MURINE LYMPHOCYTE PROLIFERATION IMPAIRED by SUBSTITUTED NEOSOLANIOLS and Calonectrlns-FUSARIUM METABOLITES ASSOCIATED with TRICHOTHECENE BIOSYNTHESIS

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ToziCOtf Vol. 29. No.9. pp. 1107-1113. 1991. 0041-{)101/91 S3.00 ....00 Prinled in Greal Britain. 'D 1991 Pergamon Preso pic

MURINE LYMPHOCYTE PROLIFERATION IMPAIRED BY SUBSTITUTED NEOSOLANIOLS AND CALONECTRlNS-FUSARIUM METABOLITES ASSOCIATED WITH TRICHOTHECENE BIOSYNTHESIS

G. S. BoNDY,1 S. P. McCORMICK,2 M. N. BEREMAND" and J. J. PEsTKAI *

IDepartment of Food Science and Human Nutrition. and Depattment of Microbiology and Public Health. Michigan State University, East Lansing. Mi 48824-1224. U.S.A.. lNonhern Regional Research Center. Agricultural Research Service. U.S. Department of Agriculture. 1815 North University Street, Peoria. IL 61604, U.S.A.

(ReceiY~ 24 December 1990: accepted 23 A.pri' 1990

G. S. BoNDY, S. P. McCORMICK. M. N. BEREMAND and J. J. PEsTKA. Murine lymphocyte proliferation impaired by substituted neosolaniols and caloe nectrins--Fusarium metabolites associated with trichothecene biosynthesis. Toxicon 29,1101-1113. 199 I.-Thecapacity ofFusarium secondary metabolites associated with trichothecene biosynthesis to inhibit murine spleen lymphocyte proliferation was evaluated and compared to that for wen known trichothecenes. Activity of these compounds was not specific for Band T lymphocytes since they inhibited [3H]thymidine (TdR) incorporation in unstimulated. Con A- and LPS-stimulated lymphocytes to the same extent. Concentrations of 8-propionyl neosolaniol and 8-butyrylneosolaniol which inhibited [JH]Tdr uptake by 50% (IDsoS) were 0.95 and 0.34 ng/mi, respectively. The IDsa for T-2 toxin was 0.26 ng/mi, indicating that there are minor aitera tions in 12, 13-epoxytrichothecene toxicity resulting from the replacement of the isovaleryl moiety on C8 of the trichothecene skeleton with other buiky acyl groups. IDsa values for 4, 15-diacetylnivalenoi. fusarenon X. deoxynivalenol and 3-acetyldeoxynivalenol were 25, 38, 120 and 1800 ng/ml, respectively. Comparatively, ID sa values for 3,15-dideacetyicalonectrin, 15-deacetylcaio nectrin. and 7,8-dihydroxycalonectrin were 390, 2700 and 2400 ng/mi, respectiveiy, indicating that the modified calonectrins had equivalent or less toxicity. Lymphotoxicity of trichothecenes thus decreased upon substitution of acyl groups at the C8 with keto or hydroxy moieties and was also dependent on the nature of substitutions at the C3, C4 and CI5 positions. Sambucinol and the trichothecene precursor trichodiene, metabolites which do not contain a 12,13-epoxide. did not inhibit lymphocyte proliferation. The resuits suggest the need for further assessment of occurrence and in vivo toxicity of Fusarium metabolites. panicuiary the substituted neosolaniols and caionectrins.

"Author to whom cOlTeSflOndence should be addressed ac Department of Food Science and Human Nutrition. Rm 236 Food Science. Michigan State University. East Lansing. MI 48824-1224, U.S.A.

1107 1108 G. S. BONDY et ai.

