RESUMO
The cancer-promoting potential of fumonisin B1 (FB1) was investigated by feeding different dietary levels (10, 50, 100, 250, 500 mg FB1/kg) to diethynitrosamine (DEN)-initiated rats for 21 days. Dietary levels containing 50 mg FB1/kg and higher, markedly increased the number and size of the placental form of glutathione-S-transferase-positive (GSTP+) foci in the liver of the rats. The cancer-promoting activity of FB1 was associated with an inhibitory effect on partial hepatectomy (PH)-induced regenerative hepatocyte proliferation, as the incorporation of 3H-labelled thymidine was significantly (P < 0.05) reduced by those FB1-containing diets that exhibited cancer promotion. In vitro studies on the mitogenic activity of epidermal growth factor (EGF) in primary rat hepatocytes further supported the in vivo data in that FB1, similar to other cancer promoters such as phenobarbital and 2-acetylaminofluorene (2-AAF), alters growth stimulatory responses in primary hepatocytes. No significant (P > 0.05) changes in the sphinganine/sphingosine (Sa/So) ratio were observed in the liver of the rats fed the lowest FB1-containing diet (50 mg FB1/kg diet) that effected cancer promotion. The present study indicated that FB1 exhibited cancer-promoting activity in the absence of adverse hepatotoxic effects and at dietary levels that failed to effect cancer initiation.
Assuntos
Ácidos Carboxílicos/toxicidade , Carcinógenos Ambientais/toxicidade , Dietilnitrosamina , Fumonisinas , Neoplasias Hepáticas Experimentais/induzido quimicamente , Fígado/efeitos dos fármacos , Micotoxinas/toxicidade , Animais , Sinergismo Farmacológico , Glutationa Transferase/metabolismo , Fígado/enzimologia , Neoplasias Hepáticas Experimentais/enzimologia , Regeneração Hepática/efeitos dos fármacos , Masculino , Ratos , Ratos Endogâmicos F344RESUMO
The mitoinhibitory effect of fumonisin B1 (FB1) on the mitogenic response of epidermal growth factor (EGF) was investigated in primary hepatocyte cultures with respect to the alterations in the omega6 fatty acid metabolic pathway. Fatty acid analyses of hepatocytes showed that EGF treatment resulted in a significant decrease in the relative levels of 20:4omega6 (arachidonic acid) and an increase in 18:2omega6 (linoleic acid). Supplementation of the hepatocyte cultures with 20:4omega6 in the absence of EGF resulted in an increase in the total omega6 and omega6/omega3 fatty acid ratio. Addition of 20:5omega3 (eicosapentaenoic acid) resulted in an increase of the relative levels of the long chain omega3 fatty acids at the expense of the omega6 fatty acids. When 20:4omega6 and 20:5omega3 was added in the presence of EGF, the mitogenic response of EGF was increased and decreased respectively. When compared to the fatty acid profiles in the absence of EGF, the decreased mitogenic response coincided with a decrease of total omega6 fatty acids and total polyunsaturated fatty acids (PUFA). In addition, the saturated and mono-unsaturated fatty acids increased and the polyunsaturated/saturated (P/S) fatty acid ratio decreased which implied a more rigid membrane structure. Addition of prostaglandin E2 (PGE2) and prostaglandin E1 (PGE1) stimulated and inhibited the mitogenic response respectively. Ibuprofen, a known cyclooxygenase inhibitor, and FB1 inhibited the EGF-induced mitogenic response in a dose-dependent manner. The mitoinhibitory effect of FB1 on the EGF response was counteracted by the addition of PGE2. FB1 also disrupts the omega6 fatty acid metabolic pathway in primary hepatocytes, resulting in the accumulation of C18:2omega6 in phospatidylcholine and triacylglicerol. The disruption of the omega6 fatty acid metabolic pathway and/or prostaglandin synthesis is likely to be an important event in the mitoinhibitory effect of FB1 on growth factor responses.
