Cycasin and its mutagenic metabolites

Mutation Research, 114 (1983) 19-58

19

Elsevier Biomedical Press

Cycasin and its mutagenic metabolites Robin W. Morgan i and George R. Hoffmann 2,, I Department of Biology, The Johns Hopkins University, Baltimore, MD 21218, and 2 Department of Biology, College of the Holy Cross, Worcester, MA 01610 (U.S.A.) (Received 4 March 1982) (Revision received 3 August 1982) (Accepted 4 August 1982)

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chemical and physical properties of cycasin and methylazoxymethanol . . . . . . . . . . . . . . . . . . . Cycads and cycad products Occurrence of cycads and their azoxyglycosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Uses of cycads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Metabolism of cyeasin and methylazoxymethanol Deglucosylation of cycasin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hydrolysis and oxidation of methylazoxymethanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Detoxification of cycasin and methylazoxymethanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Toxicology of cycasin and methylazoxymethanol Systemic toxicology and cytotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Carcinogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Teratogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic effects in bacteria Induction of mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Host-mediated assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Repair assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prophage induction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic effects in yeasts Induction of mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Induction of mitotic recombination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic effects in vascular plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic effects in Drosophila . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic effects in mammalian cells in culture Induction of gene mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Induction of chromosome aberrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Induction of sister-chromatid exchanges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA strand breakage and unscheduled D N A synthesis . . . . . . . . . . . . . . . . . . . . . . . . . .

* To whom correspondence should be addressed.

Abbreviations: MAM, methylazoxymethanol; MAMAL, aldehydic form of MAM. 0165-1110/83/0000-0000/$03.00 © Elsevier Biomedical Press

20 20 22 23 24 25 26 27 29 36 36 38 39 41 41 41 46 46 47 48 48 49

20 Genetic effects in mammals Induction of DNA strand breakage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Induction of chromosomeaberrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Induction of morphologicalsperm abnormalities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

50 50 51 51 52 52

Introduction Cycasin is one of several azoxyglycoside compounds that are produced by cycads. It is of practical interest because of possible toxicologic effects in people who use cycad products. Cycasin and its aglycone derivative methylazoxymethanol (MAM) are also of scientific interest as compounds that illustrate complex interrelationships between metabolism and toxicologic action. Adverse effects of cycasin and MAM in people were first suspected when it was realized that natives of Guam, who consumed cycad products, exhibited a high incidence of amyotrophic lateral sclerosis (Laqueur, 1977). Although a clear link between ingestion of cycasin and amyotrophic lateral sclerosis has never been established, the possibility of such an association spurred investigation into the biological effects of cycasin. In addition to neurotoxicity, evidence has accumulated on other toxicologic effects of cycads and cycad products, including carcinogenicity, teratogenicity and mutagenicity. The acute toxicity and carcinogenicity of cycasin and related compounds have been the subjects of earlier reviews (Whiting, 1963; Laqueur and Spatz, 1968; Spatz, 1969; Hirono, 1981; Laqueur, 1977; Laqueur and Spatz, 1975). This review concentrates on genetic effects of cycasin and its derivative MAM.

Chemical and physical properties of cyeasin and MAM As an azoxyglycoside, cycasin is a member of a class of compounds that includes several potent toxicants. It is a crystalline substance that contains D-glucose attached to an aliphatic azoxy structure by a fl-glucosidic linkage (Laqueur, 1977). Removal of the sugar from cycasin yields the aglycone MAM. MAM occurs not only in the structure of cycasin, but is common to all of the cycad azoxyglycosides and is responsible for their toxicologic properties (Laqueur, 1977), including their mutagenicity. Many studies of MAM have used chemically synthesized methylazoxymethanol acetate (MAM acetate) rather than MAM derived from cycads. The synthesis of MAM acetate is initiated by the oxidation of 1,2-dimethylhydrazine to azomethane and azoxymethane. Subsequent bromination of azoxymethane in the allylic position and conversion into the acetate yields MAM acetate (Laqueur, 1977; Matsumoto et al., 1965). Molecular formulas, Chemical Abstracts Service (CAS) registry numbers, and

592-62- i

Methylazoxymethanol acetate

(Methyl-ONN-azoxy)methanol acetate (ester) Methylazoxymethyl acetate

2-oxide

l-Hydroxymethyl-2-methyldiimide-

( Methyl-ONN-azoxy)methanol

(Methyl-ONN-azoxy)methyl-fl-Dglucopyranoside Methylazoxymethanol-fl-D-glucoside fl-D-Glucosyloxyazoxymethane Cycas reooluta glucoside Cykazine fl- D-Glucosyloxyazoxymethane

Synonyms

a Compiled from Merck Index (1976); Registry of Toxic Effects of Chemical Substances (1981).

590-96-5

14 901 = 0 8 - 7

Cycasin

Methylazoxymethanol

CAS Registry Number

Compound

TABLE I NAMES AND STRUCTURES OF CYCASIN, MAM AND MAM ACETATE a

C 4H s N 2 0 3

C 2H 6 N 2 0 2

C s H 16N 2 0 7

Molecular formula

OH OH

' ~ OCH 2N==NC~oH3

O

H 3 C - C OCH 2 N=: N C H 3

[(~

HOCH 2N:::::NCH 3 O

H

HOC H 2 0~~(

Structural formula

Ix.)

22 TABLE 2 CHEMICAL AND PHYSICAL PROPERTIES OF CYCASIN, MAM AND MAM ACETATE Compound

Molecular weight

Melting point

Cycasin

252.2

149oc

Methylazoxymethanol Methylazoxymethanol acetate

90.1 132.1

Boilingpoint

154°C b

a

3oc c

Decomposition Density point at 24°C

51°C (0.6 mm) d

1.208 0

45°C (0.45 mm) d

1.172 d

a Matsumoto and Strong (1963). b Merck Index (1976). c Matsumoto et al. (1965). d Kobayashi and Matsumoto (1965).

synonyms for cycasin, MAM, and M A M acetate are presented in Table 1. Chemical and physical properties of the compounds are summarized in Table 2.

Cycads and cycad products Occurrence of cycads and their azoxyglycosides Cycads are gymnosperms and, as such, are intermediate between ferns and flowering plants in the phylogenetic hierarchy. They are members of a single family, Cycadaceae, in which there are 9 genera: Bowenia, Ceratozamia, Cycas, Dioon, Encephalartos, Microcycas, Macrozamia, Strangeria, and Zamia. A key to the genera of cycads appeared in Whiting (1963). Although they were more abundant 200 million years ago than today, cycads are still well-represented in tropical and subtropical floras (Fosberg, 1964). Cycads are slow-growing plants with either short and often-branched tuberous stems or tall and seldom-branched columnar stems (Whiting, 1963). The stem typically consists of a thin layer of wood surrounding a large pith. Cycad roots, which have symbiotic nodules capable of fixing atmospheric nitrogen, are large and deep enough to endow the plant with considerable stability during storms. The pinnately compound leaves of cycads range from a few inches to 7 feet in length. Cycads are dioecious plants and bear seed-bearing cones or pollen-bearing cones at the top of the stems (Whiting, 1963). The azoxyglycoside macrozamin was first isolated by Cooper in 1941 from seeds of the Australian cycad Macrozamia spiralis. Subsequently, macrozamin has also been found in seeds of Encephalartos bakeri and Encephalartos hildebrandtii (Laqueur, 1977). Macrozamin differs from cycasin, in that the carbohydrate moiety is primeverose, a disaccharide of glucose and xylose, rather than glucose alone. Cycasin itself has been found in only 2 species of cycads: Cycas revoluta and Cycas circinalis. Besides cycasin, plants of the genus Cycas produce several other

23 azoxyglycosides, called neocycasins (Laqueur, 1977). Azoxyglycosides have been identified in the genera Cycas, Macrozamia, Encephalartos, and Zamia. According to Laqueur (1977) the remaining five genera of cycads have not been examined for azoxyglycosides. The concentration of cycasin or related compounds varies among plants and tissues but ranges from traces to 4% of the dry weight (Wogan, 1976). MAM is the aglycone of cycasin, macrozamin, the neocycasins, and all of the other cycad azoxyglycosides that have been studied. It now appears that the single compound M.AM is responsible for the biological effects of the entire family of toxic compounds from cycads. Little is known about how cycasin is synthesized in the plant. It has been suggested that cycasin, like azoxymethane, could arise through oxidation of endogenous aliphatic amines (Fiala, 1980), but the experimental evidence is limited.

Uses of cycads Cycads that have been used as food by people include species of Cycas, Encephalartos and Zamia (Whiting, 1963). Because they are deeply rooted, cycads survive harsh weather that can destroy other food sources (Laqueur, 1977). Essentially all portions of cycads, including columnar stems, tuberous stems, and leaves have been used as food. A major food use of cycads is as cycad flour. Cycads contain an edible starch that can be ground into a dry meal. The meal can be eaten in a mixture with brown sugar and coconut, used to thicken soups, or baked like a tortilla. The preparation of cycad meal is an involved process, for it has long been recognized that the plant contains poisonous substances that must be removed before consumption. Methods of preparing cycad meal vary with the locale but typically involve extended soaking of the starchy portions of the plant, followed by drying in the sun. The dried cycads are ground in a mortar to make flour. In some locations, such as many Pacific islands, large quantities of cycad starch are produced (Whiting, 1963). Between about 1845 and 1920, cycad starch was also produced commercially in the vicinity of Miami and on the Florida Keys; it was called Florida arrowroot starch and was used in infant foods, biscuits, chocolates, and spaghetti (Whiting, 1963). The industry exploited wild populations of Zamia floridana (synonym Zamia integrifolia), the stem of which has a high starch content. Processing involved grinding the stems, straining out coarse fibers, and washing out a water-soluble, red, toxic material. At the peak of arrowroot starch production, some Florida companies produced as much as 300 tons of arrowroot starch per year (Neal, 1965). The cycad starch business apparently continued on a significant scale into the 1920's (Small, 1921, 1926). Closing of the Florida arrowroot factories resulted from depletion of the plant populations. Zamia floridana, commonly called Florida coontie and Florida arrowroot, is now considered to be a threatened plant species in Florida (Ward, 1980). Although no longer collected for starch production, some collection of Zarnia for horticultural purposes continues, despite the plant's protection by state law. Besides its commercial production, cycad starch was a staple of the Seminoles and earlier native populations of southern Florida (Ward, 1980). In addition to the use of cycads to prepare flour, cycad seeds were consumed raw,

24 and cooked seeds were eaten as a boiled vegetable, added to curries, or simmered with meat and vegetables as a stew (Whiting, 1963). Cycad preparations have found various uses for medicinal purposes (Whiting, 1963). For example, cycad gum has been used as a topical medicine for snake bites and insect bites; cycad seeds, stems and buds have been used to treat ulcers, boils and wounds. Other medicinal uses of cycad preparations have included the chewing of roots to relieve coughs and improve the singing voice and treatment with extracts of leaves or megasporophylls to relieve colic or to stop bleeding. For a thorough review of the economic botany of cycads, the reader is referred to Whiting (1963).

