* Ohio State University, James Cancer Center, 1148 James CHRI, 300 W. 10th Avenue, Columbus, Ohio 432101240;
California State Polytechnic University-Pomona, College of Agriculture, 3801 W. Temple Avenue, Pomona, California 91768;
University of Nebraska Medical Center, Department of Pathology and Microbiology, 983135 Nebraska Medical Center, Omaha, Nebraska 681983135;
§ Integrated Laboratory Systems, Inc., Health Science Division, P.O. Box 13501, Research Triangle Park, North Carolina 27709;
¶ American Health Foundation, One Dana Road, Valhalla, New York 10595;
|| Division of Biochemical Toxicology, HFT-110, National Center for Toxicological Research, U.S. Food and Drug Administration, 3900 NCTR Drive, Jefferson, Arkansas 720799502; and
||| USDA, Toxicology and Mycotoxin Research Unit, P.O. Box 5677, Athens, Georgia 306045677
Received November 18, 2000; accepted January 18, 2001
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ABSTRACT |
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Key Words: apoptosis; fumonisin; mycotoxins; kidney cancer; liver cancer; regeneration; carcinogenesis.
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Introduction |
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Consumption of corn molded with F. verticillioides can lead to leukoencephalomalacia in horses, pulmonary edema in pigs, and liver and kidney toxicity in these and other species (Dutton, 1996; IPCS, 2000). Numerous mycotoxins have been identified in F. verticillioides-contaminated corn. During the past decade, FB1 has been identified as the major Fusarium mycotoxin producing these toxicities (Dutton, 1996
; Gelderblom et al., 1993
), and it has been hypothesized as a risk factor for human esophageal cancer in certain regions of southern Africa and China. Early studies showed that inclusion of F. verticillioides in the diet of rats produced significant hepatotoxicity with necrosis, fatty degeneration, bile duct proliferation and eventually fibrosis (Jaskiewicz et al., 1987
; Marasas et al., 1984
; Wilson et al., 1985
). Because of its ubiquitous presence in corn and potential for widespread exposure, the Food and Drug Administration recently evaluated FB1 in chronic bioassays in rats and mice that were performed at the National Center for Toxicological Research through the National Toxicology Program (Howard et al., 2001a
; NTP Technical Report, 2001). The results of the bioassay indicated that FB1 was carcinogenic, producing significant incidences of kidney tumors in male rats and liver tumors in female mice. Previous bioassays using male rats performed in South Africa resulted in liver tumors (Gelderblom et al., 1991
).
A major biological effect of FB1 is the induction of apoptosis in various in vitro and in vivo model systems (Tolleson et al., 1996). Apoptosis is a specialized process of cell death that is part of normal organ development and tissue maintenance, but it can also occur in response to various environmental stimuli and can be indicative of toxicity. Numerous proteins and other cellular constituents have been identified that are involved in the apoptotic process and that regulate its occurrence. These include several sphingolipids and other lipids affected by exposure to FB1 (Gelderblom et al., 2001
; Merrill et al., 2001
). To evaluate the role of apoptosis as a mode of action for carcinogenesis, with FB1 as a case study, the International Life Sciences Institute (North American Branch) convened an expert working-group in February, 1999, with members chosen based on their expertise in carcinogenesis, toxicology, pathology, biochemistry, and cell biology. Additional discussions regarding the material took place by direct communication between the members of the working group. A summary of their deliberations is presented in this manuscript.
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Cell Death and Carcinogenesis |
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As described by Majno and Joris (1995), apoptotic necrosis is morphologically identified by cytoplasmic shrinkage and karyorrhexis, in contrast to oncotic necrosis, identified by cytoplasmic swelling and karyolysis. Either cell death process can occur in individual cells or in multiple cells in clusters. Apoptosis has frequently been referred to as programmed cell death, but in reality, both apoptotic and oncotic necrosis occur through identifiable molecular biological pathways. Similarly, some have distinguished apoptotic from oncotic necrosis based on the absence of an accompanying inflammatory reaction with apoptotic necrosis, whereas in fact, inflammation can accompany either form of necrosis.
