induced c-Myc expression has also been suggested in cancer promoting effects of FB1 [19], therefore, expression of Myc/Max/Mad network oncogenes was measured after FB1 treatment. Other intracellular molecules involved in regulation of apoptosis, like caspases (caspase 3, caspase 6 and caspase 8) and Bcl-2 family pro-apoptotic (Bax and Bad) and anti-apoptotic (Bcl-2) molecules were also measured. FB1 treatment in mice caused induction of both apoptotic and anti-apoptotic genes in the liver. The induction of cytokine network and the Myc/Max/Mad complex in mice liver could play an important role in the observed FB1 toxicity.
2. Materials and methods
2.1. Animal and housing Adult male and female BALB/c mice, 7 weeks old (17–20 g body weight), were obtained from Harlan Laboratories (Indianapolis, IN). The animals were acclimated in the University of Georgia Animal Resources facility for 1 week at the targeted temperature of 23 °C and 50% relative humidity, with a 12-h light: 12-h dark cycle. Pelted feed (fumonisin-free, Teklad 22/5 rodent diet from Harlan– Teklad, Madison, WI) and fresh water were provided ad libitum. Food and water consumptions were recorded daily. The protocols for animal use followed the Public Health Service Policy on Humane Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee.
2.2. Treatment and sampling Mice (five per group per sex) were treated daily with subcutaneous injections (in a volume of 1 ml/100 g body wt.) of endotoxin-free FB1 (99.6% purity; Promec, Tygerberg, South Africa) in physiological buffered saline for 5 days; the dose per mouse being 0 (saline-treated controls) or 2.25 mg FB1/kg per day. The protocol for treatment was based on our previous studies in which FB1 produced consistent liver
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injury characterized by apoptosis in both male and female mice, and females showing greater hepatotoxixicty [6,12]. One day after the last injection, animals were euthanized with halothane, and pieces of liver tissue were obtained, quickly frozen in aliquots on dry ice and stored at − 85 °C until further analysis. The mice were sacrificed in the order alternating between treatment group and sex. All the animals were sacrificed within a 90 min interval starting at 09:00 h.
2.3. RNA isolation and RNase protection assay (RPA) RNA was isolated from frozen livers (each 70–80 mg) with TRI® reagent (Molecular Research Center, Cincinnati, OH), using a protocol described earlier [7]. Frozen tissues were grounded in liquid nitrogen in a mortar and then TRI reagent added to the mortar and tissues further homogenized. RPA was performed on samples using RiboQuant™ RPA starter kit (Pharmingen, San Diego, CA) as described in our previous study [16]. In brief, three Pharmingen template sets, one for cytokines (mCK-2b), the second for TNFa and Fas signaling molecules (mAPO-3), and a third mouse template set (containing TNFa/Myc/Max/Mad/Bcl2/Bax/Bad/Caspases) were used. The synthesis of high-specific-activity, [a32P]UTPlabeled, T7 RNA polymerase-directed, anti-sense RNA probes was done using the in vitro transcription kit and manufacturer’s directions (Pharmingen, San Diego, CA). About 50 mg of sample RNA was hybridized with the probe over night and digested with RNase A and RNase T1. The RNase-protected probes were purified and resolved on a denaturing polyacrylamide gel. The relative gene expression was quantified via densitometric imaging using UN-SCAN-IT software (Silk Scientific Inc., Orem, UT) and normalized against ribosomal protein L32 and glyceraldehydes-3phosphate dehydrogenase (GAPDH) house keeping genes.
2.4. Statistics Data from these studies were analyzed by one-way analysis of variance (ANOVA) followed by the Duncan’s Multiple Range test. The SAS computer program (SAS Institute, Cary, NC) was employed for all calculations. The level of PB 0.05 was considered statistically significant for all comparisons.
3. Results Multiple dose treatment of either male or female mice with FB1 for 5 days produced no obvious gross clinical or behavioral effects. Gene alterations in mice liver after FB1 treatment were measured by RPA. Fig. 1 shows the representative gels obtained from the three RPA templates. The data reported have been normalized to ribosomal protein L32 (accession cK02060). Identical results were obtained with GAPDH as the normalizing gene (accession c M32599, data not shown). Genes from the three template sets have been grouped into different figures based on their relevance and the range of their relative expression values.
