Mycotoxins and Other Fungal Related Food Problems

15 The Structure and Toxicity of the

Downloaded by UNIV OF TENNESSEE KNOXVILLE on December 21, 2014 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/ba-1976-0149.ch015

Alternaria Metabolites D. J. HARVAN and R. W. PERO

1

National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, N. C. 27709 The Alternaria are a common field fungi responsible for a variety of plant diseases including tobacco brown spot, tomato blight, and citrus seedling chlorosis. They have been implicated in diseases of poultry and have been demonstrated lethal to mammals. The metabolites of the Alternaria represent several structural classes: dibenzo-pyrones, anthraquinones, tetramic acids, and polypeptides. The metabolites are discussed with regard to: structure; mammalian, plant, and cytotoxicity; methods of analysis; synthesis, and biosynthetic pathways. The Alternaria are common plant pathogens which infest a wide variety of food crops. Grain crops, hay, and silage are often contaminated with this fungus, generally as a field infection (1,2). Black spot of Jap anese pear (3), tobacco brown spot (4), early blight of tomato and po tato, and citrus seedling chlorosis are all caused by Alternaria spp. (5). The toxicity of the Alternaria has been well established. Grains which had been infected with A. humicola and A. alternata (Fries) Keissler (A. tenuis, Auct. and A. longipes) (6) were believed the source of several out breaks of moldy grain toxicosis in man in the U.S.S.R. during World War II (7). Of the Alternaria isolates tested from a variety of food crops, 90% were lethal to rats when fed in a corn-rice mixture (2). Doupnik and Sobers (8) reported that 33% of the isolates tested were lethal to chicks. Slifkin and Spalding (9) found that A. mali was toxic to HeLa cells and mice in feeding studies. The Alternaria have been implicated as toxins to geese and other poultry (10, 11). Of 212 Alternaria isolates from tobacco, 60% were lethal to mice following intraperitoneal injection (12). Several workers attempted to establish a correlation between pathogenicity 1

Current address: University of Lund, Lund, Sweden. 344

In Mycotoxins and Other Fungal Related Food Problems; Rodricks, J.; Advances in Chemistry; American Chemical Society: Washington, DC, 1976.


15.

HARVAN AND PERO

ALTENUENE

Alternaria Metabolites

DEHYDRQAL7ENUSIN

ALTERSOLANOL A

Figure 1.

ALTENUSIN

345

AL7ENUIC ACID II

ALTERSOUWL Β

Structures of the metabolites of Alternaria spp.

to t o b a c c o leaves a n d t o x i c i t y o f t h e isolates ( 1 3 ) . I n these studies 7 4 % of t h e p a t h o g e n i c isolates w e r e t o x i c to c h i c k s , a n d 7 5 % o f t h e n o n p a t h o g e n i c isolates w e r e n o n t o x i c .

T h e Alternaria

possess a w i d e r a n g e o f

a n t i b i o t i c a c t i v i t y . O f 127 isolates 86 w e r e a c t i v e against e i t h e r b a c t e r i a , yeast, o r m o l d s ( 1 4 ) .

Structure of the Metabolites T h e s t r u c t u r e s o f t h e k n o w n Alternaria

metabolites are presented i n

F i g u r e s 1 a n d 2. T h e m o s t c o m m o n l y o c c u r r i n g class o f c o m p o u n d s a r e the dibenzo-pyrones a n d their derivatives: alternariol, alternariol m e t h y l ether, a l t e n u i s o l , a l t e r t e n u o l , a l t e n u e n e , d e h y d r o a l t e n u s i n , a l t e n u s i n , a n d the altenuic acids. A l t e r n a r i o l a n d t h e m e t h y l ether a r e p r o d u c e d b y m o s t A . isolates as w e l l as m a n y o t h e r Alternaria talline compounds.

alternata

species; b o t h a r e colorless, c r y s

A l t e r n a r i o l has a m e l t i n g p o i n t o f 3 5 0 ° C ( d e c ) ; t h e

m e t h y l e t h e r melts at 2 6 7 ° C . B e c a u s e o f t h e i r p h e n o l i c n a t u r e , t h e y e x h i b i t



346

MYCOTOXINS

AL7ER7TJXIN I

0

^

PHYTQftLŒRNARIN A

AL7ERT0XIN II

(qfyfo

PHY7OAL7ERNARIN Β

0 ^

PHYTOALTERNARIN C

BRASSICICOLINA

Figure 2.

Structures of the metabolites of Alternaria spp.

intense p u r p l e f e r r i c reactions a n d fluoresce b r i g h t b l u e u n d e r u v i r r a d i a tion. T h e y are p r o d u c e d i n rather large quantities accounting for u p to 1 3 % o f t h e d r y m y c e l i a l w e i g h t o f some isolates ( 1 5 ) . T h e i r biosynthesis is t h e best s t u d i e d of t h e metabolites ( 1 6 , 1 7 ) . h a v e b e e n r e p o r t e d f o r a l t e r n a r i o l (18,19),

Several synthetic methods

a n d t h e selective m e t h y l a t i o n

of a l t e r n a r i o l to t h e m e t h y l ether has b e e n d e s c r i b e d

(20).

A l t e n u i s o l a n d a l t e r t e n u o l a r e c l o s e l y r e l a t e d metabolites of A . alternata a n d i n f a c t m a y b e i d e n t i c a l c o m p o u n d s .