Assuntos
Ácidos Carboxílicos/toxicidade , Ácidos Graxos/biossíntese , Fumonisinas , Fígado/efeitos dos fármacos , Fígado/metabolismo , Micotoxinas/toxicidade , Alprostadil/farmacologia , Animais , Ácido Araquidônico/metabolismo , Carcinógenos Ambientais/toxicidade , Morte Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Dinoprostona/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Ácidos Graxos/farmacologia , Ibuprofeno/farmacologia , Ácido Linoleico/metabolismo , Fígado/citologia , RatosRESUMO
An in vitro study on the interaction and biotransformation of the [14C]fumonisin B mycotoxins was conducted, using primary rat hepatocyte cultures and subcellular enzyme preparations. At the same concentration, fumonisin B2 (FB2) exhibited a higher cytotoxicity and specific binding to primary rat hepatocytes than fumonisin B1 (FB1). However, if the effective dose level (EDL) is considered (i.e. the lowest level of toxin that binds to the hepatocytes to elicit a cytotoxic effect), FB1 and FB2 exhibited a similar cytotoxic effect. FB1 was found to be associated with both the soluble and insoluble compartments within the cell. As assessed by the radioactivity associated with the cellular preparations, very little (approximately 0.01%) FB1 and/or FB2 bound to hepatocytes. In the subsequent fractionation of the culture medium using amberlite XAD-2 and silica-gel chromatography, no metabolites were detected, indicating that the fumonisin molecule was not metabolized by primary hepatocytes. The latter aspect was confirmed by the fact that incubation of FB1 with microsomal enzyme preparations also failed to indicate any metabolism of the fumonisins by the esterases or by cytochrome P-450 monooxygenase. FB1 was also found not to be a substrate for the triglyceride hepatic endothelial lipase, nor for a lipase from porcine pancreas. This study supports further the hypothesis that the intact molecule of the fumonisins is required for biological activity.
Assuntos
Fumonisinas , Fígado/efeitos dos fármacos , Micotoxinas/farmacologia , Animais , Biotransformação , Células Cultivadas , Técnicas In Vitro , Fígado/metabolismo , Masculino , Micotoxinas/farmacocinética , Ratos , Ratos Endogâmicos F344 , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismoRESUMO
A short-term rat liver cancer initiation/promotion model was used to monitor the cancer-initiating activity of the mycotoxins fumonisin B1 (FB1), fumonisin B2 (FB2) and fumonisin B3 (FB3) as well as the N-acetyl derivatives of FB1 and FB2, and their respective hydrolysis products the aminopolyols. The induction of resistant hepatocytes, which develop into hepatocyte nodules on selection by the 2-acetylaminofluorene-partial hepatectomy promoting treatment, was taken as the endpoint for cancer initiation. When fed at a level of 1000 mg/kg diet for 21 days, only the fumonisins B were found to initiate cancer. In addition, these mycotoxins caused a marked reduction in the rat body weight during the initiating treatment. Comparative cytotoxicity studies in primary rat hepatocytes indicated that FB2 exhibited the highest cytotoxic effect followed by FB3 and FB1. In general, the fumonisin B mycotoxins exhibited a low cytotoxic effect in hepatocyte cultures, and the concentrations of FB1 and FB2 that caused a 50% (CD50) release of the total lactate dehydrogenase (LDH) were in the order of 2000 and 1000 microM, respectively. The N-acetyl derivatives also exhibited a cytotoxic effect, but were not as cytotoxic as the parent molecules at high concentrations. The respective aminopolyols exhibited a higher cytotoxicity than did the parent compounds, while tricarballylic acid (TCA) exhibited no dose-response effect despite the fact that it had a higher background cytotoxicity compared with the control. The apparent inability of the aminopolyols to act as cancer initiators could be related to a lack in absorption from the gut.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Carcinógenos/toxicidade , Fumonisinas , Micotoxinas/toxicidade , Análise de Variância , Animais , Carcinógenos/química , Células Cultivadas , Relação Dose-Resposta a Droga , L-Lactato Desidrogenase/efeitos dos fármacos , Neoplasias Hepáticas Experimentais/induzido quimicamente , Masculino , Micotoxinas/química , Ratos , Ratos Endogâmicos F344 , Relação Estrutura-Atividade , Fatores de TempoRESUMO