Metabolism of cycasin and MAM Cycasin must be metabolized before it can react with cellular macromolecules, including DNA. An essential step is its deglucosylation to form the aglycone MAM, because the biological effects of cycasin appear to be attributable to MAM. The biological activity of MAM seems to occur through the production of methylcarbonium ions from MAM or possibly from an aldehydic form of MAM (MAMAL). The putative aldehydic form may also react with macromolecules directly.

Deglucosylation of cycasin Cycad glycosides are toxic only when deglycosylated. Young and mature animals differ with respect to the enzymatic deglucosylation of cycasin (Laqueur, 1977). In rats older than 28 days, deglucosylation occurs only in the gut and is catalyzed by enzymes of the microbial flora. After intraperitoneal injection, unmetabolized cycasin is almost completely recovered in the urine; following gastric intubation, however, only 30-60% of the cycasin can be recovered over a 2-day period, the remainder probably being metabolized in the gut (Kobayashi and Matsumoto, 1965). Germfree Sprague-Dawley rats, fed a diet consisting of 0.2% cycasin in laboratory chow for 3 days, excreted 92-100% of the cycasin in urine and feces, whereas conventional rats excreted only 18-35% of ingested cycasin (Spatz et al., 1966). Relative to conventional rats, germfree animals were also found to be resistant to the systemic toxicity (including liver damage) and carcinogenicity of cycasin (Laqueur, 1964). When a normal flora was established in 2 of 4 germfree littermates and all animals were fed 0.2% cycasin, the littermates with a microbial flora suffered liver damage as did the conventional rats; the germfree littermates had normal livers despite their exposure to cycasin (Laqueur, 1964). Germfree rats colonized with Streptococcus fecalis, which produces fl-glucosidase, showed centrolobular hemorrhagic necrosis of the liver and excreted less cycasin than germfree rats colonized with Lactobacillus salivarious, which lacks fl-glucosidase. The animals colonized with microorganisms lacking fl-glucosidase activity were the same as germfree animals with respect to cycasin excretion (Spatz et al., 1967b). Unlike cycasin, MAM acetate was carcinogenic in germfree rats as well as in

25 normal rats. Whether it was administered by feeding or by intraperitoneal injection, MAM acetate (total dose of 12.5 mg/rat) caused tumors of the colon (Laqueur et al., 1967). The data indicate that the deglucosylation of cycasin by enzymes of microorganisms in the gut is a prerequisite for the toxic and carcinogenic effects of cycasin in mature animals. Mutagenicity experiments support the same conclusion. Cycasin and conjugates of MAM are mutagenic in the Ames test only when preincubated with an appropriate hydrolase (Matsushima et al., 1979), because Salmonella typhimurium lacks the necessary fl-glucosidase. In the host-mediated assay, cycasin was mutagenic in Salmonella if administered orally but not if administered intraperitoneally, intramuscularly, or intravenously (Gabridge et al., 1969). Mutagenicity tests are discussed more thoroughly later in the review. Unlike mature animals, neonatal animals deglucosylate cycasin independently of bacterial enzymes (Laqueur, 1977). The mammalian fl-glucosidase activity occurs in several tissues. The skin of fetal and neonatal rats, from 15 days prenatal to 30 days after birth, was reported to contain fl-glucosidase activity; in both conventional and germfree Sprague-Dawley rats, the activity peaked at about the time of birth and began to decline by the sixth postnatal day (Spatz, 1968). fl-Glucosidase activity in the small intestine was reported to be highest on the 15th postnatal day and decreased thereafter, being detectable for 75 more days (Matsumoto et al., 1972). Activities in skin, pancreas, liver, muscle and stomach were also found to persist, but at low levels. Tumors were induced in germfree rats by cycasin administered as late as 25-35 days after birth; therefore, the low but persistent fl-glucosidase activity may be sufficient to hydrolyze cycasin to MAM (Laqueur, 1977).

Hydrolysis and oxidation of MAM MAM need not be metabolized to have biological effects, because it is subject to spontaneous decomposition that produces reactive methylcarbonium ions. It has been suggested that the decomposition of MAM is similar to that of metabolically activated dimethylnitrosamine (Miller and Miller, 1965). Spontaneous hydrolysis of MAM occurs most rapidly at alkaline pH, with half-lives of MAM being 2.8 h at pH 10 (0.1 M sodium borate), 11.6 h at pH 7 (0.1 M sodium phosphate), and 12.3 h at pH 4 (0.1 M sodium phosphate) (Feinberg and Zedeck, 1980). MAM acetate is somewhat more stable in aqueous solution than MAM (Kobayashi and Matsumoto, 1965). Spontaneous hydrolysis of MAM results in the production of formaldehyde; whether formaldehyde contributes significantly to the biological effects of MAM under some conditions is unclear (Miller, 1964). The enzymatic cleavage of cycasin to MAM is required, but not necessarily sufficient, to explain its mechanisms of action. Further metabolic modification of MAM may also be toxicologically important; in fact, it has been suggested that a dehydrogenase dependent on NAD + or NADP + oxidizes MAM to a reactive aldehydic form, MAMAL (Grab and Zedeck, 1977; Feinberg and Zedeck, 1980). Tissues that are sensitive to the effects of MAM (i.e., liver, colon and caecum) convert NAD + into NADH when MAM is used as a substrate, whereas tissues that are relatively insensitive to MAM (i.e., jejunum and ileum) are incapable of

26 converting NAD + to N A D H in this reaction. Upon fractionation of a liver homogenate, the MAM-dehydrogenase activity coincided with alcohol dehydrogenase activity. Moreover, MAM can serve as a substrate for purified horse-liver alcohol dehydrogenase; and pyrazole, which inhibits alcohol dehydrogenase, blocks reduction of NAD + in the presence of MAM. If given 2 h before MAM, pyrazole also prevents MAM-induced lethality (Grab and Zedeck, 1977). Rats injected intraperitoneally with [laC]azoxymethane, which is metabolized in vivo to MAM, show increased urinary excretion of [laC]MAM when pretreated with pyrazole, probably because of inhibited oxidation of MAM by alcohol dehydrogenase (Fiala et al., 1978). Similarly, pyrazole inhibited the mutagenicity of MAM acetate in Salmonella typhimurium strains TA1535 or TA100 (Kanagalingam and Andrews, 1979). In assays for the formation of methylcarbonium ions from MAM and methylnitrosourea (MNU), the addition of NAD ÷ and alcohol dehydrogenase increased their formation from MAM but not from MNU, which does not require enzymatic activation. Omitting the alcohol dehydrogenase or its cofactor, or adding pyrazole, prevented the formation of methylcarbonium ions from MAM (Feinberg and Zedeck, 1980). These results provide evidence that the oxidation of MAM to MAMAL can be catalyzed by alcohol dehydrogenase, and this reaction is likely to be important in the mechanism of action of MAM. The exact role of MAMAL as a biological intermediate is unclear. The observation that disulfiram, an inhibitor of aldehyde dehydrogenase, enhances the biological activity of MAM (Zedeck et al., 1979) is consistent with MAMAL being significant in the toxicity of MAM. Nevertheless, chemical reactions involving MAMAL and macromolecules have not been demonstrated directly. Since MAMAL can also decompose to form reactive methylcarbonium ions even more readily than MAM does, the most obvious role of the MAM-dehydrogenase reaction and MAMAL is to increase the production of these ions. The metabolism of cycasin and MAM is summarized in Fig. 1. The deglucosylation of cycasin to MAM is clearly essential for its biological effects. The biological activity of MAM can proceed either via MAMAL or through its more direct generation of methylcarbonium ions. The relative importance of these alternative pathways of MAM activity is uncertain. The involvement of diazomethane as an intermediate in the production of methylcarbonium ions, as shown in Fig. 1, is also speculative.

Detoxification of cycasin and M A M Excretion of unmetabolized cycasin seems to be the primary means by which mammals minimize its effects. Deglucosylation of cycasin by enzymes of the intestinal flora or by mammalian enzymes in neonates does, however, activate it through the release of MAM. Little information is available on detoxification of MAM in mammals, either through conjugation or through the possible oxidation of M A M A L in a reaction involving aldehyde dehydrogenase. Larvae of the arctiid moth Seirarctia echo feed on cycads without obvious ill effects. Tissue extracts of feeding larvae contain cycasin, which persists through the

27 oAcohol

Cycnsin

f3-glucosidose "N . glucose

det~'omnc~

(MAIvl) NAD~ .jNADH ~ ,I --N==N-CH2OH • ' [; HCOOH H20 FEHO

[x~ :HO

~

~

"

H

(MAMAL)/ CH3--N=N-C. i "~O ~.~-6 \ /NH2--R IHO

CH2N2 (dioz~l-I:lne) N2

\

--

Nt-~--R H20 C F ~ - - N : N - - C --H -~ I II

t

N R

(methyl corbonium ion) CH3R

Fig. i. Enzymaticand spontaneousreactionsof cyeasinand MAM. Enzymaticreactionsare designatedby horizontal arrows pointing to the right; the other reactions are spontaneous. The letter R designates cellular macromolecules,including DNA, that are subject to chemicalattack. The schemeof reactions is based on the work of Feinberg and Zedeck (1980), Miller and Miller (1965) and Zedeck et al. (1979). pupa and adult stages and even into eggs produced by the adults. Although all stages of the life cycle contain an enzyme with fl-glucosidase activity, no MAM is detectable (Teas, 1967). When larvae were fed a diet containing MAM for 24h, cycasin could be found in the hemolymph. It has been postulated that the MAM was glucosylated to cycasin, retained in the hemolymph, and excreted by malpighian tubules. Furthermore, extracts prepared from larvae that were fed Zamia leaves contained an azoxyglycoside that appeared to be cycasin even though cycasin is not the prominent azoxyglycoside in Zamia (Teas, 1967). Conversion of MAM, regardless of its origin, into cycasin appears to protect these moths from the toxic effects of the cycad metabolites.

