CURRENT AWARENESS DOCUMENT
POLYNUCLEAR LACTONE-TYPE AND RELATED ALKYLATING AGENTS
CARCINOGENICITY AND STRUCTURE ACTIVITY
RELATIONSHIPS. OTHER BIOLOGICAL PROPERTIES.
METABOLISM. ENVIRONMENTAL SIGNIFICANCE.
Prepared by:
David Y. Lai, Ph.D.
Yin-Tak Woo, Ph.D., D.A.B.T.
JRB Associates/
Science Applications
International Corporation
8400 Westpark Drive
McLean, Virginia 22102
EPA Contract No. 68-02-3948
JRB Project No. 2-813-07-409
EPA Project Officer and Scientific Editor
Joseph C. Arcos, D.Sc.
Extradivisional Scientific Editor
Mary F. Argus, Ph.D.
March 1985
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5.3.1.2 Polynuclear Lactone-type and Related Alkylating Agents: Penicillium
Toxins.
5.3.1.2.1 Introduction.
The Penicillium represent another important fungal group containing
species which elaborate toxigenic as well as carcinogenic metabolites.
Several Penicillium toxins, which have been tested for carcinogenic activity,
are shown in Table XII.
Like the Aspergillus, members of the Penicillium group occur frequently
as natural contaminants of foods and feeds and have been implicated as the
causative agents in many instances of illness and death of humans and farm
animals. A case in point is the outbreak of the "yellowed rice disease" in
Japan shortly after World War II. The incident, which led to many deaths, has
been attributed to rice heavily contaminated with _P. islandicum, the mold that
produces luteoskyrin, cyclochlorotine and islanditoxin. P. viridicatum, which
elaborates ochratoxin A, citrinin, griseofulvin and penicillic acid, is one of
the major contaminants of stored corn and various types of decaying vegetation
(see rev. 1).
Early interest in the studies of griseofulvin, citrinin, patulin, peni-
cillic acid, penicillin G and rugulosin arose largely because of their poten-
tial usefullness as antibiotics. Since the discovery of aflatoxin in the
1960's, awareness of the importance of natural chemicals as environmental
contaminants has intensified; the biochemical, toxicological and human health
effects of these and other mycotoxins have attracted dramatically increased
attention in the last two decades. Several publications summarize current
knowledge of these effects of Penicilliurn toxins (2-8).
100
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Table XII
Penicillium Toxina Which Have Been Tested for Carcinogenic Activity
i°°H "
Ochratoxin A
CH3 CH3
Citrinin
CHO
H3C "'" CH,
PR toxin
HO
ff
0 OCH3
V II I ^
,C-C-C=CH-
0
II
-C-HN-
Patulin
Penicillic acid
Penicillin G
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Table XII (Continued)
H3CO 0 OCH3
3 i ii v
-c.
H.CO
Cl
Griseofulvin
CH2OH CH2CH3 CH2OH
CO-NH-CH-CH2-CO-NH-CH CH-CO-NH-CH-CO-NH-
HO-CH2-CH CO NH
NH-CO-CH-NH-CO
CH2
CH3 Cl Cl
Cyclochlorotine
I
CO-CH2-CH-NH-CO
CH2OH
•CH
CO
0
Rubratoxin B
OH 0
H
Islanditoxin
OH 0 OH
II
R 0 |H1 II
1 IJ l\\\ 0 R
Luteoskyrin R=OH
Rugulosin RrH
CI2
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5.3.1.2.2 Physicochemical Properties and Biological Effects.
5.3.1.2.2.1 PHYSICAL AND CHEMICAL PROPERTIES.
Penicillium toxins display wide variations in their chemical structure
(see Table XII) as well as in physicochemical properties. The ultraviolet,
infrared, nuclear magnetic resonance and mass spectral data of many of these
compounds have been compiled (9, 10). Some other important physical proper-
ties of Penicillium toxins are summarized in Table XIII.
Ochratoxin A is a 7-carboxy-5-chloro-8-hydroxy-3,4-dihydro-3-R-methyl
derivative of isocoumarin linked to the amino group of L-y§-phenylalanine .