INTRODUcnON

TRICHOTHECENE mycotoxins are secondary metabolites that are produced by members of Fusarium and several other fungal genera (ISHII, 1983). Trichothecenes are potent inhibi tors of protein synthesis and suppress various aspects of the immune system, including cellular immunity (CORRIER and ZlPRIN, 1987; FROMENTIN et al., 1981; PEsTKA et aL, 1987; ROSENsTEiN et ai., 1979; YAROM et aL, 1984) and resistance to infectious disease (KANAI and KONDO, 1984; TAl and PEsTKA, 1988). Inhibition of in vitro lymphocyte protein and nucleic acid synthesis has been effectively used to relate trichothecene structure to lympho-

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FIG. 1. STRUCTURES OF Fusarium SECONDARY ME1'ABOUTES. (Al Structures of trichothecene derivatives and shunt metabolites; (B) structures of trichodiene and sambucinoJ. Lymphotoxicity of Fusarium metabolites 1109 tOXICIty (COORA Y, 1984; FORSELL et ai., 1985; FORSELL and PEsTKA. 1985; PESTKA and FORSELL. 1988; THOMPSON and WANNEMACHER, 1986; TOMAR et ai., 1988). Several Fusarium metabolites have been recently identified as putative precursors and shunt products in the biosynthesis of the trichothecenes deoxynivalenol (DON or vomi toxin) and Tc2 toxin, Trichodiene (Fig. I) is a precursor of the trichothecenes that requires multiple oxygenations and cyclizations to become T-2 toxin (MCCORMICK et ai., 1990). Other metabolites are structurally based on the parent 12,13oepoxytrichothec-9--ene skeleton but are modified with oxygen functions, induding esters and alcohols, at positions 3,4\,7,8 and 15 (Fig. 1). 8o Propionyi neosolaniol (P-NEO) and 80 butyryl neosola niol (BcNEQ) have been isolated as alternative end products to T-2 toxin over produced by a Fusarium'sporotrichiodes leucine auxotroph (BEREMAND et ai.• 1988). Sambucinol. 15 deacetylca10nectrin (DECAL), 3, 15-dideacetylcalonectrin (DIDECAL), and 7,8-dihydroxycaionectrin (DHCAL) have been identified in Fusarium cuitures grown in defined medium (BALDWIN et ai., 1987; GREEHALGH et ai., 1986; LAUREN et ai., 1987). In general, little is known of the occurrence and potential toxicity of these metabolites. In one notable exception. KIM et ai., (1990) recently purified DIDECAL from a F. oxysporum culture that was isolated from corn and determined it to be more toxic than DON. The purpose of this study was to assess potential Iymphotoxicity of these newly described Fusarium metabolites in the mitogen-induced murine lymphocyte proliferation assay. For comparative purposes trichothecenes such as T-2 toxin, DON, 3-acetyldeoxy nivalenol (3-ADON), 4,15-diacetylnivalenol (4, 15cDAN), and fusarenon X (FX), were included in this study. The results indicted that although trichodiene and sambucinol had no effect in this assay, the modified neosolaniols and calonectrins inhibited murine lymphocyte blastogenesis. Lymphotoxicity of these and other trichothecenes was highly dependent on the nature of modifications to the parent 12.1300epoxytrichothec-9-ene skeleton at the C3, C4, C8 and CI5 positions.

MATERIALS AND METHODS Reagents Trichodiene. DIDECAL. DECAL. P-NEO and B-NEO were prepared from cultures of F. sporOlrichioides mutants as described by BEREMAND et ai. (1988) and MCCORMICK et ai. (1989). DON was produced in Fusarium graminearum R6576 cultures and purified by water-saturated silica gel chromatography (WITT' et ai.. 1985). FX and 3-ADON were purchased from Romer Labs, Inc. (Washington. MO, U.S.A.). 4,15-DAN was kindly provided by T. Yoshizawa (Kagawa University, Japan). Sambucinol and DHCAL were generously supplied by J.D. Miller (Agriculture Canada. Ottawa. Ontario). T-2 toxin and all other reagents. including RPMI-I640 medium. media components and mitogens. were from Sigma Chemical Co. (St. Louis. MO, U.S.A.). Fetal bovine serum (FBS) was purchased from Gibco Laboratories (Grand Island. NY. U.S.A.). Toxin purity was > 99% for ail toxins tested.