Of fundamental importance in considering the biological effects of either type of necrosis is the tissue response. The cells that die can be lost and not replaced, leading to atrophy, or the tissue can regenerate new cells to replace those lost to cell death, commonly referred to as regeneration. Cell renewal systems exist in most tissues to replace cells that die. Cell death, whether apoptotic or oncotic, is a common effect of chemical toxicity and is most often accompanied by regeneration and usually associated with development of hyperplasia. Regardless of whether there is ultimately an increase in the number of cells, or merely a replenishment of the cells that died, there is an increase in the number of cell divisions in the affected tissue (Cohen, 1998). The rate of cell division is frequently increased compared to normal tissue, but this is not always the case.
Cancer is commonly accepted as being a consequence of multiple genetic alterations arising from inherited mutations in germ cells or as a consequence of mutations in somatic cells, resulting in altered growth (Knudson, 1971). Also, although DNA replication has incredible fidelity, it is not absolute. Thus, despite extensive and efficient cellular DNA repair mechanisms, every time DNA replicates, there is potential for a mistake to occur which can go unrepaired. These mistakes are rare, estimated to occur at the rate of approximately 1 error per 1010 nucleotides per DNA replication. If the error occurs in a gene involved in the carcinogenic process, then a step is taken toward the formation of cancer. Numerous endogenous processes frequently result in cellular DNA damage, including oxidation, deamination, exocyclic adduct formation, depurination, and others. Most of these are repaired, but when unrepaired, a mutational event can result. These mistakes occur on a regular basis endogenously, and there is the potential for DNA damage, even without exposure to an exogenously ingested, DNA adduct-generating agent.
Thus, an agent can increase the risk of carcinogenesis either by damaging DNA so that there are more mistakes each time DNA replicates, by increasing the number of DNA replications, or by a combination of both processes (Cohen and Ellwein, 1990; 1991; Moolgavkar and Knudson, 1981
). If the agent, or a metabolite, forms a mutagenic adduct and causes DNA damage, it is commonly referred to as genotoxic (DNA reactive). Agents that increase carcinogenesis by increasing the number of DNA replications have been referred to by a variety of names, including non-genotoxic carcinogens.
Cell death and compensatory regeneration has become a well established mode of action for a variety of non-genotoxic chemical carcinogens targeting several tissues (Cohen, 1998). The cytotoxicity that is produced by these chemicals has been either demonstrated to be, or assumed to involve, oncotic necrosis. However, theoretically, apoptotic necrosis should also engender regeneration and have the potential for increasing the likelihood of cancer induction. Thus, on theoretical grounds, apoptotic necrosis with consequent regeneration should have the same potential to produce a carcinogenic process as oncotic necrosis plus regeneration. Those chemicals, which have been extensively studied and involve cytotoxicity via oncotic necrosis with consequent regeneration, have a non-linear dose response with respect to carcinogenicity (Cohen, 1998
). Although cytotoxicity theoretically implies a threshold response, such thresholds for non-genotoxic carcinogens continue to be the source of considerable controversy. Similar considerations are theoretically involved in interpreting the carcinogenic risk assessment of chemicals that have a mode of action involving apoptotic necrosis, rather than oncotic necrosis, with consequent regeneration.
In addition to apoptotic necrosis resulting in regeneration and increased proliferation, other mechanisms have been suggested whereby apoptosis may contribute to the carcinogenic process (Goldsworthy et al., 1996; Lowe and Lin, 2000
; Manning and Patierno, 1996
). Apoptosis of some cells could potentially result in development of a population of other cells that become resistant to apoptosis, which could accumulate heritable genetic changes during an increased life-span. Alternatively, an increased susceptibility to the signals for apoptosis in some cells, if accompanied by less efficient DNA repair in the remaining cells, could increase the cell population at risk. Apoptotic necrosis could also serve as an anti-carcinogenic mechanism by killing pre-neoplastic or neoplastic cells that develop. These different effects are not mutually exclusive, so that more than one could be affecting carcinogenesis.