The modulating role of fumonisin B1 (FB1) on lipid biosynthesis was evaluated in a short-term (21 day) experiment using male Fischer rats fed high dietary levels (50, 100 and 250 mg FB1/kg) and in a long-term (2 yr) experiment using male BD IX rats fed low dietary levels (1, 10 and 25 mg FB1/kg) of FB1. The total serum and liver cholesterol was significantly (P < 0.01) increased in the rats fed 250 mg FB1/kg diet for 21 days, while the liver phospholipids, sphingomyelin and phosphatidylethanolamine (PE) were significantly decreased (P < 0.01) and increased (P < 0.05), respectively. In the long-term study, only PE was significantly (P < 0.05) increased in all the FB1-treated animals. Fatty acid (FA) analysis of PE indicated that C18:2n-6 was significantly increased (P < 0.05 to P < 0.01) in the FB1-treated rats of the short-term study, while it was markedly (not significantly) increased in phosphatidylcholine (PC). The same pattern was observed in the PC and PE fractions of the liver of the FB1-treated rats from the long-term studies, but the changes were not significant due to the small number (three rats per group) of rats analysed. The levels of C22:5n-6 and C22:6n-3 were also markedly decreased and increased respectively in the 10 and 25 mg FB1/kg-treated groups. When the FAs were determined in the total lipids in a larger number of rats (four to six animals per group) the level of C18:2n-6 was significantly increased in the 10 (P < 0.01) and 25 (P < 0.05) mg FB1/kg-treated groups. Similar effects were noticed in plasma PC with respect to the C18:5n-6 and C22:55n-6 in both the long- and short-term treated groups, except that C20:4n-6 was also lower in both cases. The total n-6 FAs and polyunsaturated FAs were significantly (P < 0.01) and markedly reduced in PC and PE, respectively, of the rats fed the 250 mg FB1/kg diet. In the long-term experiment the n-6/n-3 ratio was significantly (P < 0.01) decreased in PE and markedly lowered in PC due to a significant (P < 0.05) increase in the n-3 FAs of both phospholipid fractions. The sphinganine/sphingosine ratio was significantly (P < 0.05) altered in the liver of the rats fed the 100 and 250 mg FB1/kg diets for 21 days, while in the long-term study no significant changes were noticed in either the liver or sera. The present data indicate that FB1 affects lipid biosynthesis in rat liver and plasma differently, depending on the dietary level and duration of treatment. Alterations to the n-3 and n-6 FA biosynthetic pathways, detected in rats fed relatively low dietary levels of FB1, are likely to be important mediators for FB1-induced effects on hepatocyte cell proliferation.
Assuntos
Ácidos Carboxílicos/farmacologia , Carcinógenos Ambientais/farmacologia , Ácidos Graxos/metabolismo , Fumonisinas , Fígado/efeitos dos fármacos , Fosfolipídeos/biossíntese , Animais , Ácidos Carboxílicos/administração & dosagem , Carcinógenos Ambientais/administração & dosagem , Colesterol/sangue , Dieta , Relação Dose-Resposta a Droga , Ácidos Graxos/análise , Fígado/metabolismo , Masculino , Tamanho do Órgão/efeitos dos fármacos , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Ratos , Ratos Endogâmicos F344 , Aumento de Peso/efeitos dos fármacosRESUMO
The effect of fumonisin B1 (FB1) on protein and lipid synthesis was evaluated in primary rat hepatocytes. FB1 did not affect incorporation of [3H]leucine into hepatocytes at either non-toxic (150 microM) or cytotoxic (500 microM) concentrations indicating that protein synthesis was not affected. However, FB1 significantly (P < 0.01 to P < 0.0001) inhibited incorporation of [14C]palmitic acid into hepatocyte cultures implying that lipid synthesis was altered. Incorporation of the radiolabel was significantly (P < 0.05 to P < 0.0001) lowered in triacylglycerol (TAG) and sphingomyelin fractions and increased in phosphatidylcholine (PC) and phosphatidylethanolamine (PEA) in both FB1 concentrations. The incorporation pattern of [14C]palmitic acid closely resembles the changes in phospholipid levels in the treated cells. The sphingolipid, sphinganine (Sa), was significantly (P < 0.0001) increased in treated cells but there was no significant difference between the toxic and non-toxic dose levels implying that the increased Sa level alone is not responsible for the in vitro toxicity. FB1 significantly (P < 0.01 to P < 0.001) decreased the level of free cholesterol within the cell, resulting in an increased PC:cholesterol ratio suggesting a more rigid membrane structure. Subsequent studies on the fatty acid (FA) profiles in PC and the neutral lipid, TAG, indicated that FB1 significantly (P < 0.05 to P < 0.0001) increased the levels of the polyunsaturated FAs C18:2n-6 and C20:4n-6 at both concentrations. The FB1-induced changes to cellular membranes, specifically those related to FA changes in the major membrane phospholipids, and the altered FA content of the hepatocytes are likely to be key events in explaining the cytotoxic effects and altered growth responses induced by fumonisins in primary hepatocytes.