Toxicology of cycasin and MAM

Systemic toxicology and cytotoxicity In the preparation of cycad meal, inadequate washing of cycad parts leads to incomplete removal of toxins; ingestion of the incorrectly prepared food can cause toxic effects in people (Laqueur, 1977). Symptoms of cycad toxicity range from nausea, vomiting and headache to convulsions and death (Laqueur, 1977; Whiting, 1963; Hirono, 1981). Jaundice has also been observed, suggesting hepatotoxicity (Laqueur, 1977). In addition to toxicity in people, adverse effects have been reported in livestock that have ingested cycads. In fact, significant economic losses have been incurred because of cattle and sheep grazing on cycads (Whiting, 1963). Several studies have examined the acute toxicity of cycasin and MAM acetate in experimental animals; LD~0 values for cycasin and MAM acetate are listed in Table 3. In rats exposed to cycasin, the first symptoms appear in the liver and

28 TABLE 3 ACUTE TOXICITY OF CYCASIN AND MAM ACETATE Compound

Species

Route of administration

LDs0 (mg/kg)

Reference

Cycasin

Rat Rat Mouse Mouse Mouse Hamster Rabbit Guinea pig Guinea pig Rattus exuluns

Intragastric Oral Oral ? Intragastric Intragastric Intragastric Intragastric ? Intragastric

562 270 500 1000 500 <250 ~30 < 20 1000 < 250

Rat

Intraperitoneal

90

Mouse

Intravenous

10

Hirono et al., 1972 RTECS, 1981 a Hirono, 1972 Merck Index, 1976 Hirono, 1972 Hirono, 1972 Hirono, 1972 Hirono, 1972 Merck Index, 1976 Hirono, personal communication Ganote and Rosenthal, 1968 RTECS, 1981

MAM acetate

a Registryof Toxic Effects of Chemical Substances (1981).

include loss of cytoplasmic basophilia and glycogen from cells that surround the central vein (Laqueur, 1977). Cytoplasmic eosinophilia, pyknotic nuclei, and focal cellular necrosis are evident within 48 h, and hemorrhage and fluid retention occur a few days later. Cytological effects of MAM acetate in the liver include segregation of nucleolar components with depletion of granular material, hypertrophy of smooth endoplasmic reticulum, formation of membrane whorls, decreases in numbers of ribosomes, loss of polysomes, and fat accumulation (Ganote and Rosenthal, 1968; Zedeck et al., 1970). Cycasin is also neurotoxic, particularly to young animals; ataxia and posterior paralysis have been reported in 80% of surviving animals when newborn C57B1/6 mice were given an intraperitoneal injection (500 mg/kg) of cycasin. Exposure of rats or mice to MAM acetate causes decreases in DNA synthesis (Zedeck et al., 1970), RNA synthesis (Zedeck et al., 1970), and protein synthesis (Zedeck et al., 1970; Lundeen et al., 1971), particularly in the livers of treated animals. MAM has been reported to react with protein in vivo (Nagata and Matsumoto, 1969) and to affect the activities of several enzymes (Table4) and the amounts of cellular lipids and membrane components (Recheigl, 1964; Williams, 1965, 1974). D N A synthesis is reported to be inhibited after exposure to MAM acetate in rabbit colon organ cultures (Mak and Chang, 1978), colon explants derived from humans (Mak et al., 1979), and a variety of cell cultures, including HeLa cells (Van den Berg and Ball, 1972; Bedford et al., 1974), primary rat kidney fibroblasts (Hoosen and Grasso, 1977) and murine lymphoma cells (Shinohara and Matsudaira, 1976). In HeLa cells the inhibition of D N A synthesis causes mitotic delay that becomes apparent only after the template has undergone one round of

29 replication; the mitotic delay leads to an accumulation of RNA and proteins and consequent increases in cell volume (Van den Berg and Ball, 1972). MAM and MAM acetate have been shown to methylate both DNA and RNA in vitro (Matsumoto and Higa, 1966) and in vivo (Shank and Magee, 1967; Nagata and Matsumoto, 1969). In the liver, methylation of guanine was greater in DNA than in RNA; liver RNA, however, was more susceptible to methylation than that in the kidney or small intestine (Shank and Magee, 1967). The alkylation product detected was 7-methylguanine; because the methodology that was used did not permit detection of other alkylation products (Culvenor and Jago, 1979), it is difficult to relate alkylation by MAM to its genetic effects, particularly since other sites of alkylation are regarded as more important than the N-7 position of guanine with respect to mutagenicity (Hoffmann, 1980). Methylation can also be detected after cycasin treatments, but MAM yields more alkylation product, probably because a larger proportion of the administered dose reaches the target organs (Shank and Magee, 1967).

Carcinogenicity The carcinogenicity of cycasin and cycads has been reviewed by Laqueur (1977) and by Laqueur and Spatz (1975). At least 4 species of cycads, namely Cycas circinalis, Cycas revoluta, Zamia floridana and Encephalartos hildebrandtiL have been shown to contain carcinogenic compounds (Laqueur, 1977). Neoplastic lesions of the liver, kidney, colon and lung were first reported in rats fed crude cycad meal (Laqueur et al., 1963). Subsequent studies have demonstrated the carcinogenicity of cycasin (Laqueur, 1964), MAM (Laqueur and Matsumoto, 1966; Laqueur et al., 1967; Hirono et al., 1968), and MAM acetate (Laqueur et al., 1967). From these studies it became clear that cycasin is the carcinogenic component of cycad meal and that MAM is the proximate carcinogen of cycasin (Laqueur, 1977; Laqueur and Spatz, 1975). In all strains of rats tested, cycasin and MAM induced tumors in the liver, kidneys, colon, lungs, brain and duodenum; although less frequently, tumors also occurred in the ear canal, peripheral nerves and urinary bladder. Administration of the compounds by chronic feeding generally leads to liver tumors, whereas acute exposures preferentially affect the kidneys. The latent period for tumor induction was approximately 6 months, regardless of the age of the animal when treatment was begun (Laqueur, 1977). Both cycasin and MAM have been reported to cross the placenta of rats and induce tumors in the offspring (Spatz and Laqueur, 1967; Laqueur and Spatz, 1973). Besides rats, cycasin has been found to be carcinogenic in mice (O'Gara et al., 1964; Hirono et al., 1969), hamsters (Laqueur and Spatz, 1975), guinea pigs (Laqueur and Spatz, 1975), rabbits (Hirono, 1972), fish (Aoki and Matsudaira, 1977), and nonhuman primates (Sieber et al., 1980). Metabolism is an important step in the carcinogenicity of cycasin. In adult animals, deglucosylation of cycasin is catalyzed by the enzyme fl-glucosidase of the microbial flora of the gut. Consequently, cycasin is noncarcinogenic in germfree animals (Laqueur, 1964; Laqueur and Spatz, 1975). Unlike cycasin, MAM induces tumors in both conventional and germfree animals (Laqueur and Spatz, 1975;

cycad meal; 5-day exposure

Guinea pig liver

Rat brain

Rat blood

Cholinesterase

Cholinesterase (plasma)

0.075% cycasin

MAM acetate of gestation

100 g

100 g

in chow

on day 15

0.2% cycasin in chow cycasin or cycad meal

cycad meal; 5 day exposure

Guinea pig liver

Rat liver Rat kidney

200-400

mg cycasin/

mg cycasin/

Rat liver

Catalase

Alkaline phosphatase

200-400

Rat liver

Adenosine triphosphatase

MAM acetate of gestation

on day 15

ACTIVITIES

Rat brain

Treatment

ON ENZYME

Acetylcholinesterase

AND ITS METABOLITES

Source

OF CYCASIN

Enzyme

EFFECT

TABLE 4

effect

Increase

(1.3-

1.9-fold)

Increase per *am cerebrum

Decrease No significant

Increase in centrolobular areas Increase

Decrease in centrolobular areas in some animals Increase in periportal regions Decrease in centrolobular areas

Increase per gram cerebrum

Effect on activity

et al., 1978

1964 1964

1964

1964

1964

Orgell and Laqueur,

Nagata

et al., 1978

1964

Rechcigl, Rechcigl,

Spatz,

Spatz,

Spatz,

Spatz,

Nagata

Reference

1964

Spatz, 1964

Lundeen et al., 1971 Spatz, 1964 Spatz, 1964

Decreased peribiliary granular esteras¢ activity Decrease in central and periportal zones Decrease Decrease Decrease in centrolobular region; by 10 days, decrease extended to midzonal region

0.075~ cycasin in chow

MAM acetate (5-30 mg/kg); intraperitoneal 0.025~-4~ cycasin in chow cycad meal; 5-day exposure

Rat liver

?