T h o m a s (20)

postulated

the altertenuol structure based m a i n l y o n t h e e m p i r i c a l formula a n d b i o s y n t h e t i c a r g u m e n t s . A l t e r t e n u o l is a c r y s t a l l i n e m a t e r i a l ( m p = 2 8 5 ° C ) w h i c h forms a triacetate ( m p — 2 4 5 ° C ) . lated altenuisol ( m p =

284°-

P e r o et a l . (21)

iso

2 7 7 ° - 2 8 2 ° C ; triacetate m p — 2 1 0 ° - 2 1 3 ° C ) a n d

p r o p o s e d this d i f f e r i n g s t r u c t u r e b a s e d o n t h e f a c t t h a t a l t e n u i s o l f a i l e d t o r e a c t w i t h a m o l y b d a t e i o n , a p r o p e r t y c o m m o n to o r t h o d i h y d r i c p h e n o l s ( 2 2 ) , b u t d i d react after d e m e t h y l a t i o n w i t h h y d r i o d i c a c i d . T h e p o s i t i o n of t h e m e t h o x y l g r o u p w a s e s t a b l i s h e d b y c o m p a r i n g t h e N M R shifts o f a l t e n u i s o l a n d its triacetate w i t h s c o p o l e t i n a n d its acetate. F u r t h e r w o r k is necessary to resolve t h e s t r u c t u r e o f these t w o m e t a b o l i t e s . A l t e n u e n e , d e h y d r o a l t e n u s i n , a n d a l t e n u s i n a r e closely r e l a t e d m e t a bolites w h o s e structures h a v e b e e n d e t e r m i n e d b y x - r a y c r y s t a l l o g r a p h y . A l t e n u e n e w a s i s o l a t e d b y s i l i c a g e l c h r o m a t o g r a p h y o f A . alternata


ex-

15.

HARVAN

Alternaria

A N D PERO

Metabolites

347

tracts a n d w a s c r y s t a l l i z e d as colorless needles, m e l t i n g p o i n t · = 191 ° C .

Its o r i g i n a l structure p o s t u l a t i o n (23)

190°-

has b e e n r e v i s e d

D e h y d r o a l t e n u s i n w a s i s o l a t e d b y t h e a d s o r p t i o n of A . alternata

(24). culture

m e d i u m on charcoal, followed b y ethanol extraction. T h e resulting solu t i o n y i e l d e d d e h y d r o a l t e n u s i n as y e l l o w plates m e l t i n g at 1 8 9 ° - 1 9 0 ° C . T h e o r i g i n a l structure p o s t u l a t i o n w a s i n c o r r e c t ( 2 5 ) c e n t l y r e v i s e d (26).

colorless p r i s m s , m e l t i n g at 2 0 2 ° - 2 0 3 ° C . Downloaded by UNIV OF TENNESSEE KNOXVILLE on December 21, 2014 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/ba-1976-0149.ch015

a n d has b e e n r e

A l t e n u s i n c r y s t a l l i z e d f r o m c h l o r o f o r m extracts as It is i n t e r c o n v e r t i b l e w i t h d e

h y d r o a l t e n u s i n b y o x i d a t i o n w i t h f e r r i c c h l o r i d e or r e d u c t i o n w i t h s o d i u m dithionite

(27).

A l t e n u i c a c i d I I is one of three i s o m e r i c acids i s o l a t e d f r o m A . alter nata (27).

I t was separated f r o m acids I a n d I I I b y its l i m i t e d s o l u b i l i t y

i n ether, a n d c r y s t a l l i z e d f r o m a q u e o u s d i o x a n e as colorless plates m e l t i n g at 2 4 5 ° - 2 4 6 ° C . A l t e n u i c a c i d I a n d I I are c o n v e r t e d i n t o a l t e n u i c a c i d I I I b y t r e a t m e n t w i t h d i l u t e s o d i u m h y d r o x i d e . T h e s t r u c t u r e of a l t e n u i c a c i d I I has b e e n e s t a b l i s h e d b y x - r a y c r y s t a l l o g r a p h y (28).

T h e structures of

a l t e n u i c acids I a n d I I I are u n d e t e r m i n e d . A s e c o n d class of metabolites are t h e a n t h r a q u i n o n e p i g m e n t s l a t e d f r o m A . solani

(29, 30).

iso

T h e pigments were isolated b y silica gel

c h r o m a t o g r a p h y of the c h l o r o f o r m extracts of the fungus.

Anthraquinones

A , B , a n d C are s u b s t i t u t e d x a n t h o p u r p u r i n s , a n d the altersolanols are p a r t i a l l y r e d u c e d a n t h r a q u i n o n e s . A l t e r s o l a n o l A is c o n v e r t e d to a n t h r a q u i n o n e Β u p o n d e h y d r a t i o n at m o d e r a t e temperatures, a n d a l t e r s o l a n o l Β furnishes a n t h r a q u i n o n e A u p o n a r o m a t i z a t i o n w i t h t h i o n y l c h l o r i d e i n p y r i d i n e . A n t h r a q u i n o n e C is i d e n t i c a l to the p r e v i o u s l y i d e n t i f i e d m a c r o s p o r i n , a m e t a b o l i t e of Macrosporium

porri

(31).