Assuntos
Carcinógenos/toxicidade , Fumonisinas , Lipídeos/biossíntese , Fígado/efeitos dos fármacos , Fígado/metabolismo , Micotoxinas/toxicidade , Animais , Radioisótopos de Carbono/metabolismo , Células Cultivadas , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Fígado/citologia , Masculino , Fluidez de Membrana/efeitos dos fármacos , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Fosfolipídeos/biossíntese , Precursores de Proteínas/metabolismo , Ratos , Ratos Endogâmicos F344 , Esfingolipídeos/biossíntese , Triglicerídeos/química , TrítioRESUMO
The fumonisins and toxins produced by Alternaria alternata f. sp. lycopersici (AAL toxins) are structurally related mycotoxins that disrupt sphingolipid biosynthesis by inhibiting the rate-limiting enzyme, ceramide synthase. Rat primary hepatocytes were exposed to fumonisin B1 (FB1), its N-acetyl analogue, FA1, its fully hydrolysed analogue, AP1 and the AAL toxins (TA and TB) at concentrations of 1 microM for 40 hr in culture. The extent to which these compounds disrupt sphingolipid biosynthesis in hepatocytes in vitro was investigated by analysing the sphingosine (So) and sphinganine (Sa) levels by HPLC. The inhibition of ceramide synthase was irreversible as the Sa:So ratio was maximally increased by FB1 after 24 hr of exposure and the subsequent removal of FB1 had no effect on the ratio as compared with the 40-hr incubation period in the presence of FB1. The Sa concentration was significantly (P < 0.01) increased in all the cultures treated with the different structurally related compounds, while only AP1 increased the So concentration significantly (P < 0.05) above the control. As AP1 was found to be less effective in disrupting sphingolipid biosynthesis it would appear that the tricarballylic (TCA) moiety is required for maximal inhibition of ceramide synthase. The presence of an amino group appears not to be a requisite for activity, since FA1 increased the Sa:So ratio to the same extent as FB1. The AAL toxins TA and TB increased the Sa concentration significantly (P < 0.01) above that of FB1 and FA1, while the Sa:So ratios were altered to the same extent. The structural requirements for the induction of cytotoxicity differ from those required for ceramide synthase inhibition as TA and TB were significantly (P < 0.05 to P < 0.01) less toxic to primary hepatocytes than FB1 at all the concentrations tested.
Assuntos
Carcinógenos Ambientais/toxicidade , Fumonisinas , Fígado/efeitos dos fármacos , Micotoxinas/toxicidade , Esfingolipídeos/biossíntese , Alternaria , Animais , Ácidos Carboxílicos/toxicidade , Células Cultivadas , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/toxicidade , L-Lactato Desidrogenase/análise , Masculino , Oxirredutases/antagonistas & inibidores , Proteína Quinase C/antagonistas & inibidores , Ratos , Ratos Endogâmicos F344 , Esfingolipídeos/análise , Esfingolipídeos/antagonistas & inibidores , Esfingosina/análogos & derivados , Esfingosina/análise , Relação Estrutura-AtividadeRESUMO
Cancer induction by the non-genotoxic mycotoxin, fumonisin B1, has been investigated by studying the mechanisms involved during cancer initiation and promotion in rat liver. Cancer initiation is effected through a toxic-proliferative response while the inhibitory effect on hepatocyte cell proliferation appears to be a key aspect determining cancer promotion. Dose-response effects of the fumonisins on the induction of early neoplastic lesions in both long- and short-term animal experiments have been established. The biphasic response of FB1 on hepatocyte proliferation will be discussed in relation to the known mechanisms of cancer induction by the genotoxic hepatocarcinogens. Recent investigations regarding the effect of the fumonisins on lipid biosynthesis and its inhibitory effect on hepatocyte growth stimulatory responses in vitro will be highlighted. Integration of our current knowledge regarding the carcinogenic potential of the fumonisins in setting a realistic and applicable risk assessment model for this non-genotoxic carcinogen will finally be addressed.