Rat fiver

Esterase (nonspecific)

Ethylmorphine-Ndemethylase

Glucose-6-phosphatas¢

Baxter and Byvoet, 1974 Spatz, 1964 Williams, 1964

No effect Decrease Decrease

MAM acetate; 1.0 mM 0.2-4~ cycasin in diet 5~ cycad meal

Rat liver nuclei

Rat liver

Rat liver

Histonelysine methyl transferase

5-Nucleotidase

•Succinic oxidase

Guinea pig fiver

Cox, 1980

No effect

0.32-32 pg MAM acetate per 100 #g purified enzyme

Rat liver

DNA methylase

Q u a n t i t y per plate

9 mg

u p to 0.13 m g

N o t specified

2.5 # g in D M S O

2.5 ~ g in D M S O

Compound

Cycasin

Cycasin

Cycasin

Cycasin

Cycasin p l a t e test

+ (Aroelor-induced) + (Aroclor-induced) + (Aroclor-induced)

TA1537

+ (Aroclor-induced)

--

T A 100 TA1535

TA98

-

TAi535 TA1537 Standard Ames

--

--

TA98 TAI00

+ / + / + / + / +/-

+/-

p l a t e test

p l a t e test

Standard Ames

hisG46 TA92 T A 1975 T A 1950 T A 1535 TAIO0

Standard Ames

+/(Aroclor-induced) + / -- ( A r o c l o r - i n d u c e d )

(Aroclor-induced)

TA1537 TA98

(Aroclor-induced)

+/-

+ / -

TA1535

hisD 130 hisC496 hisC527 T A 100

-

hisC207 hisCl51 hisC50

±

+ d

-

---

-

-

--

-

-

Result c

(1979)

W e h n e r et al.

W e h n e r et al. (1979)

(1979)

M a t s u s h i m a et al.

(1975)

M c C a n n et al.

S m i t h (1966)

Reference

STRAINS OF Salmonella typhimurium

p l a t e test

-

hisC ! 20

-

$9

hisG46

Strain b

IN HISTIDINE AUXOTROPHIC

Standard Ames

Spot test

M e t h o d of test a

MUTAGENICITY OF CYCASIN AND RELATED COMPOUNDS

TABLE 5

M A M acetate

125 ~ g

0.9-2.25 nag

MAM

Standard Ames plate test

test

hisG46

Standard Ames plate test

1.32, 13.2, 132/~g 1.32, 13.2, 132 lag 1.32, 13.2, 132 #g 1.32, 13.2, 132 #g

MAM

Spot

TAI00 TA98 TA1537 TA1538

test test test test

Spot SpOt Spot Spot

I, 2ms I, 2mg 1.32, 13.2, 132 #g

MAM

M A M acetate

hisG46 TA1535 TA1535

Spot test Spot test Spot test

up to 20.2/~g

Cycasin preincubated with fecalase

TA1535 TA1536 TA1537 TA1538

TA1535

hisG46

hisG46

hisG46 TA92 TAI975 TAI950 TAI535 TA100

Standard Ames plate test

0-5 mg c

hisG46 hisC50 hisDl30 hisC496 hisC527 hisC151 hisCl20 hisC207

Cycasin preincubated with #-glucosidas¢

test

Spot

1.5 m g

Deglucosylated cycasin

/ + (Arocior-induced) / + (Aroclor-induced) - - / + (Aroclor-induced) + (Aroclor-induced) --

-

+

m

m

m

m

m

m

m

D

B

m

+

+

+

+s

-¢-

+

+

+ + + + + +

q -

-4-

+ + +

f

Simmon (1979b)

M c C a n n et ai. (1975)

M a t s u s h i m a et al. (1979)

Jacobs (1977)

Jacobs (1977)

T a m u r a et al. (1980)

Matsushima et al. (1979)

Smith (1966)

0 - 4 0 lamoles

Methylazoxymethyl-fl D-glucosiduronic acid

Standard Ames p l a t e test

Macrozamin

2.5 lag in D M S O

Preincubation w i t h fecalase before plating

Standard Ames p l a t e test Preincubation w i t h E. coli fl-glueuronidase before plating

Preincubation 150 m i n w i t h fecalase before plating

Standard Ames p l a t e test 30 m i n p r e incubation before plating

S p o t test S p o t test S p o t test S p o t test Standard Ames p l a t e test S p o t test

Method of test a

Methylazoxyglucosiduronic acid

0 - 4 0 la m o l e s

0-50 nmoles

1.3-6.6 mg

1.3-6.6 mg

Neocycasin preincubated w i t h fecalase

MAM acetate

2 . 5 - 2 5 lag 2 . 5 - 2 5 lag 2 . 5 - 2 5 lag 10 lag 10 lag

MAM acetate

2 . 5 - 2 5 lag

Quantity per plate

Compound

TABLE 5 (continued)

TA98 TA100 T A 1535 TA1537

hisG46

hisG46

hisG46

hisG46

hisG46

hisG46

TA1535

TA1535 TA1538 T A 1538 TA1535 TA1535

Strain b

+ / +/+ / -+/--

-

-

-

-

-

-

(Aroclor-induced) (Aroclor-induced) (Aroclor-induced) (Aroclor-induced)

+ (uninduced)

+ (uninduced) --

$9

--

-

+

-

+

+ h

+ h

-

+ + --

Result c

W e h n e r et al. (1979)

T a m u r a et al. (1980)

M a t s u s h i m a et al. (1979)

T a m u r a et al. (1980)

M a t s u s h i m a et al. (1979)

Rosenkranz and Poirier (1979)

Reference

a b c d c f s h

Preincubation with fecalase before plating

hisG46

The standard Ames plate test is as described by Ames et al. (1975). Genotypes of strains with the prefix TA are given in Ames et al. (1973, 1975). The designation + includes .both weak and inconsistent positive responses. Questionable positive; see text. 0-5 mg cycasin was preincubated with 30 units//-glucosidase at 30°C for 90 min. Stronger response in u v r B + strains; see text. Stronger response in the presence of $9; see text. Stronger response with preincubation; see text.

Macrozamin

-

-

Tamura et al. (1980)

36 Laqueur et al., 1967). The relationship between the metabolism of cycasin and MAM and their toxicologic properties is considered in greater detail in the section of this review on metabolism.

Teratogenicity The teratogenicity of MAM was reported by Spatz et al. (1967a), who observed malformations of the brain, eyes and extremities in surviving fetuses of hamsters injected with MAM on the 8th day of gestation. The most pronounced teratogenic effects of MAM are a consequence of neurotoxicity during brain development. The neurotoxicity generally can be partitioned into prenatal and neonatal effects (Laqueur, 1977). Prenatal exposure of rats to MAM resulted in cerebral malformations, and even a single dose of MAM on the 14th or 15th day of gestation was sufficient to induce microencephaly; both cerebral hemispheres were affected, but the remainder of the brain and skull were apparently normal. Exposure of laboratory animals to MAM at birth caused necrosis of the cerebellar external granule cell layer and a reduction in the size of the cerebellum. Exposure on the 4th-6th day of life caused similar cerebeUar changes, but the anterior lobes were more affected than the posterior lobes; atrophy of the retina was also observed. The pattern of prenatally induced cerebral changes and neonatally induced cerebellar changes has been observed in several species (Laqueur, 1977). In the ferret, however, administration of MAM on the 38th day of gestation causes both cerebral and cerebellar abnormalities (Haddad et al., 1972). The appropriateness of extrapolating from one species to another with respect to particular teratologic effects of MAM therefore remains uncertain.

Genetic effects in bacteria

Induction of mutations Results from many experiments on the reversion of histidine auxotrophs of

Salmonella typhimurium by cycasin and related compounds are presented in Table 5. Because the mutagenicity of cycasin and MAM is intimately related to both microbial and mammalian metabolism, reference to the proposed metabolic pathways depicted in Fig. 1 will aid in evaluating the data in Table 5. With the exception of one study, cycasin has been found to be nonmutagenic in the Ames Salmonella/microsome assay in all strains both with and without an $9 metabolic activation system (Smith, 1966; Matsushima et al., 1979). The one positive test was in strain TA1535 in the presence of commercial $9 mix (Monsanto Chemical Company) prepared from the livers of Aroclor 1254-induced rats (Wehner et al., 1979). The positive result could be attributable to the presence of fl-glucosidase in the $9 preparation (Wehner et al., 1979), but this explanation is not supported by the fact that macrozamin was inactive in the same study. In any case, the preponderance of evidence is that cycasin itself is not mutagenic in Salmonella. In contrast to cycasin, MAM and enzymatically deglucosylated cycasin are mutagenic in the Ames test (Smith, 1966; Matsushima et al., 1979; Jacobs, 1977).

37 Cycasin incubated with fecalase (an extract of human feces) is also mutagenic (Tamura et al., 1980). These results are consistent with findings that cycasin is noncarcinogenic in germfree rats but carcinogenic in conventional rats, whereas MAM is carcinogenic in both. The mutagenicity data support the conclusion that the conversion of cycasin into a biologically active form requires the liberation of the aglycone from cycasin by fl-glucosidase. Most evidence is consistent with MAM being mutagenic in base-pair substitution strains like TA1535 in the absence of metabolic activation (Smith, 1966; McCann et al., 1975; Jacobs, 1977; Matsushima et al., 1979; Rosenkranz and Poirier, 1979). It is likely, however, that factors associated with treatment conditions, including the inherent instability of MAM, can lead to negative results in some tests (Simmon, 1979b). MAM is subject to spontaneous decomposition; therefore, the time-course of an experiment and the existence of conditions under which bacteria are exposed to high chemical concentrations are apt to be important factors in detecting mutagenicity. Accordingly, MAM is sometimes observed to be positive in a spot test, but not in the standard plate test (Rosenkranz and Poirier, 1979), or to be considerably more effective when a preincubation technique is used (Matsushima et al., 1979). Overall, the data indicate that MAM is a direct-acting base-pair substitution mutagen for which spurious negative results are not unlikely. MAM acetate was tested for mutagenicity in the Ames test as part of the International Program for the Evaluation of Short-Term Tests for Carcinogenicity (IPESTTC) (de Serres and Ashby, 1981). Of 11 laboratories that tested MAM acetate in the standard Salmonella plate test, 7 reported it to be mutagenic in base-pair substitution strains; a negative result was reported by 3 laboratories and a questionable result by 1 laboratory. The purpose of the IPESTTC was not to conduct thorough studies of the mutagenicity of particular compounds, but to evaluate test systems in current use. Because of the instability of MAM and the fact that small amounts of the chemical were provided to investigators, high doses were not always used and questionable results were not always repeated. Therefore, inconsistencies in the data are not surprising. The IPESTTC study underscores the effects of minor differences in laboratory techniques on the detection of the mutagenicity of MAM acetate in the Ames test. Results from a microtitre fluctuation test of MAM in Salmonella typhimurium were consistent with results from the standard plate test, in that MAM acetate increased the frequency of mutations in strain TA1535 without metabolic activation (Gatehouse, 1981). MAM acetate has also been shown to induce forward mutations that confer resistance to azaguanine in Salmonella typhimurium strain TM677, a his + derivative of strain TA1535 that carries the plasmid pKM101; exponentially growing cells were treated in suspension at concentrations up to 1000 # g / m l in the presence of a phenobarbital-induced $9 preparation (Skopek et al., 1981). Results on the mutagenicity of MAM acetate in E. coli WP2 trp- support the same conclusions as the Salmonella tests - - MAM induces base-pair substitutions. The mutagenicity, however, is reported to require the presence of an $9 metabolic activation system (Venitt and Crofton-Sleigh, 1981). The literature on the effect of metabolic activation on the mutagenicity of MAM