T e n u a z o n i c a c i d is a t e t r a m i c a c i d d e r i v a t i v e w h i c h is p r o d u c e d b y a l a r g e v a r i e t y of Alternaria oryzae

spp.

( 3 3 ) , Sphaeropsidales,

(32).

It is also a m e t a b o l i t e of

a n d s o m e Aspergilli

(34).

Pyricularia

It w a s first iso

l a t e d b y Rosett i n 1957 ( 2 7 ) , a n d its s t r u c t u r e w a s e l u c i d a t e d b y S t i c k ings i n 1959 ( 3 5 ) .

I t has b e e n f o u n d i n n a t u r a l l y i n f e c t e d r i c e p l a n t s at

2.6 m g / k g p l a n t tissue (36).

T e n u a z o n i c a c i d is a n o p t i c a l l y a c t i v e l i q u i d

w h i c h loses a c t i v i t y u p o n l o n g s t a n d i n g o r w i t h t r e a t m e n t i n a base. T h i s is a t t r i b u t e d to the f o r m a t i o n of i s o t e n u a z o n i c a c i d , a c r y s t a l l i n e s o l i d w h i c h is b e l i e v e d to b e a m i x t u r e of diastereoisomers.

Tenuazonic acid

forms a c r y s t a l l i n e salt w i t h Ν,Ν'-dibenzylethylenediamine w h i c h is a c o n v e n i e n t m e t h o d for storage of the c o m p o u n d .

T h e synthesis of

the

c o m p o u n d has b e e n d e s c r i b e d ( 3 7 ) ; a series of analogs w a s p r e p a r e d , a n d t h e i r b i o l o g i c a l activities w e r e assessed

(38).

Z i n n i o l is a p e n t a - s u b s t i t u t e d b e n z e n e d e r i v a t i v e p r o d u c e d zinniae

(42).

T h e s t r u c t u r e is b e l i e v e d to be

by

A.

l,2-bis-(hydroxymethyl)-5-

(3,3'-dimethylallyloxy)-3-methoxy-4-methylbenzene,

although

t i o n of the m e t h o x y a n d d i m e t h y l a l l y l o x y substituents is n o t

the

posi

absolutely


348

MYCOTOXINS

established (43).

It is q u i t e s i m i l a r i n s t r u c t u r e to the a n t i f u n g a l agent

q u a d r i l i n e a t i n , a m e t a b o l i t e of Aspergillus

quadrilineatus.

A l t e r n a r i c a c i d is p r o d u c e d b y strains of A . solani w h i c h are n o t e d for t h e i r specific a n t i f u n g a l a c t i v i t y . T h e c o m p o u n d is p u r i f i e d b y r e c r y s t a l l i z a t i o n f r o m b e n z e n e , g i v i n g colorless plates a n d m e l t i n g at 138 ° C 45).

(44,

Its s t r u c t u r e w a s e s t a b l i s h e d b y B a r t e l s - K e i t h a n d G r o v e i n 1959

(46). Downloaded by UNIV OF TENNESSEE KNOXVILLE on December 21, 2014 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/ba-1976-0149.ch015

A l t e n i n is a n o p t i c a l l y a c t i v e , y e l l o w l i q u i d that is p r o d u c e d b y A . kikuchiana.

I t is i s o l a t e d f r o m t h e c u l t u r e filtrate a n d p u r i f i e d b y c h r o m a

t o g r a p h y o n a l u m i n a a n d s i l i c a g e l . I t is u n s t a b l e at h i g h e r t e m p e r a t u r e s ; i t loses its b i o l o g i c a l a c t i v i t y i n t e n m i n u t e s at 8 0 ° C a n d i n 1 h r at 6 0 ° C . T h e a s s i g n e d s t r u c t u r e is b e l i e v e d to b e the m o s t p r o b a b l e of several tautomeric forms

(3).

«,)S-Dehydrocurvularin is a m e t a b o l i c p r o d u c t of A . cucumerina,

the

c a u s a t i v e agent of leaf spot o n c u c u r b i t s , p a r t i c u l a r l y m u s k m e l o n a n d watermelon (47).

I t is a colorless, c r y s t a l l i n e s o l i d , m e l t i n g p o i n t 2 3 0 ° -

2 3 2 ° C , w i t h a n o p t i c a l r o t a t i o n of — 8 5 ° . I t is also a m e t a b o l i t e o f s e v e r a l Curvularia

spp.

(48).

A l t e r t o x i n s I a n d I I are m e t a b o l i t e s of u n k n o w n structure a n d are p r o d u c e d b y A . mali ( 4 9 ) a n d A . alternata.

A l t e r t o x i n I ( C o H i O e ) is a 2

6

y e l l o w , amorphous solid w i t h an undefined m e l t i n g point; i t decomposes a t ca. 210 ° C . It fluoresces b r i g h t y e l l o w u n d e r i r r a d i a t i o n , a n d t h i s p r o p e r t y has b e e n u s e d as a m e t h o d of analysis ( 5 0 ) . A l t e r t o x i n I I ( C o H i 0 ) 2

4

is a n o r a n g e , c r y s t a l l i n e s o l i d w h i c h d e c o m p o s e s at 1 8 5 - 1 9 5 ° C . 0

