Dose-dependent fumonisin B1 hepatotoxicity and hepatocarcinogenicity, detoxification of fumonisin B1, and suppression by isoflavones of fumonisin B1-promoted hepatocarcinogenesis in rats

Three hypotheses were tested in present studies: 1. that a threshold dose of fumonisin 3^ (FB-J hepatotoxicity and tumor promotion may be below 50 ppm in a short-term diethylnitrosamine (DEN) initiation/FB-. promotion model; 2. that modifying FBt with fructose may reduce or prevent hepatotoxicity and tumor promotion in rats; 3. that isoflavones at 1 mmol/kg diet may at least partly inhibit FBT-promoted rat hepatocarcinogenesis. A short-term threshold dose of FB: hepatotoxicity and hepatocarcinogenicity was determined in rats initiated with DEN and promoted with 18, 35 and 70 ^unol FBi/kg diet (12.5, 25 and 50 ppm FBi, respectively). Increases in plasma alanine aminotransferase (ALT) activity and plasma total cholesterol (TC) concentration were found in animals fed 35 and 70 (imol FB>/kg diet, and greater hepatic prostaglandin (FG) production in the group fed 70 (imol FBi/kg diet. Placental glutathione Stransferase (PGST)and gamma-glutamyltransferase (GOT)-positive altered hepatic foci (.AHF) developed with feeding 35 and 70 (imol FBi/kg diet. The short-term threshold of FB, hepatotoxicity and hepatocarcinogenicity, therefore, was probably >18 (imol but :ification in DEN-initiated rats. Feeding 70 ^mol FB-/kg diet increased plasma .°iLT activity, plasma TC concentration, and hepatic PG synthesis. Only FB;-fed rats had PGSTand GGT-positive AHF. Liver-associated natural killer (NK) cell activity was significantly decreased in the FB:-fed rats and increased in the group fed 70 [imol FBi.-fructose/kg diet. Therefore, reacting FB-, with fructose eliminated tumor promotion by FB, while stimulating the NK cell activity. To evaluate whether isoflavones suppress FBi-promoted hepatocarcincgenesis, DEN-initiated rats were fed 1 mmol isoflavones/kg diet with 18, 35 and 70 nmol FBi/kg diet. Development of PGSTand GGTpositive AHF and increases in plasma ALT activity, plasma TC concentrati and hepatic PG production were found with feeding 70 jimol FB-Vkg diet wit or without isoflavones. Significant increases in plasma ALT activity, plasma TC, and developm.ent of PGSTand GGT-positive .AHF were shown wit.h feeding 35 (imol FB-./kg diet, but these effects were diminished by isoflavones. Therefore, isoflavones suppressed rat hepatocarcinogenesis promoted by 35 (Xmol but not 70 |xmol FB,/kg diet. GENERAL INTRODUCTION Resui~s frc.T. ~hree studies are present:ed in this disserrarion. F'-imcr-isir. 3; hepatczoxiciry and hepatccarrincger.esis in rats are investigated in. Study . Two different apcroacnes to FB: detoxif icat:Lcn are reported in Study 2 and Study 3. Study 1. Threshold Dose for Short-Term Cancer-Promotion by Dietary Fumonisin-Bi in Diethylnitrosamine-Initiated Rat Hepatocarcinogenesis Fumonisins are recently discovered "vcotcxins produced cy the predominant corn fungi Fl'Ss rijin anc crcliferazi::n. "u-Tionisin 3; iFB-., , a .Tiajcr ~eincer zz tne fu-ticn-sin family, caused equine leui-'-cencephalorrialatia '.Kellerman et al., 1390; Wilson et al., 15921 and pcrcine pulmonary edema ^Harrison et al., 1990', and was nepatctoxic and nepatocarcinogenit in rats 'Gelderblcm. et al., 153S and 1991; . The occurrence tf FB-. _n F. :r.or.lllfcr~e-czr.zariLr.azez corn was implicated in a relatively high rate cf human esophageal cancer in southern Africa 'Marasas et al., 1989; Sydenham et al., 199G). Several bicmarkers have bee.n usee to stucy F3; hepatotcxicity and hepatocarci.noge.nicity. Fum.cnisin 5-.-prtmcted rat nepatccarcincge.nesis was readily quantifiec by measuring place.ntal glutat.hione S-tra.nsferase {PGSTipositive altered hepatic foci ;.A.KF; Lebepe-Hatur et al., 1995; and gammaglutarayltransf erase '.GQT;-positive foci ;Geiderblotn et al., 1985] . Because cf the early occurrence cf PGST induction, v-yhic.h was evident withi.n 4 8 h in single hepatocytes after diethyl.nitrosa.r.i.ne i.nitiaticn (Moore et al. , 19S7;, and because of the persistence cf bot.h ?GS7 and GGT during nepatocarcinogenesIS ;.Hendrich et al., 1937) , t.hese two encvuies can serve as sensitive bicm.arkers of early stages cf hecatccarci.nogenesis. Our previous study s.ncwed tnat coth rGST— and GGT — cositive A'AT were detected after -4 wee.ks in rats fed cl com crucel-.our-f--d -nd h'ch'v cp.olesteroi (TC) level and alanine aminotransferase (ALT) activity have been associated with fumonisin hepatotoxicity and hepatocarcinogenicity. Fusarium proliferacum strain 5991-fermented corn providing 50 ppm FB. to DEN-initiated rats significantly increased plasma TC and ALT while causing hepatocellular adenomas (Hendrich et al., 1993). Similar results were obtained using highly purified FBi, and additionally, greater hepatic prostaglandin production was related to FB.