Upon acid or enzymic hydrolysis, L- A-phenylalanine and the isocoumarin acid
are formed. Ochratoxin A is quite stable in stored foods, but decomposes
readily under fluorescent light (9, 11).
Citrinin (4,6-Dihydro-8-hydroxy-3,4,5-trimethyl-6-exo-3H-2-benzopyran-7-
carboxylic acid) resembles structurally the isocoumarin derivative of ochra-
toxin A. The compound is thermally stable in hexane or ethanol, but is ther-
mally labile in acid or alkaline solution. It is also unstable under fluo-
rescent light and is inactivated by cysteine (9).
PR toxin (7-Acetoxy-5,6-epoxy-3,5,6,7,8,8a-hexahydro-3',8,8a-trimethyl-3-
oxospiro[naphthalene-2(1H),2'-oxirane]-3'-carboxaldehyde) has a eremophilane
ring system with an acetoxy group, an aldehyde, and an O^,B -unsaturated
ketone, two epoxides and three methyl groups. The compound reacts with
ammonia and free amino acids (12).
Both patulin (a furopyrone) and penicillic acid have a five-membered lac-
tone ring unsaturated in the c(,& -position to the carbonyl group. In aqueous
solution, penicillic acid is in equilibrium with the corresponding open-chain
hexanoic acid (see Table XII). Patulin is stable under acidic conditions or
101
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Table XIII
Physical Properties of Some Penicilliutn Toxins3
Toxin
Physical Form
m. p.
Optical Rotation
Solubility
Ochratoxin A
Citrinin
PR toxin
Patulin
Colorless crystals
Yellow needles
Colorless crystals
Colorless to white
crystals
Penicillic acid Colorless crystals
Penicillin G
Amorphous white powder
Griseofulvin
Colorless octahedra
or rhombs
89-95°C (benzene)0
169°C (xylene)c
178°C
155°C
87°C
220°C
= _H8
- -27.7C
Inact ive
Inactive
= +269°
17 _
[<*ID = +370C
Slightly soluble in
water; soluble in polar
organic solvents
Insoluble in water;
soluble in dilute alkali,
ethanol and dioxane
Insoluble in water,
dilute acid or alkali;
soluble in organic
solvents
Soluble in water and
polar organic solvents
Soluble in water,
ethanol, ether, benzene
and chloroform
Sparingly soluble in
water; soluble in
methanol, ethanol,
acetone, ether, chloro-
form and ethyl acetate
Insoluble in water,
petroleum or ether;
slightly soluble in
ethanol, methanol,
acetone, benzene, chloro-
form, ethyl acetate and
acet ic ac id
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Table XIII (continued)
kf 2
Toxinb
Rubratoxin B
Luteoskyrin
Rugulosin
Physical Form
Colorless crystals
Yellow, rectangular
crystals
Yellow, prism-like
crystals
Cyclochlorotine White needles
m.p.
169°C
287°C
290°C
251°C
Optical Rotation
Solubility
= -880
= +492
= -92.9C
Sparingly soluble in
water; soluble in
ethanol, ethyl acetate,
dioxane and acetone
Insoluble in water;
soluble in sodium bicar-
bonate and most organic
solvents
Insoluble in water;
soluble in sodium bicar-
bonate and most organic
solvents
Soluble in water and
n-butanol
aSummarized from IARC Monographs, Vol. 10, 1976; P.M. Scott, Penicillium Mycotoxins. In "Mycotoxin Fungi,
Mycotoxins, Mycotoxicoses, An Encyclopedic Handbook" (T.D. Wyllie and L.G. Morehouse, eds.), Vol. 1, Part 2,
Marcel Dekker, New York, 1977, p. 283; The Merck Index, 10th ed. , Merck & Co., Rahway, NJ, 1983.
See Table XII for structural formulas.
""Solvents used in crystallization.