38 in base-pair substitution strains is inconsistent. In some studies, a mammalian $9 fraction appears to enhance the mutagenicity of MAM (Matsushima et al., 1979), whereas in others it abolishes the mutagenicity of MAM (Rosenkranz and Poirier, 1979; Purchase et al., 1978). MAM is probably subject to metabolic deactivation in addition to spontaneous decomposition (see Fig. 1). Therefore, it is tempting to attribute discrepancies to differences in concentrations of enzymes involved in the metabolism of MAM. In the absence of metabolic activation, MAM does not cause reversion in strains that revert by frameshift mutation (Jacobs, 1977; Simmon, 1979; Rosenkranz and Poirier, 1979). An interesting result that is somewhat difficult to put into perspective is that positive results have been obtained in frameshift tester strains in the presence of a metabolic activation system (Simmon, 1979; Purchase et al., 1978). The evidence is not adequate to conclude whether this activity is due to a lack of specificity of MAM for base-pair substitutions or whether the explanation is more complicated, perhaps involving metabolism of MAM to a frameshift mutagen. The dependence of the mutagenicity of MAM in strain TA1538 on metabolic activation (Simmon, 1979) could suggest that a metabolite such as M A M A L is a frameshift mutagen. However, inconsistencies in the literature, nonstandardized procedures, and inadequate knowledge of the metabolic intermediates formed from MAM make firm conclusions about the frameshift mutagenicity of MAM or its metabolites tenuous. There is some evidence that S. typhimurium strains hisG46, TA92, and TA1975 are more revertible by MAM than strains that carry a deletion through the uvrB gene (Jacobs, 1977; Matsushima et al., 1979). Although the relative mutability of uvrB + and u v r B - strains (Matsushima et al., 1978; Murray, 1979) is an interesting topic, the instability of MAM and the importance of treatment conditions in its mutagenicity limit one's ability to reach conclusions on the basis of quantitative differences in reversion of the Ames strains. Factors such as the time course of the experiment, the condition of the MAM solution, and relative concentrations of MAM have varied widely among experiments and cloud the interpretation of quantitative data. Although the data are not totally consistent, they are adequate to conclude that MAM is mutagenic in bacteria. MAM induces base-pair substitution mutations in Salmonella without a requirement for metabolic activation. The data on the frameshift mutagenicity of MAM and on effects of metabolic activation are inconclusive. Host-mediated assays

An $9 metabolic activation system is not the only way that mammalian metabolism can be incorporated into microbial tests; another means is the host-mediated assay (Legator and Mailing, 1971). By this technique, the test microorganism is injected into a laboratory animal and the test chemical is given to the same animal by another route of administration. Most typically, the indicator organisms are injected into the peritoneal cavity and the test chemical is given orally or by intravenous or intramuscular injection. After an appropriate time has elapsed, the indicator organisms are recovered from the animal, and mutagenicity is measured by the usual procedures for that test system; the indicator organisms have been exposed

39 not only to the chemical itself but also to some of its mammalian metabolites. Results of tests of cycasin, MAM, and M_AM acetate in the intraperitoneal host-mediated assay are summarized in Table 6. These assays indicate that cycasin is positive in strain hisG46 when the chemical is administered orally but not when it is administered parenterally (Gabridge et al., 1969). The mutagenicity of orally administered cycasin can be abolished by treatment of mice with the antibiotic ampicillin, which destroys the bacterial flora that converts cycasin to MAM in the gut (Gabridge et al., 1969). MAM and MAM acetate are positive in the host-mediated assay whether administered orally or parenterally (Simmon et al., 1979). Results from the host-mediated assay support the conclusion that the mutagenicity of cycasin depends on its metabolism to the aglycone MAM by fl-glucosidase, a source of which is the microbial flora of the gut.

Repair assays Differential killing of DNA-repair-proficient and DNA-repair-deficient strains of bacteria is frequently used as an indicator of genetic damage. In theory, chemicals that produce reparable prelethal and possibly premutagenic lesions in DNA should induce greater killing in strains that have impaired ability to repair those lesions. In contrast, chemicals like ampicillin that kill by means other than DNA damage should not cause differential killing. Differential killing has been used as an indicator of DNA damage induced by MAM acetate in tests that compare appropriate repair-proficient strains with a polA strain of Escherichia coli (Rosenkranz and Poirier, 1979; Rosenkranz et al., 1981), uvrA polA and uvrA recA lexA strains of E. coli (Green, 1981; Tweats, 1981), and a rec- strain of Bacillus subtilis (Kada, 1981). In the E. colipolA plate test, 10/~1 of M.AM acetate produced a 28.4-mm diameter zone of growth inhibition in the DNA polymerase-I-deficientpolA strain and a zone of inhibition of 18.5 mm in the polA + strain (Rosenkranz and Poirier, 1979); MAM acetate was regarded as positive in the test. In addition, MAM acetate has been tested in the polA + strain (W3110) and the polA- strain (P3478) in a liquid suspension assay and was strongly positive; the survival index (ratio of survival of polA- to polA +) was less than 0.006 in the absence of a metabolic activation system and 0.03 in the presence of $9 prepared from Aroclor 1254-induced rats (Rosenkranz et al., 1981). The data indicate that damage to DNA by MAM acetate is reparable by a system involving the poiA + gene product (DNA polymerase I). MAM acetate was tested for differential killing in E. coli strains WP2 (wild-type repair), WP67 (uvrA, po/A), and CM871 (uvrA, recA, lexA), but the results were equivocal (Green, 1981; Tweats, 1981). Although most tests were negative, a positive result for MAM acetate in the presence of rat-liver $9 was reported'for the triple mutant (Tweats, 1981). Because these results were part of a collaborative study (de Serres and Ashby, 1981) in which little MAM acetate was available for testing, fresh solutions were not made for each experiment and the results were not confirmed. These and other limitations suggest that the results with these E. coli strains should be regarded as preliminary tests for the activity of MAM acetate. MAM acetate was positive in a repair assay that uses the rec- strain M45 of

Swiss albino mice

Swiss albino mice Swiss albino mice

Swiss albino mice

Swiss-Webster mice

Swiss-Webster mice

Cycasin (0.5 ml of 2%)

Cyeasin (0.5 ml of 2%) + ampieillin Cycasin (0.5 ml of 2%)

MAM (0.5 ml of 1%)

MAM acetate (469 mg/kg)

MAM acetate (469 mg/kg)

a IP, intraperitoneal; IM, intramuscular; IV, intravenous.

Animals

Chemical

Parenteral (IM)

Parenteral (IM)

TA1538

TAI530

hisG46

hisG46

Parenteral (IP, IM, IV) Oral

hisG46

hisG46

Bacterial strain

Oral

Oral

Route of chemical exposure a

672-fold increase above spontaneous 148-fold increase above spontaneous

lO0-fold increase above spontaneous

No increase

30-fold increase above spontaneous No increase

Result

MUTAGENICITY OF CYCASIN, MAM A N D MAM ACETATE IN THE HOST-MEDIATED ASSAY

TABLE 6

Simmon et al., 1979

Simmon et al., 1979

Gabridge et al., 1969

Gabridge et al., 1969

Gabridge et al., 1969

Gabridge et al., 1969

Reference

41 Bacillus subtilis and its repair-proficient counterpart. Germinating spores were treated with MAM acetate, and greater growth inhibition was observed in the repair-defective strain than in the strain with normal repair capability. The presence of $9 preparations from the livers of rats or fish did not eliminate the differential killing (Kada, 1981). Prophage induction A variety of mutagens and carcinogens induce lyric phage in lysogenic strains of bacteria (Heinemann, 1971; Moreau et al., 1976). The induction of phage lambda in Escherichia coli is the most commonly used phage induction test (Moreau et al., 1976). MAM acetate is active in inducing plaques of phage lambda in the lysogenic strain 58161 of E. coli (Thomson, 1981). At a concentration of 20 #g/ml, the induction index (ratio of chemically induced induction to spontaneous induction) for MAM acetate was 25. Since the lambda repressor is cleaved by the SOS repair-induced recA gene product, the effect of MAM acetate probably involves damaging cellular DNA in such a way that SOS repair is induced. The results of the bacterial phage induction and repair assays are summarized in Table 7, along with the results of tests of cycasin and MAM in other test systems.

Genetic effects in yeast Induction of mutations MAM acetate has been reported to be mutagenic in strain XV185-14C of Saccharomyces cerevisiae (Mehta and von Borstel, 1981). This strain detects reversions at several loci by back mutation, as well as by second site mutations; the mutagenicity did not require metabolic activation. Induction of mitotic recombination MAM acetate induces mitotic crossing-over and mitotic gene conversion in Saccharomyces cerevisiae. Mitotic crossing-over was detected at the ade2 locus in strain D3; the recombinogenicity was enhanced by the addition of a metabolic activation system prepared from the livers of Aroclor 1254-treated rats (Simmon, 1979a). MAM acetate was also reported to induce mitotic crossing-over at the ade2 locus of log-phase cells of S. cerevisiae strain T2 (Kassinova et al., 1981), but the effect was detected only in the absence of $9. It is unclear why $9 abolished the recombinogenicity of MAM acetate in this study. MAM acetate is reported to induce mitotic gene conversion at the trp5 locus in strain D7 of S. cerevisiae without metabolic activation (Zimmermann and Scheel, 1981). Increasing the period of preincubation of MAM acetate with growing cells enhanced the recombinogenicity (Zimmermann and Scheel, 1981).