6

I t is

c l o s e l y r e l a t e d t o A l t e r t o x i n I a n d is p r o b a b l y t h e d e h y d r o d e r i v a t i v e . U n d e r i r r a d i a t i o n i t appears as a d a r k , q u e n c h i n g spot. B o t h c o m p o u n d s a r e h i g h l y a r o m a t i c m a t e r i a l s , possessing n e i t h e r m e t h o x y l n o r c a r b o n m e t h y l g r o u p s . T h e y b o t h h a v e c a r b o n y l absorptions at 1650 c m " w h i c h 1

i n d i c a t e s h y d r o g e n - b o u n d i n g b y adjacent h y d r o x y l s . T h e i r s t r u c t u r e is b e i n g investigated b y x-ray crystallography. B r a s s i c i c o l i n A is a m e t a b o l i t e i s o l a t e d f r o m A . brassicicoh. m a t e r i a l is a colorless o i l , w i t h [ « ]

D

=

The

2 0 . 1 ° . Its e l e m e n t a l c o m p o s i t i o n

has b e e n r e p o r t e d to b e C o H i 0 . T h e c o m p o u n d possesses anti-yeast 2

a n d m i l d anti-bacterial activity

3

9

(51).

P h y t o a l t e r n a r i n s A , B , a n d C are p r o d u c e d b y A . kikuchiana

Tanaka.

P h y t o a l t e r n a r i n A is a colorless s o l i d ; Β is a y e l l o w fluorescent l i q u i d , a n d C i s a colorless s o l i d , m e l t i n g at 2 3 5 ° C ( 5 ) .

A l l three give p o s i t i v e n i n

h y d r i n , L i e b e r m a n , a n d X a n t h o p r o t e i c tests, b u t n e g a t i v e F e h l i n g a n d B i u r e t reactions ( 5 2 ) . Synthetic

Methods

A l t e r n a r i o l w a s first s y n t h e s i z e d b y c o u p l i n g 2 - b r o m o - 4 , 6 - d i m e t h o x y b e n z o i c a c i d w i t h 3 , 5 - d i h y d r o x y t o l u e n e i n t h e presence of b a s i c c o p p e r


15.

HARVAN

sulfate.

The

A N D PERO

Alternaria

resulting material was

methylated

i d e n t i c a l to a l t e r n a r i o l t r i m e t h y l ether ( 1 8 ) . a l t e r n a r i o l has r e c e n t l y b e e n

described

m e t h y l orsellinate w a s c o n d e n s e d triketone derivative.

349

Metabolites giving a

compound

A m o r e elegant synthesis of

(19).

The

d i b e n z y l ether

of

w i t h d i l i t h i o a c e t y l a c t o n e to g i v e

a

T h e triketone was carboxylated w i t h l i t h i u m d i -

i s o p r o p y l a m i d e a n d esterified w i t h d i a z o m e t h a n e .

R e m o v a l of the b e n z y l

p r o t e c t i v e groups c a u s e d spontaneous c y c l i z a t i o n to a c h r o m a n d e r i v a t i v e .


T r e a t m e n t of the c h r o m a n w i t h s o d i u m acetate g a v e a l t e r n a r i o l i n 5 2 % y i e l d ( F i g u r e 3 ). A l t e r n a r i o l p r e s u m a b l y is f o r m e d b y a n a l d o l c o n d e n s a tion between

positions t w o

a n d seven, f o l l o w e d

by

dehydration

and

l a c t o n i z a t i o n . S i n c e a l t e r n a r i o l c a n b e s e l e c t i v e l y m e t h y l a t e d to a l t e r n a r i o l m e t h y l ether, this constitutes a synthesis of t h e m e t h y l ether as w e l l ( 2 0 ) . B y use of t h e a p p r o p r i a t e b e n z o i c a c i d analogs this m e t h o d c o u l d also p r o v i d e s y n t h e t i c routes to a l t e n u s i n , d e h y d r o a l t e n u s i n , a n d a l t e r t e n u o l . Tenuazonic

a c i d is s y n t h e s i z e d

by

condensing

L-isoleucine w i t h

d i k e t e n e . T h e p r o d u c t is m e t h y l a t e d w i t h d i a z o m e t h a n e a n d c y c l i z e d b y r e f l u x i n g w i t h the s o d i u m m e t h o x i d e i n b e n z e n e .

T h e c o m p o u n d is p u r i

fied b y r e c r y s t a l l i z a t i o n of its c o p p e r ( 3 7 ) or its N , N ' - d i b e n z y l e t h y l e n e d i a m i n e salt ( 3 4 ) . A l t e n i n has b e e n p r e p a r e d b y t h e c o n d e n s a t i o n of e t h y l g l y o x a l a t e w i t h 3-acetoxy-acetylacetone

i n the presence

of p o t a s s i u m a m i d e

(3).

T h e s y n t h e t i c m a t e r i a l w a s i d e n t i c a l to the n a t u r a l l y o c c u r r i n g c o m p o u n d i n p h y t o p a t h o l o g i c a c t i v i t y as w e l l as

spectroscopy.

T h e o n l y a n t h r a q u i n o n e m e t a b o l i t e t h a t has b e e n m a c r o s p o r i n m o n o m e t h y l ether ( 5 4 ) .

s y n t h e s i z e d is

T h e method involved condensing

a-resorcylic a c i d w i t h 3-methoxy-4-methylbenzoic

acid b y heating w i t h C

Figure 3.