-promoted hepatocarcinogenesis in rats (Lu et al., 1997). Fumonisin B, has recently been listed as a class 2B carcinogen (International AqencY Research on Cancer, 1993) . Development of appropriate animal models of carcinogenesis is critical to understanding the probable FB; carcinogenicity in humans. Because nitrosamines often contaminate .human diets (National .Academy of Sciences, 1981) , and FB; is almost always found in corn-based human foods, a 2-stage rodent model combining DEN to initiate and FB; to promote carcinogenesis may approach tc some extent the reality of .human carcinogenesis (Hendrich et al., 1997). One advantage of utilizing this model is that the cancer promoting potential of FB; can be readily quantified by PGSTand GGT-positive .AHF. Our DEN initiation/FB; promotion model, i.e. initiation of rats with DEN (15 mg/kg body weight) followed by 50 ppm FB; promotion for 4 weeks, can induce rat hepatocarcinogenesis rapidly, requires relatively small amou.nts of FB;, and may mimic the natural e.nvironment where humans are exposed to a combination of chemical carcinogens. Although FB; was non-mutagenic in the Salmonella mutagenicity assay (Gelderblom and Snyman, 1991) and lacked genotoxicity based on unscheduled DNA synthesis assay in primary rat hepatocytes (Norred et al., 1992), it seemed to be a complete carcinogen either at 50 ppm long-term (18-24 months) or at 250 ppm. short-term (3 weeks) (Gelderblom et al., 1991 and 1994). As a potent promoter in the DEN initiation/FB; promotion .model, fumonisin B; was effective to promote hepatocarcinogenesis at a dietary level of 50 ppm in 4-wee!c studies (Hendrich ec ai., 1993; Lu et al., 1997). It was unknown, however, whether 50 ppm was the cancer-promoting threshold dose of FB; short-term. This exposure level to FBi may still be an order of magnitude higher than the natural occurrence of FB:. in corn-based human foods and ani.mal feeds, which commonly contained 0.2-3 ppm FB: (Hopmans and Murphy, 1993; Murphy et al., 1993; Sydenham et al., 1991). In Study 1 using the short-term DEN initiation/FB-. promotion model, it was hypothesized that FB; would still e.xert hepatotoxic and cancerpromoting effects at dietary concentrations lower than 50 ppm. The dietary levels of FBi were lowered to 25 and 12.5 ppm to approac.h more closely the natural occurrence of FB-., to establish dose-response effects of FB-. on hepatotoxicity and hepatocarcinogenesis in rats, and to ascertain a shorrterm cancer-promoting threshold for FB;. Study 2. Reaction with Fructose Detoxifies Pumonisin Bi While Stimulating Liver-Associated Natural Killer Cell Activity in Rats It was hypothesized in this study that modifying FB; with fructose would reduce or prevent FB; hepatoto.xicity and promotion of hepatocarcinogenesis in rats. Efforts were devoted to detoxifyi.ng FB; in several ways. Thermostability of FB. proved to be great, because nearly 85s of FB; was recovered after different heat treatments: 75°C for 135 min., 100°C for 45 min., and 125°C for 5 min. (Dupuy et al., 1993). Treatment of fumonisin-contaminated corn with 21 ammonia for 4 days, a process that deto.xified aflatoxin B;, led to slight reduction in the concentration of FB; without decreasing the toxicity in rats (Norred et al., 1991). Hydrolyzed FB;, which was produced by boiling F. proiiferacum-contaminated corn in 1.2% calcium hydroxide solution for 1 h, was similar in toxicity to FB; when the nutritional status of rats was adequate (Hendrich et al., 1993). In vitro toxicity studies on several analogs of FB; showed that the analogs containing FB;'s amine group and the tricarballylic side chains were more to.xic than FB-., whereas the analog containing only the