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Gene alterations in cytokine network were observed in both males and females after FB1 treatment. FB1 treatment in males caused increased expression of TNFa, IL-1a, IL-1b, IL-1Ra, IL-6, IL-10, IL-18 and IFNg in liver (Fig. 2). In males maximum increase was noted in TNFa (33-fold) followed by IL-1Ra (18-fold), IFNg (12-fold), and IL-6 (11-fold). Table 1 displays calculated fold-increase in expression of cytokine genes, which were significantly altered after FB1 treatment either in males or females. FB1 treatment of female mice caused increased expression of TNFa, IL-1a, IL-1Ra, IL-6, IL-10 and IFNg (Fig. 2). In females, maximum increase was noted in IL-6 followed by TNFa and IFNg. In general, control females showed higher basal levels of most cytokines compared with control males, of which expression of IL-1a, IL-1Ra, IL-10, and IFNg was significantly greater in females than males (Fig. 2). Expression of IL-12 p40 was increased in males but unaltered in females after FB1 treatment (Fig. 3). Females also showed higher basal levels of IL-12 p40 compared with males. IL-12 p35 (Fig. 3) and macrophage migration inhibitory factor (MIF, data not shown) expression was unaltered in both males and females after FB1 treatment. Despite significant differences in basal levels of IL-1a, IL-1Ra, IL-10 and IFNg between male and female, their absolute amounts following FB1 treatment were nearly identical.
Fig. 1. Representative gel from an RNase protection assay. Three template sets were used (A) for cytokines (mCK-2b), (B) for TNFa and Fas signaling molecules (mAPO-3), and (C) for apoptosis signaling molecules (a custom template). A non-hybridized probe set (P) was run as a size marker. The gene names are listed on the left side of each probe set. Treatment groups are indicated as (1) male, saline-treated, (2) male, FB1-treated, (3) female, saline-treated and (4) female, FB1-treated. These bands were obtained from exposure at different times. The efficiency of exposure is linear for several orders of magnitude.
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Fig. 2. Modulation of cytokine expression in male (A) and female (B) mice liver after FB1 treatment. RPA was performed with liver RNA from individual animals. The relative mRNA expression is indicated and normalized against L32. Results are expressed as mean 9 standard error (S.E.; n =5) *, Indicates significantly different from the control group at P B0.05 and a, indicates significantly different from the corresponding male group at PB0.05.
Signaling molecules involved in TNFa apoptotic pathway were increased in both males and females after FB1 treatment. Expression of TNFR 55 increased 40% over control after FB1 treatment (Fig. 4). No alterations were observed in expression of receptor interacting protein (RIP) with FB1 (Fig. 4). FB1 treatment increased TNF receptor associated death domain (TRADD) gene expression 70% over control (Fig. 5). Observed increases in expression of apoptosis and signaling genes, which were significantly altered after FB1 treatment, are shown in Table 2. FB1 treatment caused no alteration in Fas signaling molecules, namely Fas-associated protein factor (FAF, Fig. 4), Fas and Fas-associated death domain (FADD, Fig. 5). Expression of FasL and Fas-associated phosphatase (FAP) could not be detected in any of the liver samples (Fig. 1). No alterations were observed in the expression of TNF-related apoptosis-inducing ligand (TRAIL) after FB1 treatment (Fig. 5). Gene expression of c-Myc, B-Myc, Max and Mad, involved in the Myc/Max/ Mad network were all activated after FB1 treatment, without any gender differences
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Table 1 Expression of cytokines after FB1 treatment in mice liver Gene
GenBank accession number
Fold increase over respective controls
Significance
Male
Female
TNFa IL-1a IL-1b IL-1Ra
M11731 X01450 M15131 M57525
33.3* 5.4* 3.7* 17.8*
4.6* 2.3* 1.6 2.9*
IL-6 IL-10 IL-12 p40 IL-18 IFNg
J03783 M37897 M86671 D49949 K00083
11.0* 4.0* 3.7* 1.6* 11.8*
5.0* 2.5* 1.5 1.0 3.8*
Pro-inflammatory (hepatotoxic) Pro-inflammatory Pro-inflammatory (hepatotoxic) Anti-inflammatory (cancer promoter) Pro-inflammatory Anti-inflammatory Induce IFNg Induce IFNg Activate Kupffer cells (hepatotoxic)
*, Indicates FB1 treatment caused significant increase in gene expression compared with control group at PB0.05. See Figs. 2 and 3 for relative high expression of many cytokines in females than in males.