Biogenetic type synthesis of

«0

alternariol


350

MYCOTOXINS

c o n c e n t r a t e d s u l f u r i c a c i d a n d b o r i c a n h y d r i d e . M e t h y l a t i o n of this m a t e r i a l gave m a c r o s p o r i n m o n o m e t h y l ether. A l t h o u g h t w o isomers w e r e possible, o n l y t h a t one c o r r e s p o n d i n g to n a t u r a l l y - o c c u r r i n g m a c r o s p o r i n w a s isolated.

Biosynthetic Pathways


T h e biosynthesis of a l t e r n a r i o l has b e e n discussed i n r e l a t i o n to the p o l y k e t i d e hypothesis ( 5 6 ) .

This theory maintains that a long poly-β

c a r b o n y l c h a i n is f o r m e d b y h e a d - t o - t a i l condensations of acetate m a l o n y l - C o A units.

with

T h e r e s u l t a n t c h a i n m a y t h e n c y c l i z e b y a l d o l or

C l a i s s e n - t y p e reactions a n d l e a d to t h e o b s e r v e d p r o d u c t s .

T h e theory

i n v o l v e s o n l y s l i g h t m o d i f i c a t i o n of t h a t b e l i e v e d to b e r e s p o n s i b l e for f a t t y - a c i d synthesis. T h e e n z y m e c o m p l e x r e s p o n s i b l e for a l t e r n a r i o l synthesis has b e e n purified b y gel

filtration

o n Sephadex G-25 (16).

T h e enzyme utilizes

a c e t y l - C o A a n d m a l o n y l - C o A as substrate b u t is i n h i b i t e d b y excessive concentrations of e i t h e r reagent. O p t i m u m v e l o c i t y w a s o b s e r v e d w i t h a r a t i o of m a l o n y l p a n t o t h e i n e to a c e t y l - C o A of 6:1 w h i c h is t h e r a t i o of m a l o n a t e to acetate f o u n d i n a l t e r n a r i o l . T h e o p t i m u m a c t i v i t y w a s o b s e r v e d at p H 7.8 at 2 8 ° C ( 1 7 ) .

It was also d e m o n s t r a t e d t h a t the a d d i

t i o n of S - a d e n o s y l m e t h i o n i n e to the r e a c t i o n m i x t u r e c a u s e d the f o r m a t i o n of a l t e r n a r i o l m e t h y l ether. T h e d e g r a d a t i o n of a l t e r n a r i o l m e t h y l ether c o u l d a c c o u n t for most o f the other d i b e n z o - p y r o n e m e t a b o l i t e s ( F i g u r e 4 ) . A l t e n u s i n w o u l d b e the result of h y d r o x y l a t i o n f o l l o w e d b y r e d u c t i v e o p e n i n g of t h e l a c t o n e ring.

D e h y d r o a l t e n u s i n a n d altenuene c o u l d arise f r o m a l t e n u s i n b y

o x i d a t i o n a n d r e d u c t i o n . T h e o x i d a t i o n of the c a r b o n - m e t h y l g r o u p f o l l o w e d b y d e c a r b o x y l a t i o n w o u l d l e a d to a l t e r t e n u o l . O x i d a t i o n of the c a t e c h o l g r o u p i n g of a l t e n u s i n — i n a m a n n e r analogous to the d e g r a d a t i o n of c a t e c h o l to m u c o n i c a c i d a n d t h e n to m u c o n o l a c t o n e (56)—would

lead

to the f o r m a t i o n of a l t e n u i c a c i d . T e n u a z o n i c a c i d is f o r m e d b y the N - a c e t o a c e t y l a t i o n of i s o - l e u c i n e followed by enzymatic cyclization (57).

I t has b e e n d e m o n s t r a t e d t h a t

a d d i n g v a l i n e or l e u c i n e to c u l t u r e s of the f u n g u s p r o d u c e s the c o r r e s p o n d i n g t e t r a m i c a c i d d e r i v a t i v e b u t t h a t p h e n y l a l a n i n e is n o t u t i l i z e d

(58).

Analytical Methods F e w a n a l y t i c a l m e t h o d s h a v e b e e n r e p o r t e d for the Alternaria metab olites. A l t e r n a r i o l , the m e t h y l ether, a n d altenuene h a v e b e e n a n a l y z e d b y gas c h r o m a t o g r a p h y as s i l y l ethers ( 5 9 ) . T h e s i l y l ethers a r e c h r o m a t o g r a p h e d o n 3 % O V - 1 7 at 1 0 0 ° - 2 5 0 ° C at 8 ° / m i n . U n d e r these c o n d i -


15.

Alternaria

HARVAN AND PERO

Metabolites

351

Acetyl-CoA

+

alternariol


6 Malonyl-CoA

alternariol methyl ether

•OCH

3

dehydroaltenusin I I.) oxidn. redn.

2

)

C

0

altertenuol

2

altenuene altenuic acid Figure 4.

Possible biosynthetic

pathway for the dibenzo-pyrone the Alternaria

metabolites of

tions altenuene has a r e t e n t i o n t i m e of 2 3 m i n , a l t e r n a r i o l 2 7 m i n , a n d t h e m e t h y l ether, 29 m i n . T h i s m e t h o d has also b e e n u s e d f o r s e v e r a l c o m m o n l y o c c u r r i n g f u n g a l m e t a b o l i t e s s u c h as stearic a c i d , p a l m i t i c a c i d , s u c c i n i c a c i d , e r y t h r i t o l , a n d m a n n i t o l . I t is also u s e f u l f o r s o m e m e t a b olites of other f u n g i , e.g., k o j i c a c i d , p e n i c i l l i c a c i d , a n d p a t u l i n

(60).