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in chloroform, but is unstable in alkaline solutions or in methanol. The
secondary alcohol group of the hetniacetal moiety of patulin may be esterified
to form a monoacetate, benzoate or cinnamate. Various derivatives of patulin
can also be formed involving the carbonyl group (9, 13). Unlike patulin,
penicillic acid is stable under either acid or alkaline conditions. The
reaction of penicillic acid with phenylhydrazine or excess diazomethane yields
a pyrazoline derivative. Both patulin and penicillic acid react readily with
sulfhydryl-containing amino acids or proteins (9, 13).
Penicillin G (benzylpenicillinic acid) consists of a thiazolidine ring
linked to a beta-lactam ring, to which a benzyl side chain is attached.
Removal of the benzyl side chain chemically or by the action of amidase yields
6-aminopenicillanic acid (see rev. 14).
Griseofulvin (7-Chloro-4:6:2 f-trimethoxy-6'-methylgris-2'-en-3 :4'-dione)
is a polycyclic chlorine-containing compound. Acid hydrolysis of griseofulvin
gives griseofulvic acid which, upon further hydrolysis with 0.5 H sodium
hydroxide, yields norgriseofulvin and decarboxygriseofulvic acid (15).
Rubratoxin B is a substituted analog of byssochlamic acid in which the
ethyl group is replaced by a 6-carbon 0( ,K -unsaturated lactone. Oxidation of
rubratoxin B with chromic acid in acetone at 0°C yields monoketone deriva-
tives. The toxin is stable in sodium bicarbonate (16).
Luteoskyrin is a substituted bis-polyhydroxydihydroanthraquinone. Reac-
tion of leuteoskyrin with 60% sulphuric acid yields islandicin and irido-
skyrin. Rugulosin, also an anthraquinone, is chemically related to luteo-
skyrin. Both toxins can chelate divalent cations such as magnesium and cal-
cium ions (see rev. 17).
102
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Cyclochlorotine is a water-soluble cyclic pentapeptide containing resi-
dues of a dichloroproline, 06-aminobutyric acid, serine, /J-amino- fl-phenyl-
butyric acid, and serine. The compound gives a positive result in biuret
test, but is negative in the ninhydrin reaction (cited in 10).
Islanditoxin is a cyclic peptide isomeric with cyclochlorotine. The
physicochemical properties of islanditoxin resemble those of cyclochlorotine.
5.3.1.2.2.2 BIOLOGICAL EFFECTS OTHER THAN CARCINOGENICITY.
Toxic Effects. The presence of fungi in foods and animal feeds has long
been incriminated in outbreaks of human diseases and poisonings of poultry,
swine and cattle. The common deleterious effects on farm animals include
reduced feed intake, decreased weight gains and lower production of egg and
milk. Consumption of large doses of mycotoxins generally results in animal
deaths. The LDcQ values of some Penicillium toxins in laboratory rodents are
shown in Table XIV. Among all compounds, the "yellowed rice" toxins cyclo-
chlorotine and islanditoxin are the most potent ones; rubratoxin B is also
extremely toxic to the rat and mouse when administered intraperitoneally. In
a chick embryotoxicity test, the lethal doses for several Penicillium toxins
are: ochratoxin A, 0.1 ^ug; PR toxin, 0.1 pg; rubratoxin B, 0.1 ,ug; patulin,
1.0 /ug; citrinin, 10 .ug; penicillic acid, 10.ug; and griseofulvin, 100 ug
(34). As many of these mycotoxins may occur simultaneously in mold-
contaminated foods and feeds, the possibility of toxic interaction is receiv-
ing increasing attention. A synergistic effect between the acute toxicities
of ochratoxin A and citrinin (35-37) and between the acute toxicities of
ochratoxin A and penicillic acid (27, 35, 38) in rodents has been reported.
Pathological observations indicate that different organ system may be
characteristically affected by particular mycotoxins. Ochratoxin A and
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1 of 2
Table XIV
Acute Toxicity of Some Penicillium Toxins
Toxin3
Ochratoxin A
Citrinin
PR toxin
Patulin
Penicillic acid
Penicillin G
Griseofulvin
Rubratoxin B
Luteoskyrin
Species and Route
Rat, oral
Rat , s .c . , i.p.