Chlorophyll and m o r p h o l o g i c a l alterations R o o t -t i p cytogenetics

R o o t tip cytogenetics

Sex-finked recessive lethats

DNA strand breakage

Reversion o f h i s t i d i n e auxotrophs R e v e r s i o n of h i s t i d i n e auxotrophs Reversion of h i s t i d i n e auxotrophs Reversion of h i s t i d i n e auxotrophs Reversion o f h i s t i d i n e auxotrophs Reversion o f h i s t i d i n e auxotrophs

Salmonella typhimurium

Salmonella typhimurium

Salmonella typhimurium

Salmonella typhimurium

Phaseolus vulgari$

Allium cepa

Zamia integrifolia

Drosophila melanoSaster

Rat, several tissues

Salmonella typhimurium

Salmonella typhimurium

Salmonella typhimurium

Salmonella typhimurium

Salmonella typhimurium

Cycasin

Cycasin

Cycasin

Cycasin

Cycasin

Cycasin

Cycasin

Cycasin

Cycasin

Maerozamin

Macrozamin

Macrozamin

Maerozamin

D e g l u c o sy la t e d cycasin D e g l u c o s y la t e d cycasin

Salmonella typhimurium AnY~tr~h~

Reversion o f h i s t i d i n e

Reversion o f h i s t i d i n e auxotrophs Reversion o f h i s t i d i n e auxotrophs Reversion of h i s t i d i n e auxotrophs

Salmonella typhimurium

Cycasin

MAM

Reversion of h i s t i d i n e auxotrophs Reversion o f h i s t i d i n e auxotrophs Reversion o f h i s t i d i n e auxotrophs

Salmonella typhimurium

Cycasin

Salmonella typhimurium

Test s y s t e m

O r g a n i s m o r cell type

$9

$9

N.A.

Host-media t e d assay Host-mediated assay

$9

$9

N.A.

N.A.

N.A.

N.A.

N.A.

N.A.

N.A.

Adult feeding Oral

N.A.

N.A.

N.A.

IP. IM, IV

Oral

N.A.

N.A.

N.A.

N.A.

Route of exposure a

IN GENETIC TOXICOLOGY

Metabolic activation system

OF THE EFFECTS OF CYCASIN, MAM AND RELATED COMPOUNDS

Compound

SUMMARY

TABLE 7

Base-pair s u b s t i t u t i o n s

Frameshift mutations

Base-pair s u b s t i t u t i o n s

Frameshift mutations

Base-pair s u b s t i t u t i o n s

Frameshift mutations

Basa-pair s u b s t i t u t i o n s

Single s t r a n d b r e a k s

Mutations

Chromosome aberrations

Chromosome aberrations

Putative m u t a t i o n s

Base-pair s u b s t i t u t i o n s

Base-pair s u b s t i t u t i o n s

Frameshift mutations

Base-pair s u b s t i t u t i o n s

Frameshift mutations

Base-pair s u b s t i t u t i o n s

End point measured

TESTS

+

--

+

-

-

-

--

+

-

+

4-

+

--

+

--

--

--

--

Result b

See T a b l e 5

See T a b l e 5

See T a b l e 5

See T a b l e 5

See T a b l e 5

See T a b l e 5

See T a b l e 5

Teas and Dyson, 1967 P a r o d i et al., 1978

T e a s et al., 1965; Porter & Tess, 1971 Porter a n d Teas, 1971

M o h , 1970

See T a b l e 6

See T a b l e 6

See T a b l e 5

See T a b l e 5

See T a b l e 5

See T a b l e 5

Reference

$9

Reversion o f t r y p t o p h a n auxotrophs

p o l l r e p a i r assay

rec r e p a i r a s s a y rec r e p a i r a s s a y I n d u c t i o n o f ~ lysogen

Reversion o f a u x o t r o p h y

Reoombinogenicity in strain D3 R e e o m b i n o g e n i c i t y in strain D3

R e c o m b i n o g e n i c i t y in strain T2

Salmonella typhimurium

Salmonella typhimurium

Salmonella typhimurium

Salmonella typhimurium

E$cherichia call W P 2

E$cherichia cali

Escherichia coli

Escherichia coli

Escherichia coil

Bacillus subtili$ Bacillas $ubtili$ Escherichia coil ( ~ )

Saccharomycer cere~isiae

Saccharomyce$ cerevi$iae

Saccharomyces cerevisiae

Saccharomycer cerevisiae

MAM

M A M acetate

M A M acetate

M A M acetate

M A M acetate

M A M acetate

M A M acetate

M A M acetate

M A M acetate M A M acetate MAM acetate

M A M acetate

M A M acetate

M A M acetate

M A M acetate

acetate

A z a g u a n i n e - r e s i s t anee

Salmonella typhimurium

M A M acetate

MAM

$9

Reversion of histidine auxotrophs Reversion o f h l s t i d l n e auxotrophs Reversion o f h i s t i d i n e auxotrophs

Salmonella typhimurium

M A M acetate

-

$9

-

-

-$9 -

N.A.

N.A.

N.A.

N.A.

N.A. N.A. N.A.

N.A.

N.A.

uorA p o l l and uvrA recA lexA r e p a i r assays u~rA recA lexA repair $9 assay

N.A.

N.A.

N .A.

IM

IM

Oral

N.A.

N.A.

N.A.

N.A.

-

Host-mediated assay Host-mediated assay Host-mediated assay

$9

-

-

$9

p o l l r e p a i r assay

Reversion of histidine auxotrophs R e v e r s i o n of h i s t i d i n e auxotrophs Reversion o f h i s t i d i n e auxotrophs

Salmonella typhimurium

M A M acetate

N.A.

$9

Salmonella typhimurium

N.A.

-

MAM

Reversion o f h i s t i d i n e auxotrophs Reversion of histidine auxotrophs

Salmonella typhimurium

MAM

See T a b l e 5 See T a b l e 5

Base-pair s u b s t i t u t i o n s Frameshift mutation

See T a b l e 6

Frameshift mutations

S i m m o n , 1979a

M i t o t i c crossing-over M i t o t i c crossing-over

K a s s i n o v a et aL, 1981

Mehta and von Borstel, 1981 S i m m o n , 1979a Mutation

M i t o t i c crossing-over

K a d a , 1981 K a d a , 1981 T h o m s o n , 1981

R o s e n k r a n z et al., 1981 G r e e n , 1981, Tweats, 1981 T w e a t s , 1981

Differext tial killing D i f f e r e n t i a l killing Pha ge i n d u c t i o n

D i f f e r e n t i a l killin 8

D i f f e r e n t i a l killing

D i f f e r e n t i a l killing

Differential killing

Base-pair s u b s t i t u t i o n s

S k o p e k et al., 1981 Venitt and Crofton-Sleigh, 1981 Rosenkranz and Poirier, 1979; R o s e n k r a n z et al., 1981

See T a b l e 6

Base-pair substitutions

Mutations

See T a b l e 6

Base-pair s u b s t i t u t i o n s

See T a b l e 5

See T a b l e 5

Base-palr substitutions

Frameshift mutation

See T a b l e 5

Frameshift mutations

Sex-linked recessive lethais

Drosophila melanogaster

Drosophilamelanogaster

Chinese hamster V79 cells

Chinese hamster V79 cells Chinese hamster V79 cells Chinese hamster fibroblasts

Chinese cells Chinese cells Chinese ovary

Chinese hamster ovary cells H u m a n leukocytes

H e L a cells

H u m a n skin cultures

R a t hepatocyte cultures

H e L a ceils

MAM

M A M acetate

M A M acetate

M A M acetate

M A M acetate

M A M acetate

M A M acetate

M A M acetate

M A M acetate

M A M acetate

M A M acetate

M A M acetate

M A M acetate

M A M acetate

hamster cells

hamster D O N

hamster D O N

Sex-linked r e c ~ s i v e lethals

Saccharomyces cerevisiae

M A M acetate

M A M acetate

Recombinogenicity in strain 1"2 Recombinogenicity in strain D 7

Saccharomyces cereoisiae

M A M acetate

Unscheduled D N A synthesis Unscheduled D N A synthesis Unscheduled D N A synthesis

D N A strand b r e a k a g e

Sister-chromatid exchange

Sister-chromatid exchange

Sister-chromatid exchange

Sister-chromatid exchange

C y t o g e n e t i c analysis

Cytogenetic analysis

6-Thioguanine-resistance

6-Thioguanine-resistance

Ouabain-resistance

Test system

Organism or cell type

Compound

T A B L E 7 (continued)

--

-

-

-

-

$9

-

-

-

-

$9

$9

Metabolic activation system

N.A.

N.A.

N.A.

N.A.

N.A.

N.A.

N.A.

N.A.

N.A.

N.A.

N.A.

N.A.

N.A.

feeding

Adult feeding Adult

N.A.

N.A.

Route of exposure a

D N A repair

D N A repair

D N A repair

Single strand breaks

SCE

SCE

SCE

SCE

C h r o m o s o m e breaks

C h r o m o s o m e aberrations

Mutations

Mutations

Mutations

Mutations

Mutations

M i t o t i c gene conversion

M i t o t i c crossing-over

End point measured

+

+

+

-

+

+

+

+

+

+

+

+

+

+

+

+

-

Result b

M a r t i n and M c D e r m i d , 1981

Lake et al,, 1978 Williams, 1976

Perry a n d T h o m s o n , 1981 Evans et al., 1977 Van den Berg, 1974

A b e a n d Sasaki 1977 A b e a n d Sasaki, 1977 Perry and T h o m s o n , 1981

K n a a p et aL, 1981 K n a a p et al., 1981 l s h i d a t e and O d a s h i m a , 1977

1978

Lankas, 1979: L a n k a s et al.,

al., 1981

1967: Vogel et

Teas a n d Dyson, 1967 Teas a n d Dyson,

Kassinova et al., 1981 Zimmermann, 1981

Reference

H u m a n fibroblasts

Syrian h a m s t e r fibroblasts

R a t liver

Rat, several tissues

R a t , several tissues Rat

Mouse

M A M acetate

M A M acetate

M A M acetate

M A M acetate

MAM M A M acetate

M A M acetate

Morphological s p e r m abnormalities

D N A strand b r e a k a s e Cytogenetics-liver cells

D N A strand b r e a k a s e

-

--

-

--

-

D N A strand b r e a k a s e

-

synthesis Unscheduled D N A synthesis

$9

Unscheduled D N A

Unscheduled D N A synthesis

IP

IP IV

IP

IP

N.A.