T h i n l a y e r c h r o m a t o g r a p h y has b e e n u s e d r e c e n t l y t o detect a l t e r n a r i o l a n d the m e t h y l ether i n g r a i n samples (61). T h e m e t h o d w a s of v a l u e i n d i s t i n g u i s h i n g b e t w e e n a l t e r n a r i o l , zearelenone, a n d aflatoxin i n i n f e c t e d grains.

T e n u a z o n i c a c i d has b e e n

analyzed b y a

m e t h o d (62) a n d a gas c h r o m a t o g r a p h i c m e t h o d

spectrophotometric

(63).

Toxicity C r u d e Alternaria

extracts a r e l e t h a l to m i c e ( i p i n j e c t i o n ) a t 3 0 0

m g / k g ( 5 0 ) . S i m i l a r levels of toxicosis o c c u r i n rats w i t h o r a l dosage. T h e m a j o r m a m m a l i a n t o x i n is b e l i e v e d to b e t e n u a z o n i c a c i d (64).

Sev

e r a l investigators d e m o n s t r a t e d t h e t o x i c i t y o f t e n u a z o n i c a c i d to m i c e (38, 6 5 ) , rats, dogs, m o n k e y s a n d g u i n e a p i g s

(65).

S o d i u m t e n u a z o n a t e is h i g h l y i n h i b i t o r y t o t h e h u m a n a d e n o c a r c i -


352

MYCOTOXINS

n o m a ( H A d l ) i n t h e e g g host system at 0.1 m g / e g g a n d t o x i c to t h e T h e D-allo a n d D-isomers as w e l l as s t r u c

e m b r y o at 0.48 m g / e g g ( 3 4 ) .

t u r a l analogs of t e n u a z o n i c a c i d , s u b s t i t u t e d i n the carbon-five p o s i t i o n , w e r e m u c h less a c t i v e against t u m o r s .

Analogs substituted on nitrogen

showed an increased antibacterial activity but a reduced antitumor activ i t y . A g a i n s t Bacillus

analogs s u b s t i t u t e d at carbon-five w e r e

megaterium,

a b o u t as a c t i v e as the p a r e n t c o m p o u n d .


D-sodium tenuazonate were equally active

L- a n d

A g a i n s t B. megaterium, (38).

S o d i u m t e n u a z o n a t e a n d isotenuazonate w e r e e q u a l l y effective against enteroviruses ( E C H O - 9 , C o x s a c k i e Β ), r e s p i r a t o r y viruses [ p a r a i n f l u e n z a 3, ( H A - 1 ) , S a l i s b u r y H G P ] , v a c c i n i a , herpes s i m p l e x H F , a n d Ί Γ v i r u s at 1 0 0 - 5 0 0 / x g / m l .

S o d i u m tenuazonate w a s effective against p o h o v i r u s

M E F 1 , whereas s o d i u m isotenuazonate w a s not.

N e i t h e r was a c t i v e i n

tissue c u l t u r e against p o l y o m a v i r u s or i n m i c e against A s i a n i n f l u e n z a , rabies, or F r i e n d leukemia

(66).

T e n u a z o n i c a c i d i n h i b i t s t h e i n c o r p o r a t i o n of a m i n o acids i n t o p r o t e i n in vivo i n S p r a g u e - D a w l e y rats a n d in vitro i n E h r l i c h Ascites t u m o r cells. I t is b e l i e v e d t h a t t e n u a z o n i c a c i d interferes w i t h the release of n e w l y f o r m e d p r o t e i n f r o m t h e ribosomes thus p r e v e n t i n g t h e ribosomes a c c e p t i n g a m i n o acids f r o m transfer R N A (67).

from

T e n u a z o n i c a c i d is also

k n o w n to b l o c k p e p t i d e b o n d f o r m a t i o n i n p r o t e i n synthesis of h u m a n t o n s i l a n d p i g - l i v e r ribosomes

(68).

T e n u a z o n i c a c i d has a p r o t e c t i v e effect o n in vivo c e l l d e a t h of intes t i n a l c r y p t e p i t h e l i a l cells after exposure to l-β

D-arabinofuranosylcytosine,

n i t r o g e n m u s t a r d , or x - i r r a d i a t i o n . A t 3 0 - 7 5 m g / k g i n m a l e rats t e n u a zonic a c i d inhibits leucine incorporation into protein a n d t h y m i d i n e i n corporation into D N A but not u r i d i n e incorporation into R N A . T h e pro t e c t i v e effect w a s a t t r i b u t e d to the i n h i b i t i o n of p r o t e i n synthesis

(69).

S e v e r a l other metabolites h a v e b e e n e x a m i n e d for c y t o t o x i c i t y 70) a n d t e r a t o g e n i c i t y (50).

(50,

A l t e r n a r i o l a n d the m e t h y l ether e x h i b i t e d a

s y n e r g i s t i c effect against b a c t e r i a a n d as teratogens. A l t e n u e n e , a l t e n u i s o l , a n d the altertoxins w e r e a l l active against H e L a cells, w i t h I D from 0.5-28 ^ g / m l

5 0

values

(50).