Several cellular components, such as specific proteins, have been identified that can induce apoptosis (Brenner and Kroemer, 2000). In addition, it is clear that various sphingolipid metabolites are signaling molecules in pathways that regulate apoptosis and cell survival (Brenner and Kroemer, 2000
; Merrill et al., 1997
). Ceramide synthase is a key enzyme in sphingolipid metabolism and in the production of various sphingolipids involved in apoptosis. FB1 has been identified as an inhibitor of this enzyme, leading to significant alterations in sphingolipid metabolism in both kidney and liver (Merrill et al., 1997
; Wang et al., 1991
). It was not surprising, therefore, that apoptosis was identified in various in vitro cellular systems exposed to FB1, or that apoptosis was identified in the kidney and liver in rodents administered FB1. Thus, a chain of events linking FB1-induced ceramide synthase inhibition, disrupted sphingolipid metabolism, the induction of apoptosis, and the development of kidney and liver tumors in rodents can be theoretically drawn, raising the issue of the role of apoptosis as a mode of action in carcinogenesis.
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Fumonisin B1 Carcinogenesis in Rodents |
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An experiment involving administration of purified FB1 (stated to be greater than 90 percent pure) fed at a dose of 50 ppm in the diet for 2 years to BD-IX male rats, resulted in hepatotoxicity and carcinogenicity (Gelderblom et al., 1991). Hepatotoxicity, regenerative nodules, and cholangiofibrosis were present by 6 months, with cirrhosis and hepatocellular carcinomas being present at 1826 months. Ten of 15 rats that died or were killed between 18 and 26 months of the experiment had hepatocellular carcinoma in contrast to none in the controls.
The diet used in the above experiment (Gelderblom et al., 1991) was composed predominantly (75% by weight) of cornmeal. This diet was likely associated with marginal deficiencies of a number of vitamins, such as thiamin, riboflavin and vitamin E, and also contained low levels of vitamin B12, folate, biotin, choline, and methionine when compared to a diet such as AIN-76. Some of these dietary alterations are known to be associated with hepatotoxicity and hepatocarcinogenicity in rats. In addition, the animals that were fed 50 ppm FB1 gained considerably less weight during the entire course of the experiment (approximately 12 to 25 percent less weight gain compared to controls) indicating that the selected dose of 50 ppm exceeded guideline levels for a maximum tolerated dose (MTD). A repeat study using lower doses (
25 ppm in the diet) of FB1 did not produce hepatocarcinogenicity (Gelderblom et al., 1997).
Utilizing the Solt and Farber (1976) hepatocyte selection model, Gelderblom et al. (1992) found little or no evidence of initiating activity for FB1. They did find that administration of 1000 ppm of FB1 in the diet increased the number and size of the GGT-positive foci in male F344 rats. However this dose was also associated with severe toxicity and growth retardation. A subsequent study (Gelderblom et al., 1996) evaluated FB1 administered in the diet between 10 and 500 ppm for 21 days following administration of 200 mg of diethylnitrosamine (DEN). An increased number and area of GGT and glutathione S-transferase, placental form (GSTP) foci were observed at 250 and 500 ppm. The sphinganine:sphingosine ratios in the livers of non-hepatectomized rats were increased at FB1 doses of 50 through 500 ppm. Another study (Gelderblom et al., 1996
) examined zero to 500 ppm of FB1 administered for 21 days followed by partial hepatectomy. The rats were sacrificed 24 h later. 3H-thymidine labeling index was decreased in partially hepatectomized rats.
The National Toxicology Program (NTP) at the National Center for Toxicological Research (NCTR) facilities in Jefferson, Arkansas, recently completed a 2-year bioassay in rats and mice on FB1 (Howard et al., 2001a; NTP Technical Report, 2001). The chemical was characterized as having greater than 96 percent purity, and it was administered in NIH-31 diet, which was ascertained as having contamination with FB1 less than 60 ppb. The chemical was administered in the diet at doses of 0, 5, 15, 50, or 150 ppm FB1 to groups of 48 male rats, or 0, 5, 15, 50, or 100 ppm FB1 to groups of 48 female rats for 105 weeks. Groups of 48 male and 48 female mice were fed NIH-31 diet containing 0, 5, 15, 80, or 150 ppm in the males, or 0, 5, 15, 50, or 80 ppm in the females. F344/N Nctr BR rats and B6C3F1/Nctr BR (C57BL/6N x C3H/HeN MTV) mice were used from the NCTR breeding laboratory. The animals followed the standard NTP protocol for evaluation of carcinogenicity, and the doses were chosen based on 28- and 90-day dose range-finding studies. Additional animals were used for interim sacrifices for evaluation of cell proliferation, apoptosis, hematology, clinical chemistry, urinalysis, and tissue sphingolipid parameters.