(Fig. 6). Among these four genes the induction of c-Myc was highest, followed by B-Myc, Mad and Max. FB1 treatment caused induction of pro-apoptotic Bcl-2 family genes, Bax (Fig. 4) and Bad (Fig. 6). Expression of anti-apoptotic gene Bcl-2 was induced only in females after FB1 treatment (Fig. 6). In males, the Bcl-2 expression did increase two-fold after FB1 treatment, but was not significant because of variation within animals.
Fig. 3. Alterations in IL-12 p35 and IL-12 p40 cytokine expression in male (A) and female (B) mice liver after FB1 treatment. RPA was performed with liver RNA from individual animals. The relative mRNA expression is indicated and normalized against L32. Results are expressed as mean 9 S.E. (n = 5) *, Indicates significantly different from the control group at PB 0.05 and a, indicates significantly different from the corresponding male group at PB0.05.
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Fig. 4. Alterations in the expression of TNFR55, RIP, FAF, caspase 6 and Bax in male (A) and female (B) liver after FB1 treatment. RPA was performed with liver RNA from individual animals. The relative mRNA expression is indicated and normalized against L32. Results are expressed as mean 9 S.E. (n = 5) *, Indicates significantly different from the control group at P B0.05.
FB1 treatment caused increased expression of caspase 3 in both males and females (Fig. 5). The expression of caspase 6 (Fig. 4) and caspase 8 (Fig. 5) was unaltered after FB1 treatment. This assay was capable of detecting less than 50% increase in gene expression (for TNFR 55). Only very few genes with fold increase greater than 1.5 were shown unaltered.
4. Discussion FB1-induced hepatotoxicity in male and female BALB/c mice is accompanied by induction of several pro-inflammatory cytokines in liver. Expression of TNFa signaling molecules was also induced after FB1 treatment with no involvement of Fas death receptor activated pathway. Both pro-apoptotic and anti-apoptotic classes of intracellular signaling molecules were induced after FB1 treatment.
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The induction of cytokine network in liver is important because liver has a complete innate immune system consisting of at least Kupffer cells and hepatic natural killer (NK) cells [20]. Kupffer cells are an essential immunological component of host defense, which can induce proinflammatory cytokine like TNFa and IL-1. IL-18, another cytokine produced by Kupffer cells can induce IFNg production by Th1 cells in presence of IL-12 [21]. TNFa, IL-12 and IL-18 can also activate NK cells to produce IFNg. This IFNg through a positive feedback can further activate the Kupffer cells to produce more IL-12 and TNFa [22]. IL-12 is a disulphide linked heterodimeric cytokine consisting of p35 and p40 kDa subunits. Out of the two subunits, p40 is mostly regulated whereas p35 is expressed in an almost constitutive fashion [23]. This could be the reason why we see changes only in IL-12 p40 expression after FB1 exposure and not IL-12 p35 expression. The induced cytokines after FB1 treatment may play an important role in the severity of the observed liver toxicity in mice. In our previous study, females were shown to be more sensitive to FB1 toxicity, showing greater increase in circulating alanine aminotransferase and more apoptotic cells in liver after FB1 treatment [6].
Fig. 5. Alterations in Fas, FADD, TRADD, TRAIL, Caspase 8 and Caspase 3 expression in male (A) and female (B) mice liver after FB1 treatment. RPA was performed with liver RNA from individual animals. The relative mRNA expression is indicated and normalized against L32. Results are expressed as mean 9 S.E. (n= 5) *, Indicates significantly different from the control group at PB 0.05.
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Table 2 Expression of apoptosis and signaling genes after FB1 treatment in mice liver Gene
GenBank Fold increase over accession number respective controls
Significance
Male
Female
1.4* 1.7* 5.4*
1.4* 1.8* 4.7*
TNFR 55 TRADD B-Myc
L26349 AA013699 W18410
c-Myc
X00195
51.4*
29.5*
Max
M63903
2.0*
1.6*
Mad Bcl-2 Bax Bad Caspase 3
L38926 M16506 L22472 L37296 U49929
4.1* 2.1 5.1* 1.8* 2.6*
3.9* 4.6* 3.2* 1.9* 1.5*
TNFa signaling receptor TNFa signaling adapter protein Inhibit growth (hormonally regulated) Activate transcription, proliferation and apoptosis (cancer promoter) Neutral, heterodimerizes with c-Myc or Mad Repress transcription Anti-apoptotic Pro-apoptotic Pro-apoptotic Effector caspase (execute apoptosis)
*, Indicates FB1 treatment caused significant increase in gene expression compared with control group at PB0.05.