T h e t o x i c i t y of t h e r e m a i n i n g Alternaria

metabolites has not

been

s t u d i e d extensively. N o d a t a exist for a l t e r t e n u o l , a l t e n u s i n , d e h y d r o a l t e n u s i n , a l t e n u i c a c i d , or t h e a n t h r a q u i n o n e p i g m e n t s . B r a s s i c i o l i n A has l o w a n t i b a c t e r i a l a c t i v i t y b u t f a i r antiyeast a n d a n t i f u n g a l p r o p e r t i e s . T h i s a n d other m e t a b o l i t e s of A . brassicicola

n e e d f u r t h e r s t u d y since

strains of this t y p e w e r e the most consistently a c t i v e against b a c t e r i a , m o l d s , a n d yeast ( 1 4 ) .

E v e n t h o u g h α , β - d e h y d r o c u r v u l a r i n has n o t b e e n

i n v e s t i g a t e d for a n t i b i o t i c or p h y t o t o x i c a c t i v i t y , i t is s i m i l a r i n s t r u c t u r e to zearelenone, a Fusarium

m e t a b o l i t e w h i c h has strong estrogenic a c t i v

i t y , a n d m a y b e of interest i n studies of t h a t t y p e .


15.

HARVAN

Alternaria

A N D PERO

353

Metabolites

Phytotoxicity S e v e r a l Alternaria ity (5). fruits.

m e t a b o l i t e s h a v e b e e n associated w i t h p h y t o t o x i c

T e n t o x i n is r e s p o n s i b l e for s e e d l i n g chlorosis of c o t t o n a n d c i t r u s T h e p u r i f i e d c o m p o u n d interferes w i t h c h l o r o p h y l l f o r m a t i o n i n

m a n y h i g h e r p l a n t species. M o s t d i c o t y l e d o n s are sensitive w i t h t h e e x c e p t i o n of t o m a t o a n d t h e C r u c i f e r a e ( 7 1 ) . the a l g a Euglena


filamentous

gracilis,

T h e c o m p o u n d does n o t affect

b a c t e r i a , the yeast Saccharomyces

or

cerevisiae,

f u n g i at concentrations t h a t cause 1 0 0 % chlorosis of c u c u m

b e r cotyledons. A l t e n i n is c a p a b l e of c a u s i n g b l a c k spots o n s u s c e p t i b l e Japanese p e a r at 2 Χ

10" m g / m l . T h e a c t i v e m o i e t y is b e l i e v e d to b e t h e e n e d i o l c a r 5

bonyl grouping

(3).

Z i n n i o l is r e s p o n s i b l e for leaf spot a n d s e e d l i n g b l i g h t of z i n n i a , s u n flower,

a n d m a r i g o l d . P l a n t s w i t h severe i n f e c t i o n often w i l t a n d d i e . A t

500 p p m z i n n i o l i n h i b i t s the g e r m i n a t i o n of z i n n i a , t o m a t o , l e t t u c e , w a t e r m e l o n , a n d c a r r o t seeds. A t 1000 p p m i t causes c o m p l e t e w i t h e r i n g of c u t seedlings of w a t e r m e l o n , s q u a s h , s p i n a c h , beet, t o m a t o , oat, c o r n , p e a , and bean. /*g/disc

T h e c o m p o u n d is m i l d l y i n h i b i t o r y to A c t i n o m y c e t e s at 485

(42).

A l t e r n a r i c a c i d w a s i s o l a t e d f r o m c u l t u r e s of A . sohni,

the fungus

r e s p o n s i b l e for e a r l y b l i g h t of tomatoes a n d potatoes, a n e c o n o m i c a l l y significant p l a n t disease i n t h e U n i t e d States (44, 72).

A t 5-10 /xg/ml it

causes w i l t i n g a n d d e a t h to seedlings of r a d i s h , c a b b a g e , m u s t a r d , a n d carrot.

A t 2 - 2 0 μg/m\ i t causes n e c r o t i c lesions i n t o m a t o a n d p o t a t o

shoots. I t also possesses s o m e a n t i f u n g a l a c t i v i t y , i n h i b i t i n g t h e g e r m i n a t i o n of Absidia,

Myrothecium,

f t g / m l i t i n h i b i t s t h e rate of Pénicillium

a n d Stachybotrys

at 0 . 1 - 1 . 0 f t g / m l . A t 200

g e r m i n a t i o n of Botrytis,

Fusarium,

and

(5).

A l t e r n a r i o l m e t h y l ether has also b e e n s h o w n to cause chlorosis i n t o b a c c o leaves (73).

W h e n solutions of t h e m a t e r i a l are i n j e c t e d i n t o

t o b a c c o leaves, c h l o r o t i c zones, p r o p o r t i o n a l i n size to c o n c e n t r a t i o n , w e r e f o r m e d w i t h i n 48 h r . T h e l e v e l of a c t i v i t y w a s b e t w e e n 1 0 - 2 5 / A g / m l .

It

w a s also o b s e r v e d t h a t t h e m e t h y l ether w a s p r o d u c e d b y g r o w i n g A . alternata

o n t o b a c c o substrate. T h e c o m p o u n d w a s r a p i d l y m e t a b o l i z e d

a n d w a s n o n d e t e c t a b l e 72 h r after i n j e c t i o n i n t o l i v i n g tissue. T h i s is s u p p o r t e d b y the f a c t t h a t o t h e r w o r k e r s f a i l e d to find a l t e r n a r i o l or t h e m e t h y l ether i n extracts of n a t u r a l l y m o l d y t o b a c c o

(74).