The relevant tumor findings from the NTP bioassay are presented in Table 1. In rats, the only tumors induced were in the kidney, with significant incidences only in males. In mice, there were significantly increased incidences of liver tumors in females but not males. No renal lesions or tumors were seen in mice.
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In mice, the incidence of hepatic tumors was not significantly increased in males, but adenomas and carcinomas were increased at 50 and 80 ppm doses in female mice. Spontaneous tumors occurred in the male mice at predicted rates in response to the approximately 20% caloric restriction that occurred.
In summary, FB1 has been identified as carcinogenic in male rat kidney (NTP Technical Report, 2001) and liver (Gelderblom et al., 1991) and in female mouse liver (NTP Technical Report, 2001). The reasons for differences between the results of carcinogenicity tests in the 2 rat bioassays could be due to a variety of differences in the respective laboratories, including differences in rat strain (BD-IX vs. F344/N Nctr), diet, and purity of FB1, amongst others. Regardless, the FB1 doses that resulted in tumors in these rodent bioassays were comparable (
50 ppm). It is also apparent that a carcinogenic effect is detected only at doses
50 ppm in the female mouse and
50 ppm in the male rat. There is also a distinct difference between males and females in their response to FB1. It is unclear if this is due to hormonal differences between sexes. The sex differences in the carcinogenic effects of FB1 are similar to some other chemicals that are carcinogenic in rodents, such as methyl tert-butyl ether, unleaded gasoline, hexachloroethane, chlordane, and 1,1,2-trichloroethane (Moser et al., 1997
). However, even for those chemicals, the exact mechanistic basis for this difference is unknown.
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DNA Reactivity of Fumonisin B1 |
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There has been some limited evidence that FB1 can produce DNA damage indirectly by producing cellular oxidative damage (Sahu et al., 1998; Yin et al., 1998
). Increased lipid peroxidation was reported in the livers of rats fed FB1, and in primary hepatocytes treated with FB1 (Abel and Gelderblom, 1998
), as evidenced by: increased thiobarbiturate acid reactive substances (TBARS); increased TBARS in cultured hepatocytes associated with cytotoxicity and protected by alpha-tocopherol. Increased lipid peroxidation and DNA strand breaks have also been induced by FB1 in isolated rat liver nuclei (Sahu et al., 1998
). However, the chemical structure of FB1 does not suggest pro-oxidant activity. Some of the findings suggesting cellular oxidative damage may be due to toxicity rather than a direct cellular effect of the chemical. Also, a recent study involving the feeding of FB1 at 250 ppm to rats (Lemmer et al., 1999
) concluded that FB1 appears to induce liver toxicity independently from effects on lipid peroxidation, although FB1 did potentiate the effect of iron on lipid peroxidation.
The weight of evidence strongly suggests that FB1 and other fumonisins do not produce DNA damage directly and are not DNA-reactive.
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Fumonisin B1 Toxicity |
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Ninety-day feeding studies have demonstrated that apoptosis of individual renal tubular cells was produced by FB1 in male rats at doses of 9 ppm, and in female rats, apoptosis was produced only at the highest dose of 81 ppm (Bucci et al., 1998
; NTP Technical Report, 2001; Voss et al., 1995
). In a detailed review of the kidney histopathology in the NTP 2-year carcinogenicity bioassay, Dr. Gordon Hard observed that evidence of nephrotoxicity, including apoptosis and regeneration, was sustained for the full period of FB1 exposure at the doses
50 ppm in male rats. The sequence of events in the NTP bioassay and related studies clearly demonstrated that administration of FB1 to male F344/N Nctr rats produced significant amounts of apoptosis followed by active regeneration, hyperplasia, and ultimately, renal tumors in male rats at doses of 50 and 150 ppm. A similar but less pronounced effect was seen in female rats at 100 ppm.