In the current study, females also showed higher basal expression levels of IL-1a, IL-1Ra, IL-10, IL-12 p40, and IFNg cytokines in liver compared with males, suggesting the possible role of these increased cytokines in making the females more sensitive to FB1 induced toxicity. Various cytokines are involved in different experimental mouse models of hepatotoxicity. Neutralizing antibodies to TNFa or IL-1 partially prevented liver damage in mice, initiated by hepatotoxic doses of acetaminophen [24]. TNFa, IFNg and IL-12 were involved in liver toxicity known as Shwartzman reaction caused by repeated lipopolysaccharide challenge [25]. IFNg over-expressed mice also develop chronic active hepatitis [26]. TNFa and IL-6 are involved in alcoholic liver disease [18]. TNFa and IFNg have also been related to concanavalin A-induced hepatitis [27]. IL-1Ra was the primary gene that showed altered expression in mouse hepatocellular carcinoma relative to normal liver [28]. IL-1Ra is a natural antagonist of IL-1, where its expression has been clearly correlated with an increase in the proliferating cell nuclear antigen index in hepatocellular adenomas in mice [28]. In FB1 toxicity its increased expression could support a micro-environment for promoting tumors. IL-1Ra expression was increased 18-fold in males and three-fold in females after FB1 treatment. Females also had higher basal levels of IL-1Ra (7.5-fold) as compared with males. TNFR 55 and Fas are two important death receptors whose activation by TNFa and FasL, respectively, has been shown in various liver diseases. FB1 toxicity caused increased expression of TNFR 55 and TRADD involved in TNFa signal transduction pathway, with no alteration in molecules involved in Fas pathway. Expression of TNF type 1 receptor associated protein 2 was also induced in
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FB1-sensitive CV-1 cells but repressed in FB1-resistant COS-7 cells demonstrating the involvement of TNFa-induced death pathway in FB1-induced apoptosis [29]. TNFR 55 is heavily expressed in hepatocytes and Kupffer cells. The expression of TNFR 55 was strongly enhanced in hepatitis B, hepatitis C, autoimmune hepatitis and especially alcohol-induced hepatitis [18]. Signaling via the TNFR 55 is extremely complex, leading to both cell death and cell survival signals [30]. The apoptotic pathway after TNF binding to TNFR 55 involves receptor oligomerization, leading to recruitment of the adapter protein TRADD. TRADD recruits with its self another adapter protein FADD of the Fas signaling pathway, which can activate the caspase 8. An alternative apoptotic pathway by TNFR 55 also involves binding of TRADD with RIP [30]. The RIP expression was unaltered after FB1 treatment. Another death receptor ligand, TRAIL, which could play a role in the liver toxicity [18] showed no change with FB1. Fas/FasL receptor interactions also play an important role in induction of apoptosis in liver. The hepatocytes in the liver appear to be especially susceptible to Fas mediated death, because injection of anti-Fas induces massive injury to liver and not elsewhere [31]. Fas signaling pathway was not involved in the FB1
Fig. 6. Alterations in c-Myc, B-Myc, Max, Mad, Bad and Bcl-2 expression in male (A) and female (B) mice liver after FB1 treatment. RPA was performed with liver RNA from individual animals. The relative mRNA expression is indicated and normalized against L32. Results are expressed as mean 9 S.E. (n=5) *, Indicates significantly different from the control group at PB 0.05.