N.A.

N.A.

Morphological sperm abnormalities

Single s t r a n d breaks C h r o m o s o m e aberrations

Single strand breaks

Single strand breaks

D N A repair

D N A repair

D N A repair

-

+ +

+

+

-

-

--

T o p h a m , 1980

Cox, 1975 Z e d e c k et aL, 1974

Kanagalingam & Balls, 1975

D a m j a n o v et al., 1973;

Stich, 1976

Stich, 1976

Martin and M c D a r m i d , 1981

• N.A., not applicable; IP, intraperitoneal injection: IM, intramuscular injection; IV, intravenous injection. b T h e results for tests in Salmonella typhimueium are those that we consider to represent the bulk of evidence in multiple tests. Results of individual studies are recorded in T a b l e 5 for in vitro tests a n d T a b l e 6 for host-mediated assays. All other entries in this t a b l e represent the results of the individual studies cited. T h e symbol ± designates inconclusive results.

H e L a cells

M A M acetate

46

Genetic effects in vascular plants Cycasin is reported to induce chlorophyll mutations and morphological alterations in the bean Phaseolus vulgaris (Moh, 1970). Dry seeds were treated with cycasin for 24 h; plants grown from the seeds were scored for chlorophyll mutations and alterations in morphology or growth pattern, which may have genetic origin. The percentage of cycasin-treated plants with alterations was 46-62%, and no alterations were observed in the controls. Seeds of Phaseolus vulgaris have fl-glucosidase activity capable of releasing MAM from cycasin. Although evidence of the heritability of the alterations was not presented, the results are consistent with the induction of genetic damage by the aglycone of cycasin; more information on the nature of the alterations would be required, however, for firm conclusions to be reached. Chromosome aberrations, including deletions, chromatid bridges, and chromosome bridges, are reported to occur at elevated frequencies in root tip cells of Allium cepa treated with cycasin (Teas et al., 1965). Cycasin (0.5 m g / m l ) was poured over layers of filter paper and onion seeds were sown on the paper. Frequencies of aberrations in the controls were 3 and 4 per 100 cells when the roots were 0.7 cm and 1.5 cm in length, respectively. The corresponding frequencies of aberrations in onions grown in cycasin were 10 and 33 per 100 cells. It was postulated that the induction of aberrations may be attributable to hydrolysis of cycasin by Allium cepa fl-glucosidase; MAM itself was not tested. Cycasin has been shown to induce chromosome aberrations in the roots of Zamia integrifolia. This result is interesting because tissues of Zamia contain azoxyglycosides and have fl-glycosidase activity (Porter and Teas, 1971). The clastogenicity of cycasin has been tested simultaneously in Allium and Zamia in an attempt to uncover a mechanism that could protect Zamia from its own metabolite (Porter and Teas, 1971). Although the effect of cycasin in Allium may be somewhat greater than in Zamia, the reported difference is small. Uncontrolled factors, including differences in the mitotic cycles of the two plants and differences in root morphology that can affect chemical penetration, also complicate the comparison between species. Although a cellular compartmentalization that separates cycasin from fl-glucosidase has been proposed as a means of protection of Zamia from its own metabolite (Porter and Teas, 1971), the evidence in support of this hypothesis is weak.

Genetic effects in Drosophila The ability of cycasin and its aglycone to induce sex-linked recessive lethals in Drosophila melanogaster was evaluated in the Muller-5 test (Teas and Dyson, 1967; Wtirgler et al., 1975). Cycasin, MAM or MAM acetate was added to the food of adult flies. Males were mated 2 - 4 days to exhaust mature sperm; then mated to Muller-5 virgin females. Thus, genetic effects in spermatids and spermatocytes were tested. Flies ingesting 0.76-9.3 #g cycasin did not have a higher frequency of

47 sex-linked recessive lethals than did control flies, the frequencies being 0.37 and 0.36% respectively. The frequency of sex-linked recessive lethals in flies that ingested 0.09-0.17/tg MAM was 4.4% and that in flies that ingested 0.047-0.11 #g MAM acetate was 2.16%. Homogenates of Drosophila melanogaster and of yeasts, which are present in fly food, contained no detectable fl-glucosidase activity (Teas and Dyson, 1967), explaining the negative result with cycasin. The effect of MAM acetate on the frequency of sex-linked recessive lethals was also evaluated by Vogel et al. (1981) in wild-type flies and in a repair-deficient strain. MAM acetate was a strong mutagen in both strains. The results support the conclusion that the aglycone MAM induces germ-cell mutations in Drosophila, whereas cycasin itself does not.

Genetic effects in mammalian cells in culture

Induction of gene mutations MAM acetate increases the frequency of ouabain-resistant mutants in Chinese hamster V79 cells (Lankas, 1979). Lankas (1979) reported that the frequency of mutants increased exponentially with the duration of exposure to 48 # g / m l MAM. When plates were scored one week after exposure to MAM, control cultures had a mutation frequency of less than 10 per 10 6 survivors, whereas cultures exposed to MAM for 22 h had a mutation frequency of about 1000 per 106 survivors. Lankas (1979) proposed that the complex kinetics of induction may be ascribable to a combination of increased damage and saturation of repair systems at high doses. MAM acetate has also been reported to increase the frequency of ouabain-resistant mutants in Syrian hamster embryo cells (Jonmaire, 1978). The induction by MAM acetate of ouabain-resistant mutants in V79 cells is reported to be enhanced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and to a lesser extent by phorbol, although the cytotoxicity of MAM acetate was not affected by the addition of phorbol esters (Lankas et al., 1977; Lankas et al., 1980). When cells were exposed to 24/~g/ml MAM acetate for 42 h prior to the addition of 1 # g / m l TPA or 0.59 # g / m l phorbol, mutation frequencies per 10 6 survivors were 261 with MAM acetate alone, 3.2 with TPA alone, 3.5 with phorbol alone, 439 with MAM acetate plus TPA, and 390 with MAM acetate plus phorbol; plates were scored after 10 days incubation in the presence of promoter. Similarly, a variety of alkane tumor-promoting agents such as n-decane, n-dodecane and n-tetradecane have been reported to enhance mutagenesis of Chinese hamster V79 cells to ouabain resistance (Lankas et al., 1978). However, the complexity of interactions between tumor promoters and in vitro mutation assays (Kinsella, 1981; Thompson et al., 1980) cautions against reaching simple conclusions regarding the effects of promoters on frequencies of mutants induced by MAM acetate in cell cultures, particularly when the reported effects are small. MAM acetate has also been reported to induce mutations to 6-thioguanine (6-TG) resistance in V79 cells (Knaap et al., 1981). The induction of 6-TG resistance was more pronounced in the presence of a rat-hver metabolic activation system than in its absence. Interpreting the effect of $9 on MAM-induced mutagenesis or mutant

48 recovery is not straightforward, however, because $9 appeared to reduce the survival of the cells at the higher concentrations of M A M acetate. The studies of ouabain resistance and 6-TG resistance are consistent in supporting the conclusion that M A M acetate is a mutagen in cultured Chinese hamster cells without metabolic activation. The data are not sufficient, however, to reach conclusions about the kinetics of induction or about how $9 mixtures affect the mutagenicity. The induction of ouabain-resistant mutants supports the conclusion from bacterial tests that M A M acetate induces base-pair substitution mutations. Ouabain resistance is conferred by an alteration in the specificity of the membrane ATPase involved in the transport of sodium and potassium ions (Barrett et al., 1978). In this system, mutations that lead to an appropriate minor alteration of the gene product are detected, but mutations that eliminate or fully inactivate the gene product are lethal. The system is therefore expected to be effective for the detection of base-pair substitutions but not of frameshift mutations (Arlett et al., 1975). In contrast, resistance to 6-TG is conferred by forward mutations in the gene that encodes the enzyme hypoxanthine-guanine phosphoribosyltransferase ( H G P R T ) ; any mutation that causes the elimination or pronounced reduction of H G P R T activity should confer the mutant phenotype.

Induction of chromosome aberrations M A M acetate induces chromosome aberrations in Chinese hamster fibroblasts (Ishidate and Odashima, 1977). M A M acetate (9.5 × 10 -4 M) dissolved in saline was added to 3-day-old cultures; after a 48-h incubation, cells were treated with colcemid for 2h, trypsinized, and fixed. Of 100 metaphase cells examined, 23% had chromosome aberrations, including chromatid or chromosome breaks, translocations, and achromatic lesions. In the controls, 2% of the cells had chromosome aberrations. N o data were presented on the distribution of anomalies or the average number of aberrations per cell, but an incidence of aberrant cells greater than 20% was considered (Ishidate and Odashima, 1977) to indicate a strong clastogenic effect. A dose-dependent increase in the number of chromosome breaks per cell is reported in the pseudodiploid D O N Chinese hamster cell line (Abe and Sasaki, 1977). M A M acetate was dissolved in saline and added to cultures three hours after seeding of cells. After incubation for 26 h, cells were treated with colchicine for 2 h, collected, and fixed. Frequencies of chromosome breaks per cell were 0.06 in the control and 0.09, 0.18 and 1.68 at M A M acetate doses of 10-6, 10 -5 and 10 -4 M, respectively. All media used in this study contained 5-bromodeoxyuridine (BrdUrd); and although an effect of BrdUrd on the clastogenic activity of M A M acetate cannot be precluded, there is no particular reason to suspect one.