Since statistically significant morphologic tumorigenic effects on the kidney appear to be restricted to the male rat, FB1 binding to 2u-globulin was evaluated. No effect on
2u-globulin in the male rat kidney was identified by immunohistochemistry (Bucci et al., 1998
; Howard et al., 2001a
,b
; NTP Technical Report, 2001). The morphologic alterations in rat kidney following FB1 administration also are not those usually observed following administration of chemicals having an effect on
2u-globulin. In addition, although the renal effect was statistically significant in the male rat kidney, there were also hyperplastic and neoplastic changes in the kidney of a few female rats in the bioassay, but not at a statistically significant level (Howard et al., 2001a
,b
; NTP Technical Report, 2001).
In the rodent liver, the changes appear to be more complex. There is no question, given the elevated liver enzyme activities in the serum, that liver toxicity occurred in rodents. Morphologically there was evidence of apoptotic and oncotic necrosis and changes indicative of regeneration (Gelderblom et al., 1991; NTP Technical Report, 2001; Voss et al., 1993
). This has been reported in the studies from South Africa and those performed at NCTR. However, in the rat, the changes reported in the studies in South Africa were significantly more pronounced and extensive, and were accompanied by the eventual development of hepatocellular carcinomas and cholangiocarcinomas (Gelderblom et al., 1991
). The acute liver toxicity reported in the South African experiments was evident as apoptotic (reported as single-cell necrosis) and oncotic necrosis, accompanied by increased hepatocellular proliferation (Gelderblom et al., 1991
). There were marked changes in hepatic architecture and formation of regenerative nodules. These changes progressed to cholangiofibrosis, cirrhosis, and to rat hepatocellular tumor development (Gelderblom et al., 1991
). In a subsequent experiment (Lemmer et al., 1999
), these regenerative changes were evaluated in rats fed 250 ppm of FB1 for 4 weeks. Histopathological findings included apoptosis, necrosis, oval cell proliferation, the appearance of hepatocytes staining positive for GST (pi), and the progressive development of fibrosis and regenerative nodules. Accompanying these changes were increased expression of
-fetoprotein, hepatocyte growth factor (HGF), transforming growth factor-
(TGF-
), and significantly increased levels of TGF-ß1 and c-myc. Increased cyclin D1 expression has also been shown to be increased (Ramljak et al., 2000
).
In mice, FB1 did not show evidence of hepatic toxicity at doses 27 ppm after 90 days of feeding (NTP Technical Report, 2001; Voss et al., 1995
). However, there was evidence of centrilobular and occasional mid-zonal apoptosis and accompanying increased mitoses. These changes were mild and confined to females fed 81 ppm. Additional evidence of hepatotoxicity included elevation of serum enzymes. In the bioassay performed at NCTR (Howard et al., 2001a
; NTP Technical Report, 2001), hepatocellular apoptosis was significantly increased at 2 years, at doses of 50 and 80 ppm FB1 in female mice, but was not statistically significant in males at any dose. Hepatocellular hypertrophy was significantly increased at doses of 15, 80, and 150 ppm in the males, and at 50 and 80 ppm in the females at 2 years. There was an increased incidence of hepatocellular neoplasia in the females at 50 and 80 ppm but not in the males.
In both the rat kidney and liver, the subchronic microscopic lesions, organ weight differences, and sphinganine and sphingosine elevations were reversible 3 weeks after the return from FB1-contaminated to control diet (Voss et al., 1998).
In summary, there is a relatively close correlation between induction of apoptosis in rat kidney, rat liver, and mouse liver and the induction of neoplasia in these organs in various studies, although there were significant differences between studies performed at different institutions. Apoptotic necrosis was accompanied by regeneration. In kidney, the necrosis appears to be entirely apoptotic, whereas in liver it appears to be a combination of apoptotic and oncotic necrosis. Regardless of the pathway by which necrosis was produced, regeneration followed.