Lymphocyte preparation Spleens were collected from B6CJFl (C57BL/6 x CJH/HeN) female mice. teased apan with tissue forceps in HBSS (Hanks Balanced Salt Solution) and centrifuged at 450 x g for [0 min. Erythrocytes were lysed by resuspending spleet! cells in 0.83% ammonium chloride for 2 min at room temperature. Cells were centrifuged again and resuspended in RPMI-[640 medium supplemented with [0% FBS. [00 Ulml penicillin. 100 ug/ml streptomycin. 5 x lO-l M 2·mercaptoethanol. 25 mM HEPES buffer. I mM sodium pyruvate and 0.1 mM nonessential amino aCIds.

Mitogen-induced blastogenesis Mitogen-induced blastogenesis was performed in triplicate in 96-weil tissue culture plates (Cormng Glass Works. Coming, NY. U.S.A.). Spleen lymphocytes were added to each well in 2oo.ul aliquots containing I x 10; cells. To evaluate the effect oftoxins on various lymphocyte subsets. cells were incubated in the presence of the T 1110 G. S. BONDY et ai.

TABLE 1. EJ'FEcrs OF Fusarium TOXINS ON nrYMlOlNE INCORPORATION INTO LYMPHOCYTES

IDll)(ngjmj)" Toxin Con A LPS Unstimulated Combined meant

T-2 toxin 0.26±0.OS 0.30±0.04 0.19±0.07 0.26±0.03 P-NEO 0.8S±0.67 1.30±0.9S 0.69±G.46 0.95±O.37 B-NEO 0.19±O.09 0.67±0.31 0.21 ±O.09 O.34±O.12 DECAL lOOO±400 2100±26O 6300±3800 2700± 1100 DIDECAL 72±40 560±36O 520±350 390± 160 DHCAL 1300±530 3000±9lO 2800±220 2400±41O lOON 64±34 200±64 68±21 120±30 .4. IS-DAN 30±2 24±2 21±4 2S±2 3-ADON 2200± ISO 1600±420 1400±500 1800±220 FX 39±5 54±13 22±S 38±6

"Data are eJq)ressed as Mean ± SEM of 1I > 3 lOll) values. lOll) values were calculated graphically by plotting toxin concentration V$ %eH] thymidine incorporation. Each toxin concentration was tested in triplicate for a single 1Dll) determination. tFor ead:l toxin there were no statistical differences between the mean ID~ for Con A-stimulated. LPS stimulated. or unstimulated cells using the Kruslcal-Wallace test for several independent samples. Hence, IDlQS for both mitogen-stimulated and unstimulated lymphocytes were combined for an overall mean lOll) value.

lymphocyte mitogen concanavalin A (Con A) at 5.ug,lml or the the B lymphocyte mitogen lipopolysaccharide (LPS) at 2S .ug,lml. Toxins were dissolved in ethanol (DON. DECAL. DIDECAL. DHCAL, P·NEO. B-NEO T-2 toxin) dimethyl sulfoxide (trichodiene. sambucinol). or water (FX. 4,IS-DAN. 3-ADON), and diluted with either RPMI-I640 medium or water. The maximum amount of solvent used to deliver a mycotoxin represented less than I% of the final culture volume. Solvent control cultures accompanied all toxin-treated cultures for comparative purposes. Lymphocyte cultures were incubated at 37"C in a 5% CO2 humidified atmosphere for 72 hr. For the final 6 hr. I .uCi of (JH]-thymidine (TdR: ICN RadiochemicaJs. Irvine. CA, U.S.A.) was added to each well. Cultures were harvested on filtermats (Skatron Inc.. Sterling. VA, U.S.A.) using a TiterteJc semiautomatic cell harvester (Skatron Inc.. Sterling. VA. U.S.A.). and [lHl-TdR incorporation was evaluated using a liquid scintillation counter. Untreated control cultures contained on average 3200 dpm/cu1ture. Mitogen-stimulated control cultures contained on average 391.000dpm/cu1ture (Con A) and 134.000dpm/cu1ture (LPS), indicating that mitogen stimulation of blastogenesis was effective. Results were expressed as the peromtage of thymidine uptake in mitogen or unstimulated control lymphocytes in the absence of tl'xin. Inhibitory doses that caused 50% inhibition of spleen lymphocyte blastogenesis (IDso.l were determined graphically. Differences in (JH]TdR incorporation in Con A. LPS and unstimulated lymphocytes were analyzed by the Kruskal-Wallace test for several independent samples iJSing' the Microcomputer Statistical Program (C.S. Sciences. Michigan State University).