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hepatotoxicity, because expression of Fas signaling molecules (Fas, FADD and FAF) remained unaltered after FB1 treatment. Hepatocytes constitutively express Fas and may upregulate expression of this receptor in a variety of liver diseases like viral hepatitis or alcohol induced liver disease [32]. Fas expression has been shown to be induced and upregulated by inflammatory cytokines such as IL-1 [33]. At the same time Fas expression was also reduced in particularly aggressive forms of hepatocellular carcinoma [34]. FB1 treatment caused an induction of Myc/Max/Mad network in both male and female mice. The Myc, Max and Mad proteins form a network centered on Max, which forms transactivating complexes when associated with c-Myc but repressive complexes when bound to Mad [35]. The increase in c-Myc expression after FB1 treatment was much greater than the increase in Mad, suggestive of c-Myc dominated response. Induced expression of c-Myc after FB1 exposure has also been reported earlier [19]. The ability of overexpressed c-Myc to facilitate proliferation and inhibit terminal differentiation can thus be involved in the cancer promoting effects of FB1. Overexpression of cyclin D1 protein has also been suggested as a mechanism for FB1-induced hepatocarcinoma [36]. There is conflicting data in the litrature on how c-Myc regulates cyclin D1 expression, some suggesting down-regulation and others up-regulation of cyclin D1 by c-Myc [37]. Bcl-2 family of genes represents another class of proteins, which are involved in the modulation phase of apoptosis, shifting the balance either to cell survival or cell death. Overexpression of Bad, Bax and Bcl-XS enhance apoptosis process whereas Bcl-2 and Bcl-XL expression inhibits apoptosis [38]. In this study, FB1 treatment caused induction of Bax and Bad in both sexes, whereas Bcl-2 expression was increased only in females after FB1 treatment. The Bcl-2 induction in females could be a protective feedback outcome, in response to the greater toxicity observed in females compared with males. A western blot analysis of mouse liver showed expression of Bcl-XL and Bcl-XS proteins with no detectable levels of Bcl-2 [39], thus suggesting that expression of Bcl-2 might be of lesser significance in mouse liver than Bcl-XL. Bcl-2 was also shown not to be an effective inhibitor of FB1-induced apoptosis in CV-1 cells [11]. The caspases are the final executioners of apoptosis. They are expressed as inactive proenzymes in the cytosol and are sequentially activated by proteolysis during apoptosis [40]. Initiator caspases (caspases 8, 9 and 10) activate downstream effector caspases (caspases 3, 6 and 7). Expression of caspase 3 was increased after FB1 treatment in both males and females, whereas caspase 6 and caspase 8 remained unaltered. Expression of caspase 8, being an initiator caspase in TNFa signaling pathway, was increased after 8 h of acute FB1 treatment [16]. FB1 treatment in CV-1 cells also caused cleavage of caspase 8 [11]. In the present study the liver samples were analyzed 24 h after last treatment of FB1 and caspase 8 may be induced at earlier times and then its expression reduced by I-Flice, an endogenous dominant-negative form of caspase 8 [41]. Many genes like caspases and Bcl-2 are activated by proteolytic cleavage. Their activity can be regulated both at the mRNA level as well as at the protein level. More information regarding their protein alterations is required to better understand their involvement in FB1-induced toxicity.
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A generalized activation of a variety of genes by FB1 may suggest an effect on transcription factors like nuclear factor-kB (NF-kB), activating protein-1, or nuclear factor of activated T cells, which are known to bind to regulatory elements of TNFa promoter, thus influencing its expression [42]. TNFa induction can activate the cytokine network in liver, thus leading to stimulation of multiple intracellular signaling pathways. However, an increased translocation of NF-kB to nuclei may not be relevant in FB1 hepatotoxicity, because such increase of NF-kB noted in mice carrying a human TNFa gene resulted in a reduced hepatotoxic response [43]. Since many of these genes are also known to play a protective role along with their cytotoxic effects, their induction could also be a possible outcome of liver regeneration. Cytokine network induction was also noted after short term (acute) FB1 treatment, thus suggesting these effects to be directly related to the liver damage and not to regeneration [16]. Although in the present study, c-Myc and Bcl-2 family genes were induced after FB1 treatment, their expression was unaltered in liver after an acute FB1 treatment [16]. This suggests the possible role of c-Myc and Bcl-2 family genes in liver regeneration, after FB1 toxicity. The results of the current study show an induction of cytokine network in liver after FB1 treatment. FB1-induced expression of TNFa, IL-1, IL-6 and IFN g play a role in the observed liver toxicity, whereas FB1-induced expression of IL-1Ra maybe involved in hepatocyte proliferation. TNFa death receptor pathway was activated by FB1 with no alterations in Fas signaling pathway. FB1 treatment caused increased expression of c-Myc, B-Myc, Max and Mad oncogenes, with a c-Myc-dominated response, suggesting c-Myc’s involvement in FB1-induced hepatocyte proliferation. Both pro-apoptotic and anti-apoptotic Bcl-2 family genes along with caspase 3 expression were increased after FB1 treatment. Further studies are required to understand the importance of individual altered gene in toxicity caused by FB1.
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