FB1 has a variety of toxic effects in other species in addition to rats and mice (NTP Technical Report, 2001). Hepatotoxicity and renal toxicity are common features in most species evaluated, including rabbits, horses, pigs, baboons, and vervet monkeys, after administration of either purified FB1 or cultures of F. verticillioides (IPCS, 2000). In rabbits, pigs, and horses, the liver changes resemble those seen in rats and mice, including the presence of apoptosis. In the livers of baboons and vervet monkeys, hepatotoxicity was expressed as centrilobular necrosis with bile duct proliferation and fatty degeneration, occasionally with development of fibrosis. In addition, purified FB1 has been identified as the cause of equine leukoencephalomalacia, apparently secondary to cardiovascular effects (Constable et al., 2000a). In pigs, cardiovascular effects appear to be expressed as pulmonary edema (Constable et al., 2000b
; Smith et al., 2000
).
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Fumonisin B1 Effects on Sphingolipids |
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In addition to the effects of the disruption of sphingolipid metabolism on apoptosis and cell proliferation, FB1 has been shown to alter other lipid pathways that also can affect apoptosis and cell proliferation (Merrill et al., 1997; 2001
; Riley et al., 1998
; 2001
). Many of these effects on lipid metabolism could be a consequence of the effect of FB1 on sphingolipid metabolism. Such effects include altered fatty acid and glycerophospholipid metabolism, altered expression of cytokines such as TNF-
, alterations of expression of hormones or growth factors or their receptors, or direct or indirect effects on protein kinases, phospholipases, and cyclooxygenases. Effects on interactions of epithelial cells with the extracellular matrix, by inhibition of glycosphingolipids regulating cell recognition and adhesion, could also be a pathway leading to apoptosis.
Numerous regulatory pathways involving sphingolipids have been identified that can influence the apoptotic process (Merrill et al., 1997; 2001
; Riley et al., 1998
; 2001). Control of apoptosis is a complex, non-linear process that is cell specific and dependent on a variety of feedback mechanisms (Green and Reed, 1998
; Brenner and Kroemer, 2000
). Thus, disruption of sphingolipid metabolism and associated effects on glycerophospholipid and fatty acid metabolism could lead to multiple alterations in pathways controlling cell proliferation and cell death.
In Sprague-Dawley and Fischer 344 rats, New Zealand white rabbits, and BALB/c and other mouse strains, disruption of sphingolipid metabolism in liver and kidney occurs at FB1 doses below those that produce morphologic evidence of injury (Voss et al., 2000). When liver pathology is observed, there is a close correlation between the incidence and severity of the pathology and the increase in free sphinganine, indicative of disrupted sphingolipid metabolism (Delongchamp and Young, in press; Riley et al., 1994
). In rat kidney, sphinganine concentrations increase rapidly and are seen at doses not producing morphological evidence of toxicity. Urinary sphinganine levels also rise and are closely correlated with the severity of nephrotoxicity as determined by microscopic tissue examination (Howard et al., 2001b
; NTP Technical Report, 2001; Riley et al., 1994
).
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Fumonisin B1 Toxicokinetics |
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There have also been investigations of the kinetics of FB1 following administration by either the intraperitoneal or intravenous routes (Norred et al., 1993; Shephard et al., 1992
, 1994
). However, since FB1 exposure in humans is via oral ingestion, these studies involving systemic administration may not be relevant to usual human exposures. Nevertheless, following systemic administration, approximately 60% of FB1 is excreted in bile within four h.
FB1 does not appear to accumulate in the tissues, but low levels appear to persist in the kidney and liver in the rat (Norred at al., 1993). The effects of FB1 on accumulation of free sphingoid bases and toxicity are reversible (Voss et al., 1998; Wang et al., 1999
), although the elevation of free sphingoid bases is persistent in kidney for several days after cessation of exposure to FB1 (Wang et al., 1999
). FB1 metabolites have not been identified; most of the administered FB1 can be accounted for as the unmetabolized chemical (NTP Technical Report, 2001).
In other mammalian species, including the non-human primate (vervet monkey), swine, cattle, and horses, the kinetics appear to be similar to those in the rat and indicate a low gastrointestinal absorption, rapid clearance, biliary excretion of absorbed FB1, and accumulation of minor amounts of the administered dose in liver and kidneys (Dutton, 1996; IPCS, 2000; NTP Technical Report, 2001). Non-human primate studies (Shephard et al., 1994
) produced evidence that gut microflora are capable of removing one or both tricarballylic acid groups from the molecule, but no evidence for hepatic metabolism has been reported. There is a paucity of data on the physiological and pharmacokinetic fate of FB1 under chronic administration conditions.