Phytoalternarins A , B , a n d C have been isolated b y chromatography o n a l u m i n a c o l u m n s . P h y t o a l t e r n a r i n A has t h e same host s p e c i f i c i t y as t h e f u n g u s itself. A c o r r e l a t i o n is o b s e r v e d b e t w e e n tissue age a n d o p t i m u m temperature for symptom development between the fungus

and

p u r e t o x i n . P h y t o a l t e r n a r i n s Β a n d C are t o x i c to s u s c e p t i b l e v a r i e t i e s of Japanese p e a r ( 5 ) .


354

MYCOTOXINS

Summary T h e i m p o r t a n c e o f t h e Alternaria not yet been

firmly

established.

toxins as e n v i r o n m e n t a l h a z a r d s has

H u m a n p o p u l a t i o n s w o u l d n o t b e ex

p e c t e d to r e c e i v e a c u t e l y t o x i c doses i n h i g h l y - d e v e l o p e d nations.

How

e v e r i n u n d e r d e v e l o p e d areas t h e r e is a greater p o s s i b i l i t y o f h u m a n e x p o s u r e t o i n f e c t e d f o o d crops.

M o r e i m p o r t a n t is t h e p o s s i b i l i t y o f effects


a r i s i n g f r o m c o n t i n u e d l o w - l e v e l exposure to t h e metabolites. T h e l i m i t e d f e t o t o x i c i t y d a t a i m p l i e s some c o m b i n e d a c t i v i t y f o r a l t e r n a r i o l a n d t h e m e t h y l ether, b u t t h e m a j o r i t y o f t h e metabolites h a v e n o t b e e n tested. T h e a u t h o r s f e e l t h a t c o n t i n u e d r e s e a r c h is necessary to evaluate t h e p o t e n t i a l h a z a r d s o f t h e Alternaria

toxins p a r t i c u l a r l y a t t h e c h r o n i c l e v e l o f

exposure.

Literature Cited

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33.

Christensen, C. M., Cereal Chem. (1951) 28, 408. Christensen, C. M., et al., Cancer Res. (1968) 28, 2293. Sugiyama, N., et al., Bull. Chem. Soc., Jap. (1966) 39, 2470. Lucas, G. B., "Diseases of Tobacco," p. 228, Scarecrow, New York, 1959. Templeton, G. E., in "Microbial Toxins," Vol. VII, (S. Kadis et al., Eds.), pp. 169-192, Academic, New York, 1972. Lucas, G. B., Tob. Sci. (1971) 15, 37. Joffe, A. Z., Bull Res. Counc. Isr., Sect D. (1960) 9, 101. Doupnik, B. Jr., Sobers, Ε. K., Appl. Microbiol (1968) 16, 1596. Slifkin, M . K., Spalding, J., Toxicol. Appl. Pharmacol. (1970) 17, 375. Forgacs, J., et al., Am. J. Vet. Res. (1958) 19, 744. Forgacs, J., et al., Avian Dis. (1962) 6, 363. Hamilton, P. B., et al., Appl. Microbiol. (1969) 18, 570. Sobers, Ε. K., Doupnik, B., Jr., Appl. Microbiol. (1972) 23, 313. Lindenfelser, L. Α., Ciegler, Α., Dev. Ind. Microbiol. (1969) 10, 271. Thomas, R., Biochem. J. (1961) 78, 748. Gatenbeck, S., Hermodsson, S., Acta. Chem. Scand. (1965) 19, 65. Sjoland, S., Gatenbeck, S., Acta. Chem. Scand. (1966) 20, 1053. Raistrick, H., et al., Biochem. J. (1953) 55, 421. Hay, J. V., Harris, T. M., J. Chem. Soc., Chem. Comm. (1972) 953. Thomas, R., Biochem. J. (1961) 80, 234. Pero, R. W., et al., Tetrahedron Lett. (1973) 12, 945. Pridham, J. B., Ed., "Methods in Polyphenol Chemistry," p. 120, Macmillan, New York, 1964. Pero, R. W., et al., Biochem. Biophys. Acta. (1971) 230, 170. McPhail, A. T., et al., J. Chem. Soc., Chem. Comm. (1973) 682. Coombe, R. G., et al., Aust. J. Chem. (1970) 23, 2343. Rogers, D., et al., J. Chem. Soc., Chem. Comm. (1971) 393. Rosett, T., et al., Biochem. J. (1957) 67, 390. Williams, D. J., Thomas, R., Tetrahedron Lett. (1973) 9, 639. Stoessl, Α., J. Chem. Soc., Chem. Comm. (1967) 307. Stoessl, Α., Can. J. Chem. (1969) 47, 767. Suemitsu, R., et al., Bull. Agric. Chem.Soc.Jap. (1959) 23, 547. Kinoshita, T., et al, Ann. Phytopathol. Soc. Jap. (1972) 38, 397. Iwasaki, S., et al., Tetrahedron Lett. (1972) 1, 13.



15.

34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74.

HARVAN

AND

PERO

Alternaria

Metabolites

355

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RECEIVED November 8, 1974.


Mycotoxins and Other Fungal Related Food Problems

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