Mouse, oral
s.c. , i.p.
i .v.
Rabbit, i.v.
Guinea pig , s.c.
Rat, oral
i .v .
i.p.
Mouse, oral
i.p.
Rat, oral
s.c.
i.p.
Mouse, oral
s.c.
i.p.
Hamster, oral
s.c.
i.p.
Mouse, oral
s.c.
i .v .
i.p.
Mouse, i.v.
Rat, i.v.
Rat , oral
i.p.
Mouse, oral
s.c.
i.p.
Mouse, oral
s.c.
i .v .
i.p.
LD5Q (mg/kg)
28
67
110
35
38
19
37
115
8.2
11.6
72
5.8
55
11
10
48
10
7.5
31.5
23
10
600
110
250
70
168
400
400
0.36
400
6.8
2.6
221
147
6.6
40.8
Reference
18
19
20
19
20
19
19
21
22
21
12
22
23
23
23
24
24
24
25
25
25
26
26
26
27
28
8
29
29
8
30
29
17
17
17
17
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Table XIV (continued)
2 of 2
Toxin3
Rugulosin
Cyclochlorotine
Islanditoxin
Species and Route
Rat, i.p.
Mouse, i.p.
Mouse, oral
s .c .
i .v.
Mouse, oral
s .c.
i .v .
LD5Q (mg/kg)
44
55
6.55
0.48
0.34
6.5
0.47
0.3
Reference
31
31
32
32
32
Cited in
Cited in
Cited in
33
33
33
See Table XII for structural formulas
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citrinin are primarily nephrotoxic, causing kidney enlargement, tubular
necrosis and disruption of renal functions in varying animal species (39, 40;
rev. 9). These two toxins have been suspected to be the etiologic agents of
porcine nephropathy in Denmark (41). There is also epidemiologic evidence
suggesting that these two mycotoxins nay be involved in the endemic "Balkan
nephropathy," a renal disorder of approximately 20,000 people living along the
Danube River in Romania, Bulgaria, and Yugoslavia (42, 43).
Chu and associates (44, 45) have studied the relationships between the
structure and toxicity of ochratoxin A and its derivatives in ducklings.
Whereas the methyl ester and the ethyl ester (ochratoxin C) of ochratoxin A
are as toxic as ochratoxin A, the dechlorinated analog (i.e. , ochratoxin B) of
ochratoxin A, the 4-hydroxylated ochratoxin A (i.e. , ochratoxin D) and the
hydrolysis products of ochratoxin A (i.e., ochratoxin ££,) and of ochratoxin B
(i.e. , ochratoxin A ) are much less toxic. On the basis of these findings,
Chu and coworkers (44, 45) suggested that the presence of a chlorine atom
and/or a phenolic hydroxyl group in the dissociated form are important for the
toxicity of these compounds. It was noted that the higher the pK value, the
less toxic the compound; for instance, the pK value of ochratoxin A and some
of its derivatives are: ochratoxin A, 7.07; ochratoxin C, 7.14; ochratoxin B,
7.95, and ochratoxin
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disease mimic those of acute beriberi and are characterized by vomiting,
convulsions, ascending paralysis, and respiratory arrest (46).
In the mouse, the liver is also a target organ of griseofulvin, a potent
porphyrogenic and antimitotic agent which produces various types of liver
damage (47). -Like the well-known spindle poison colchicine, griseofulvin
inhibits the assembly of microtubule and disrupt the mitotic apparatus of the
cell (e .g_. , 48). The antimitotic effect is due to the interaction of the
toxin with the sulfhydryl group of either tubulin (49) or other microtubule-
associated proteins (50, 51).