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Fumonisin B1 and Esophageal Cancer |
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However, many concerns regarding the epidemiology of the relationship between increased FB1 consumption and the development of esophageal cancer have been identified (Craddock, 1992). For example, FB1 is but one of many contaminant toxins from Fusarium that are present in corn, and there are other species of fungi also present (Shephard et al., 1996
). In addition, contamination with other known carcinogenic compounds, such as nitrosamines and polycyclic aromatic hydrocarbons, are also high in these same regions (Burrell et al., 1966
; Craddock, 1992
; Lu et al., 1986
). Importantly, several nutritional deficiencies have also been identified in these populations that could contribute to the high esophageal cancer rates, particularly in the high esophageal-incidence areas of South Africa (van Rensburg et al., 1985
). These include not only specific vitamin deficiencies such as vitamins A and C and riboflavin, but importantly, there appears to be a zinc deficiency as well (van Rensburg et al., 1986
). A study in 11 male baboons not administered FB1 demonstrated that when riboflavin was omitted from their diet, esophageal lesions appeared, including hyperplasia with numerous mitotic figures (Foy and Kondi, 1984
). Zinc deficiency has been identified as a critical factor in esophageal carcinogenesis in both human epidemiologic investigations and in animal models (Craddock, 1992
; Fong et al., 1998
; Newberne et al., 1997
).
An additional confounding factor is the practice in the high-incidence areas of the Transkei to brew beer with contaminated corn resulting in extremely high levels of fumonisin (Marasas, 1995). Alcohol itself is a known risk factor for esophageal cancer, as are the high levels of nitrosamine contamination that can be present in some of these alcoholic beverages (Anderson et al., 1996
; Newberne et al., 1997
; Rogers et al., 1995
; Siglin et al., 1995
).
Increased incidences of esophageal cancer have also been identified in areas in northeastern Italy, where the consumption of corn has been associated with these neoplasms (Franceschi et al., 1990). However, these populations also appear to have markedly increased exposure to alcohol and tobacco, known risk factors for esophageal cancer. The effect of maize consumption on the increased incidence of esophageal cancer in Italy was significantly elevated only in those individuals who consumed excessive levels of alcohol and might have also been related to dietary insufficiencies, such as niacin and riboflavin.
Studies in animals have not clarified the relationship of FB1 to esophageal cancer. Culture material from F. verticillioides, strain MRC 826, significantly enhanced nitrosamine-induced esophageal carcinoma in BD-IX rats (van Rensburg et al., 1982). However, when this rat strain was co-administered purified FB1 with nitrosamine, there was no effect of FB1 on esophageal cancer incidence (Wild et al., 1997). In separate animals, purified FB1 was administered as 5 mg per kg by gavage for 4 weeks; there was an increase in the sphinganine/sphingosine ratio in the kidney and a slight increase in the liver, but there was no statistical increase in this ratio in esophageal tissue.
Overall, the combination of epidemiologic and animal studies provides some evidence that exposure to corn highly contaminated with F. verticillioides might be related to esophageal cancer; however, FB1 itself does not appear to be an esophageal carcinogen.
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Implication for Risk Assessment |
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Liver and kidney toxicity secondary to FB1 administration appears to be a relatively common response across species, not only in rats and mice, but in rabbits, horses, pigs, and non-human primates (NTP Technical Report, 2001; Voss et al., 2000). Such toxicity, however, appears to occur at relatively high doses, usually measured in hundreds of micrograms of FB1 per kilogram body weight per day. Human exposure in the U.S. and Europe is generally at levels of micrograms or less per kilogram per day. In high consumption areas such as the Transkei in southern Africa, the consumption of FB1 can be much higher, approaching hundreds of micrograms per kilogram per day.
There are now three chronic bioassays with purified FB1 which have produced a carcinogenic effect in rodents. An estimated no observed effect level (NOEL) for renal tumors is between 0.9 and 3.0 mg/kg/day for FB1, and for female mouse liver tumors an estimated NOEL is 2.27.5 mg/kg/day based on lifetime exposure (NTP Technical Report, 2001; Howard et al., 2001a).