RESULTS AND DISCUSSION For each of the Fusarium toxins tested, inhibitory doses causing a 50% reduction of [,HJTdR (IDSO> incorporation were the same for Con A- and Lpg-stimulated lymphocytes (Table 1). Since Con A stimulated primarily T lymphocytes (STITES, 1982) and LPS stimulated primarily a lymphocytes (RaIn, 1988), lack of differences in response to mitogens indicates that all of the toxins tested were capable' of inhibiting both a and T lymphocyte subsets. This is further supported by the ability of the trichothecenes tested to inhibit proliferation in unstimulated cultures, where neither a nor T ceUs were preferen- . tially stimulated, to the same degree as Con A or LPS-stimulated cultures. Of the Fusarium toxins examined, the following toxins inhibited murine spleen lympho cyte blastogenesis (in order of most to least toxic): T-2 toxin. a-NEO, P-NEO. 4,15-DAN, FX, DON. DIDECAL, 3-ADON, DHCAL ana DECAL (Table I). Trichodiene and sambucinol did not inhibit murine spleen lymphocyte blastogenesis at concentrations up to 1 x 105 ngfmL Lymphotoxicity of Fusarium metabolites III I

The ID so for T-2 toxin in murine spleen lymphocytes was 0.26 ng/ml (Table I). This was within the range of previous reports, in which T-2 toxin reduced eHlTdR incorporation by 50% in murine spleen lymphocytes at concentrations of 2.5 ng/ml (HOLT et ai., 1988) and 0.1--0.2.5 ng,lml (ROSENSTEIN and UFARGE-FRAYSSINET, 1983), and in human peri pheral blood lymphocytes at concentrations of 1.5 ng/ml (COORAY, 1984) and 0.5-2.5 ng/ml (FORSELL et ai., 1985). P-NEO and BeNEO, with IDsoS of 0.95 and 0.34 ng,lml, respectively, differ from T-2 toxin at the C8 position (Fig. IA). Although BeNEO was similar in toxicity to T-2 toxin. P-NEO was approximately 3.7 times less toxic than T-2 toxin. indicating that changes at the isovaleryl moiety influence immunotoxicity in this assay. The high degree of toxicity for these three compounds compared .. to compounds containing either a keto group (DON, FX, 4,15-DAN), a hydroxyl group (DReAL), or a hydrogen (DIDECAL, DECAL) at the C8 indicated that these bulky acyl groups imparted a significant degree of Iymphotoxicity. It is possible that increased lipophilicity due to the presence of acyl groups at the C8 position contributes to the greater toxicity of T-2 toxin. P-NEO and B-NEO compared to the other toxins tested. The IDso value for DON in this assay was 120 ng/m1 (Table I), which is comparable to published reports of 140-160 ng/m1 in human lymphocytes (FORSELL and PEsTKA. 1985), 70 ng/mJ in rat peripheral blood lymphocytes (MILLER and ATKINSON, 1986), and 90-220 ng/m1 in rat and human peripheral blood lymphocytes (ATKINSON and MILLER. 1984). In contrast the ID so for FX in murine spleen lymphocytes was 38 ng/ml (Table I), which was two fold higher than the ID so of 18 ng/ml seen in human peripheral blood lymphocytes (FORSELL and PEsTKA, 1985). The IDsoS for 4,15-DAN and 3-ADON were 25 ng/rnA and 1800 ng/mi. respectively (Table 1). Dependence of Iymphotoxicity on the C4 substituent is similar for group A trichothecenes (T-2 toxin and metabolites) and group B (8 e keto) trichothecenes (DON, FX, 3-ADON, 4,15-DAN). FORSELL and PEsTKA (1985) found that addition of an acetyl at the C4 position of HT-2 toxin to become T-2 toxin results in a 2.3-fold increase in toxicity. Likewise, substitution of the hydrogen at C4 of DON with an acetyl group on FX results 'in a three-fold increase in toxicity (Table I). Toxicity of the group B trichothecenes was further increased by substitution of the hydroxyl group at CI5 of FX with an acetyl group at the C15 position of 4,15-DAN, . resulting in a 50% increase in toxicity from an ID so of 38 ng/ml for FX to 25 ng/ml for 4,15 e DAN (Table I). However. the presence of an acetyl group at the C15 position of 8-ketotrichothecenes does not necessarily imply increased toxicity. For example. IS-ADON, which has· an acetyl group at the CI5 position but which lacks the C4 acetyl group found on 4,15-DAN, is 1.7-fold less toxic to human lymphocytes than DON (FORSELL and PEsTKA, 1985). Toxicity of group B trichothecenes was also strongiy dependent on substitution at the C3 position. Replacement of the C3 hydroxyl' on DON with a C3 acetyl on 3-ADON resulted in a IS-fold decrease in toxicity. This is similar to the 17.5-fold decrease in toxicity of 3-ADON compared to DON using Vero ceHs in a protein synthesis inhibition assay (THOMPSON and WANNEMACHER. 1986). In contrast. 3-ADON was only five-fold less toxic than DON using rat and human peripheral blood lymphocyte blastogenesis assays (ATKINSON and MILLER, 1984). 3-ADON was also only five-fold less toxic than DON' using rat spleen lymphocytes in a protein synthesis inhibition assay (THOMPSON and WANNEMACHER. 1986). These differences indicate that although most in vitro assays rank 3-ADON as less toxic than DON, the relative toxicities of DON and 3-ADON depend on the ceHs used in the assay. Among the calonectrins tested, DIDECAL, DHCAL and DECAL were less toxic than 1112 G. S. BONDY et al.