Induction of sister-chromatid exchanges The induction of sister-chromatid exchanges (SCE) by M A M acetate is reported from experiments in cultured Chinese hamster cells and human leukocytes (Abe and Sasaki, 1977; Evans et al., 1977). M A M acetate dissolved in saline was added to cultures of Chinese hamster D O N cells 3 h after the seeding of cells. The cells were

49 then incubated in the presence of the chemical for 26 h before colchicine treatment, harvesting, and fixation. Untreated cells had an average of 7.67 SCE per cell; cells exposed to MAM acetate at concentrations of 10 -6, 10 -5 and 10-4M had 8.36, 12.87 and 51.8 SCE per cell, respectively (Abe and Sasaki, 1977). MAM acetate is also reported to induce SCE in Chinese hamster ovary cells, both in the presence and in the absence of $9 prepared from livers of Aroclor 1254-treated rats (Perry and Thomson, 1981). MAM acetate increased the frequency of SCE in human short-term leukocyte cultures. The mean frequency of SCE in 9 control cultures was 5.32 per cell, with a range of 2.93-6.09 per cell. The mean frequency of SCE in treated cultures was 10.73, the range being 6.08-14.84 SCE per cell (Evans et al., 1977).

DNA strand breakage and unscheduled DNA synthesis No single-strand breaks, measured by alkaline sucrose gradient centrifugation, were detected in the DNA of HeLa cells treated with MAM acetate at a dose of 500 mg/ml (3.8 mM) for 1 h. In contrast, methyl methanesulfonate, at a comparably cytotoxic dose, did cause breaks in template DNA (Van den Berg, 1974). However, DNA synthesis on the MAM acetate-treated template was abnormal, with the defect apparently being in the ligation of newly synthesized DNA fragments. The occurrence of DNA repair is commonly used as an indication of chemically induced DNA damage (San and Stich, 1975). Repair synthesis associated with excision can be detected as unscheduled DNA synthesis (UDS) in cells in which replicative DNA synthesis has been inhibited. UDS, measured by the incorporation of tritiated thymidine (3H-TdR) into acid precipitable material, is induced in primary human skin cultures by MAM acetate (Lake et al., 1978). When scheduled (i.e., replicative) DNA synthesis was inhibited with hydroxyurea, cultures treated with MAM acetate at concentrations between 10 and 400 # g / m l incorporated up to 2.5 times more radioactivity than did control cultures. The criticism has been raised that the measurement of amounts of radioactive material incorporated may not be as effective an indicator of UDS as the autoradiographic detection of 3H-TdR incorporation into cell nuclei. Residual replicative DNA synthesis could confuse the results by the former method, but would be readily detected as heavily labeled nuclei in autoradiography. Such replicative nuclei could be excluded from the results. In the case of MAM acetate, positive results are reported in the UDS test by both methods of detection. Williams (1976) has shown that MAM acetate induces UDS, detected autoradiographically, in primary rat hepatocyte cultures. MAM acetate has also been found to induce UDS in HeLa cells; its activity was eliminated by the presence of a rat-liver $9 metabolic activation system (Martin and McDermid, 1981). Although one negative result for the induction of UDS by MAM acetate has been reported (Stich et al., 1976), the bulk of evidence indicates that MAM acetate induces UDS in several cell systems.

50 Genetic effects in mammals

Induction of DNA strand breakage MAM acetate is reported to induce single-strand breaks in liver DNA of rats treated with the chemical by intraperitoneal injection (Damjanov et al., 1973). The sedimentation of DNA toward the top of alkaline sucrose gradients was dose-dependent over the dosage range from 1 to 25 mg/kg. No liver necrosis was observed at these doses. By 24 h after injection, DNA sedimented more toward the bottom of the gradient; however, even 14 days after treatment, gradient profiles did not mirror control profiles. Thus, although MAM acetate-induced damage is reparable, repair is incomplete or very slow. An association between slow or incomplete repair and hepatocarcinogenesis has been suggested (Sarma et al., 1973). Sedimentation on alkaline sucrose gradients was also used to detect single-strand breaks in DNA from surface, mid-villus, and crypt cells of the intestine of male C F N rats that were treated with 35 m g / k g MAM acetate by intraperitoneal injection (Kanagalingam and Balis, 1975). D N A isolated 2 4 - 4 0 h after treatment sedimented more rapidly, indicating that damage was being repaired. Repair occurred more slowly in the surface cells than in crypt cells, although damage was greater in the surface cells. MAM acetate was more damaging to D N A of the colon than to DNA of the jejunum. In a study that used similar methodology, it was found that MAM (25 m g / k g ) induced strand breakage in D N A in the liver, and to a lesser extent in the lungs and kidneys, of rats (Cox, 1975). MAM acetate treatment of immunocompetent bone marrow and spleen cells resulted in D N A fragmentation, determined by alkaline sucrose gradient sedimentation; maximum damage was detected 6 h after treatment and persisted for more than 12 h (Preston et al., 1978). Like MAM, orally administered cycasin induces strand breakage in rat DNA; breakage was detected 4 h after exposure of male Wistar rats to cyca~in (Parodi et al., 1978). At dosages in the ranges of 56.2-178 m g / k g and 178-562 mg/kg, the initial rates of alkaline elution of D N A from liver nuclei were increased 15-30% and 30-60%, respectively. The increase was smaller for kidney and colon nuclei and was not significant for lung; liver therefore appears to be more susceptible to strand breakage by MAM than does lung, colon, or kidney. In C57B1/6 mice, cycasin treatment led to dose-dependent D N A fragmentation only in the liver. In both rats and mice, repair of liver D N A damage was still incomplete 18 h after treatment (Cavanna et al., 1979). Induction of chromosome aberrations Intravenous administration of MAM acetate (35 mg/kg) induced mitotic abnormalities in the livers of rats (Zedeck et al., 1974). Upon examination of 5000-8000 nuclei per liver, 3 of 10 treated rats had occasional abnormalities 96 h after treatment, while no control rats exhibited such abnormalities. By 7 days after treatment, 9 of 11 treated rats had abnormal mitoses. The average percentage of abnormal mitoses in the nine rats was 10.6%. Of 10 saline-treated control rats, none exhibited mitotic abnormalities. The abnormalities detected in MAM-treated animals

51 consisted of chromosomal bridges and occasional acentric fragments (Zedeck et al., 1974; Zedeck and Sternberg, 1975).

Induction of morphological sperm abnormalities MAM acetate failed to induce morphological sperm abnormalities in mice (Topham, 1980). F 1 hybrid males from a cross of CBA males and BALB/c females were given daily intraperitoneal injections for 5 days at individual doses up to 25 mg/kg. Negative results in this assay may reflect an inability of MAM acetate to reach the testes (Topham, 1980). It would be premature, however, to conclude that MAM acetate is not a mammalian germ-cell mutagen on the basis of this isolated test; this is particularly true because no data are available from any mammalian test for well-defined genetic effects in germ cells, and MAM acetate does induce mutations in germ cells of Drosophila melanogaster.

Summary Cycasin is one of several azoxyglycosides that are produced by plants of the cycad family. The toxicologic properties of cycasin, including mutagenicity and carcinogenicity, are caused by its aglycone derivative MAM. The biological effects of other azoxyglycosides from cycads also seem to be attributable to the liberation of MAM by cleavage of the sugar moiety from the parent compounds. Cycasin can therefore serve as a model compound for all cycad azoxyglycosides, because MAM seems to be the active component in all of them. The distribution of enzymes that release MAM from cycasin explains some of the variation in results obtained in genetic and toxicologic studies. Specifically, the glycosidic linkage in cycasin is not cleaved in the tissues of mature mammals but is cleaved by the enzymatic activities of the microbial flora of the mammalian gut. Consequently, cycasin is carcinogenic when administered orally but not when given parenterally. Unlike mature mammals, newborn animals do have the capability to cleave the glycosidic linkage in cycasin independently of microbial enzymes. Cycasin is typically found to be nonmutagenic in tests that are conducted in vitro, except when the compound is preincubated with an enzyme preparation that cleaves its glycosidic linkage; the standard rat liver $9 metabolic activation system does not have this metabolic capability. In the host-mediated assay, cycasin is mutagenic in microbial tests when the chemical is given to the host animals orally but not when given by other routes of exposure. This result is consistent with the requirement for activation by enzymes of the microbial flora of the gut. Cycasin is similarly found to be active in other tests in which it is deglucosylated. Results on the genetic effects of cycasin and MAM are summarized in Tables 5, 6, and 7 of this review. Unlike cycasin, MAM and its commercially prepared form M_AM acetate are mutagenic in a variety of genetic toxicology tests. Occasional negative results are also obtained, however, probably for technical reasons. The data indicate that MAM induces base-pair substitution mutations. The scientific literature is inconsistent on the induction of frameshift mutations by MAM. In addition

52 to point mutations, M A M has been shown to induce mitotic recombination, sisterchromatid exchanges and c h r o m o s o m e aberrations in several test systems. Although M A M is mutagenic in m a n y systems, there is still considerable uncertainty regarding the details of its mechanisms of action. The mutagenicity of M A M does not require exogenous metabolic activation, but a variety of effects of metabolic activation systems on the genetic activity of M A M have been reported. The lack of a requirement for a metabolic activation system does not necessarily mean that M A M reacts with D N A directly. Reactions that are likely to be important in explaining the mutagenicity of M A M are summarized in Fig. 1 of this review. Most evidence is consistent with M A M giving rise to m e t h y l c a r b o n i u m ions that methylate D N A . In addition to their illustrating interesting interrelationships between metabolism and toxicity, cycasin and M A M are of interest as environmental mutagens. C y c a d products are still used as food by people in some parts of the world. Although the use of cycad foods has often been associated with the development of methods for removing toxic c o m p o u n d s in preparation of the food, the occurrence of significant h u m a n exposures to azoxyglycosides cannot be excluded. In fact, effects of h u m a n exposures to azoxyglycosides have been reported, as have economic losses due to c o n s u m p t i o n of cycads by livestock. Nevertheless, data on the extent of h u m a n exposure and its consequences are quite limited.

Acknowledgements We thank Mr. John W a s s o m and his staff at the Environmental M u t a g e n I n f o r m a t i o n Center, O a k Ridge, TN, for computerized literature searches; Dr. K.R. L a n g d o n for information on the economic b o t a n y of cycads; and Dr. I. H i r o n o for unpublished toxicology data. We also thank Drs. J. Gentile and M.D. Shelby for reviewing the manuscript. The capable secretarial work of Mrs. Shirley F o n g Ash and Mrs. L i n d a H o f f m a n n is gratefully acknowledged.

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