As indicated in the proposed cancer risk-assessment guidelines of the U.S. Environmental Protection Agency, mechanistic information should be included in any risk assessment when available. The possibility of DNA reactivity (genotoxicity) is of paramount importance. Based on the non-genotoxicity of FB1 and its apparent mode of action involving toxicity and regeneration, a non-linear extrapolation to low doses and to humans is appropriate. Toxicity to the liver appears to be the most consistent end point across species, but toxicity (as evidenced by apoptosis) to the kidney appears to be slightly more sensitive, at least in the male rat. Although the toxic effects appear to be non-linear, the question as to whether these represent true biological thresholds or not remains unclear. There have been no biological effects that we are aware of demonstrated in vivo at a dose of less than 1 ppm in any species, except for erratic effects on the growth of castrated male pigs (Rotter et al., 1996). The growth effects were clearly present at 1 ppm. Overt toxicity in kidney is associated with quite high levels of sphinganine. However, it is unclear how disruption in sphingolipid metabolism in the various tissues leads to toxicity and what aspects of altered lipid metabolism contribute to the carcinogenic response. There continues to be a need for more detailed understanding of the effects of FB1 on lipid metabolism, particularly with respect to its effects on apoptotic and oncotic necrosis in specific tissues. For example, what specific alterations in lipid metabolism lead to the induction of apoptosis and/or oncotic necrosis? What is the quantitative relationship between the preneoplastic and neoplastic effects?
Kuiper-Goodman et al. (1996) have reviewed various toxic endpoints and conducted a risk assessment based on these observations. Based on a sampling of food products and dietary patterns, they estimated an average intake of FB1 of <0.089 µg/kg body weight for 511-year-old children and less in adults, well below levels estimated to pose a health risk. The conservative assumptions on the dietary intake of FB1 have led to the assessment that the level of FB1 in Canadian diets is between 1100 and 42,000 times lower than the no-observed-adverse-effect level (NOAEL) determined for FB1 in rodents, horses, pigs, and monkeys. Similar estimates for exposure were observed for the general population in the Netherlands (de Nijs et al., 1998) and in Scandinavian countries (Nordic Council of Ministers, 1998
). Individuals requiring a gluten-free diet, including patients with celiac disease or sprue, were identified as being at increased risk because of increased corn consumption. Nevertheless, even those individuals are estimated to consume considerably less than 1 µg/kg body weight per day, well below consumption levels in the highly contaminated regions of southern Africa (Marasas et al., 1997). More recently, Kodell et al. (2001) provided a risk assessment based on liver tumorigenesis in the female mouse (NTP Technical Report, 2001), utilizing a biologically based model that included parameters for cell deaths and cell births. Similar to the actual results in the 2-year bioassay, this model concluded that tumors would occur only at the highest doses of FB1 administered, supporting a non-linear, margin of exposure approach to extrapolation of this data for human risk assessment.
Several gaps in our knowledge of the effects of FB1 in rodents and in humans limit our ability to precisely extrapolate from animal models to humans. In addition, the human epidemiology data relating FB1 exposure to esophageal cancer has several confounding factors, including a lack of detailed exposure analysis in some studies and the confounding of numerous other potential contributors to the carcinogenic process.
There are also several gaps in our knowledge with respect to research in animals and our understanding of the cell biologic effects of FB1 treatment. Although extensive research has detailed the effects of FB1 on sphingolipid metabolism, the precise alterations that occur in various tissues in different species, including humans, remain to be clarified. In particular, the critical alterations leading to the induction of apoptotic or oncotic necrosis in different cell types is yet to be delineated, and the relationship of these alterations to dose and other environmental factors, especially nutritional, are yet to be defined. More quantitative information over several time periods, and the effects of different doses of FB1 on apoptotic and oncotic necrosis and regeneration are required. Lastly, the renal tumors produced in the male rat kidney are highly unusual, the sarcomatoid variant being a unique response so far described in the rat kidney. Is there an effect of FB1 on differentiation processes in the rat kidney that could produce such an unusual response? Might other FB1-induced effects work in concert to ultimately affect carcinogenesis?
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Summary |
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed. E-mail: scohen{at}unmc.edu.
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REFERENCES |
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