Assuntos
Carcinógenos Ambientais/toxicidade , Doença Hepática Induzida por Substâncias e Drogas , Fumonisinas , Neoplasias Hepáticas Experimentais/induzido quimicamente , Micotoxinas/toxicidade , Animais , Humanos , Micotoxinas/metabolismo , Micotoxinas/farmacocinética , Ratos , Fatores de RiscoRESUMO
The mutagenic behaviour of two potentially carcinogenic mycotoxins produced byFusarium moniliforme was investigated in theSalmonella mutagenicity test using tester strains TA97a, TA98, TA100, and TA102. The mutagenic response obtained with fusarin C (1, 5, and 10µg/plate) against tester strains TA98 and TA100 in the presence of microsomal activation confirmed previous observations on the mutagenic behaviour of this mutagen while that obtained against TA97a is reported for the first time. No dose-response relationship could be detected for the concentration levels (0.2, 0.5, 1, 5, 10 mg/plate) tested for FB1, FB2, and FB3 against any of the tester strains used in either the plate incorporation and / or the pre-incubation tests. A cytotoxic effect was obtained at concentration levels of 5 and 10mg/plate in the absence of the microsomal activation mixture. From the studies it became evident thatF moniliforme produces two compounds, a mutagenic compound, fusarin C which has been shown to lack carcinogenic activity in rats and the non-mutagenic fumonisin B mycotoxins of which FB1 is known to be responsible for the hepatocarcinogenicity of the fungus in rats.
RESUMO
Dose response studies regarding the cancer initiating potential of fumonisin B1 (FB1) were conducted as a function of time. Feeding studies over 21 days indicated that FB1 induced a feed refusal effect in rats at dietary levels of 250, 500 and 750 mg FB1/kg diet. This effect was overcome after 14 days and the feed intake profiles reached a level which was equivalent to that of the controls after 21 days. Based on the feed intake records the effective dosage level (EDL) for cancer initiation over a period of 21 days is 14.2 < EDL < 30.8 mg FB1/100 g body wt. This is equivalent to a daily intake of 0.7 < EDL < 1.5 mg FB1/100 g body wt. Over a period of 14 days the amount of FB1 required for cancer initiation is 23.3 < EDL < 33.3 mg [corrected] FB1/100 g body wt. The latter values were markedly higher than the EDL values obtained in a gavage study where a fixed amount of FB1 was dosed to rats over 14 days (5.39 < EDL < 11.56 mg FB1/100 g body wt). The dietary level of FB1 required for cancer initiation is dependent on the duration of exposure as a dosage of 29.7 mg/100 mg body wt over 7 days did not initiate cancer whilst a similar dose (30.8 mg/100 body wt) over 21 days did. FB1 effectively delayed hepatocyte cell proliferation when fed at a dietary level of 250 mg FB1/kg (the lowest dietary level tested to effect cancer initiation over 21 days) or by a single gavage dose of 5 mg FB1/100 g body wt 6 h following partial hepatectomy. This inhibitory effect of FB1 on cell proliferation appears to be the reason why the fumonisins are slow cancer initiators. The present data suggest that a balance exists between the compensatory cell proliferation due to the hepatotoxicity induced by FB1 and the inhibitory effect on the subsequent hepatocyte cell proliferation. Therefore, a threshold level for cancer initiation exists which, as a function of time, will be determined by the dosage used and the subsequent inhibitory effect on cell proliferation.
Assuntos
Carcinógenos Ambientais/toxicidade , Fumonisinas , Neoplasias Hepáticas Experimentais/induzido quimicamente , Micotoxinas/toxicidade , Lesões Pré-Cancerosas/induzido quimicamente , Animais , Divisão Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Ratos , Ratos Endogâmicos F344RESUMO
The inhibitory effect of fumonisin B1 (FB1) on epidermal growth factor (EGF)-induced DNA synthesis in primary rat hepatocytes was investigated by monitoring the incorporation of [3H]thymidine in the DNA. A pulse-labelling technique was adapted to determine the incorporation of the radioactivity in the DNA (S-phase) quantitatively. FB1 inhibits the EGF-induced DNA synthesis up to 90% when incorporated at concentrations of 150 to 300 microM for a period of 44 h. A continued presence of FB1 is required to exhibit this inhibition as (i) the subsequent removal of FB1 resulted in a reversal of the effect, (ii) a higher stimulatory response in EGF-treated hepatocytes was found when the exposure period of hepatocytes in FB1 was reduced, and (iii) pretreatment of hepatocytes with FB1 only slightly reduced (not significantly) DNA synthesis induced by EGF. Whilst the growth inhibitory effect of FB1 was not associated with a cytotoxic effect, binding studies using [125I]EGF indicated that the growth factor-receptor interaction was not altered. No relationship was found between the disruption of the sphingolipid biosynthesis by FB1 and (i) the mitoinhibitory effect on the EGF response and (ii) the cytotoxicity of FB1 in primary hepatocytes.