DON, with IDsoS of 390, 2400 and 2700 ng/ml, respectively (Table 1). In contrast, KIM et al. (1990) found that DIDECAL orally administered to rats was more toxic than DON. It is likely that the involvement in whole animal studies of events other than inhibition of DNA synthesis, for example toxin metabolism and rate of transport, is responsible for differences in toxin potencies compared to in vitro ceil systems (THOMPSON and WANNEMACHER, 1986). As with the group B trichothecenes, Iymphotoxicity among the calonectrins was also dependent on the nature of the C3 substituent. Substitution of the C3 hydroxyl group ofDIDECAL with an acetyl group ofDECAL resulted in a seven-fold decrease in toxicity (Table 1). Nevertheless, esterase activity in liver and intestinal micro flora could readily convert DECAL to DIDECAL and thus increase its toxicity in vivo. Trichodiene is an intermediate in the biosynthesis of trichothecenes (SAVARD et ai., 1989). The absence of a 12,13-epoxy group at this stage in trichothecene biosynthesis is a likely reason for the lack of inhibitory activity seen in this assay system, since opening of the 12,13-epoxide group is known to abolish biological activity (UENO, 1977). Based on this assay, use of the plant growth regulator ancymidol, which blocks trichothecene production and causes trichodiene accumulation, may be a useful chemical agent for controlling trichothecenes in agricultural commodities (DESJARDINS et al., 1987). Proliferation of murine splenic lymphocytes in vitro was signiftcantly impaired by substituted neosolaniols and calonectrins. This is especially important because these compounds may be concurrently present in foods with DON and T-2 toxin. The results underscore the necessity for both a thorough assessment of the nat~l occurrence of these trichothecenes, as wen as further in vitro and in vivo structure-activity studies to determine their relative t9xicity and Iymphotoxic effects.

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