Patulin is classified principally as a neurotoxin (8, 52). However, the
compound also produces pulmonary edema, hepatic necrosis and gastrointestinal
hyperaemia in the rat, mouse and hamster (23-25, 53). Oral administration to
humans has been reported to result in nausea and stomach irritation. Applica-
tion of ointment containing 1% patulin to the human skin caused edema (cited
in 9). Studies in mice showed that the toxicity of patulin is enhanced by
treatment with SKF-525A, indicating that the parent compound, not a metabo-
lite, is the toxic form of this mycotoxin (53). On the other hand, penicillic
acid, which causes a generalized necrosis of hepatocytes and various histo-
pathological lesions of the kidney and thyroid gland in mice, is possibly be
metabolized into a more toxic intermediate(s), since the acute toxicity of
penicillic acid in the mouse is increased by pentabarbital and 3-methylchol-
anthrene pretreatment but decreased by SKF-525A (54).
PR toxin causes edema in the lung and direct damage to the liver, kidney
and heart in mice, rats and cats (12, 22). Comparison between the chemical
structures and the biological properties of some eremophilane compounds
related to PR toxin suggested to Moule et al. (55) that the aldehyde group in
105
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position 12 rather than the two epoxide moieties, or the acetyl group on the
eremophilane ring is associated with the toxic effects. This is supported by
the finding that PR imine, an analog of PR toxin without the aldehyde group in
12, is considerably less toxic in the mouse than the parent compound (12, 55).
Penicillin G possesses potent antimicrobial activity against gram-
positive and gram-negative cocci, gram-positive bacilli, spirochetes, actino-
mycetes and psittacosis virus. In some individuals receiving sodium penicil-
lin G for treatment of infectious diseases, local and generalized allergic
reactions, convulsions, bronchospasm and nephropathy may occur (see rev. 14).
Mutagenic Effects. The mutagenicity of Penicillium toxins has been
tested in Salmonella typhimurium, Bacillus subtilis, Escherichia coli,
Aspergillus nidulans, Saccharomyces cerevisiae and several mammalian systems
(Table XV).
According to the data from the studies using S. typhimurium strains TA98,
TA100, TA1535 and TA1538, only PR toxin shows positive results in one study
(61) using strain TA98 with the addition of S-9 mix; all other toxins were
negative with and without S-9 mix (8, 56, 57, 60, 61, 64, 66, 75, 77, 83).
However, considerable concern has been voiced regarding the sensitivity and
adequacy of such screening systems for these toxins since these tester strains
detect only reverse mutations representing only limited types of genetic
alterations. Indeed, Stark et al. (83) showed the mutagenicity of rugulosin
and a photoproduct of luteoskyrin (lumiluteoskyrin) in_£. typhimurium strain
TM677 which detects forward mutations. The. mutagenesis assay was carried out
in suspension at low concentrations for long exposure periods. Addition of
rat liver microsomes to the assay system diminished the mutagenicity. In
1982, a new Salmonella tester strain, TA97, was developed to replace strain
106
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Mutagenicity of Some Penicillium Toxins
Toxin3
Ochratoxin A
Citrinin
PR toxin
Patulin
Penicillic acid
Penicillin G
Griseofulvin
Rubratoxin B
Luteoskyrin
Rugulosin
Cyclochlorotine
Salmonella
typhfmurium
- (56,57)c
- (56,57,60,61)
+ (61,63)d
- (64)
- (56-59,66)
- (57,60,61)
- (75)
- (56,57,61,77,78)
- (56,60)
- (57,61,83)
+e (83)
- (61,83)
- (8)
Bacillus
subtilis
- (58)
+ (58)
+ (58)
+ (58)
+ (58)
- (76)
- (58,76)
- (58)
+ (58)
+ (58)
n.t.f
Saccharomyces
cerevisiae
- (56)
- (56)
+ (65)
+ (67)
- (56)
- (56)
n.t.
- (56)
- (56)
+ (84)
+ (84)
n.t.
Chromosomal
Aberrations
- (59)
+ (62)
n.t .
+ (59,68-70)
+ (59,74)
n.t .
- (77)
+ (81)
- (59)
n.t .
n.t.
Other
Tests
n.t .
-g (62)
-8 (64)
+8 (71)
-8 (68)
-h (72,73)
n.t .
+k (76)
-h (79)
-^ (80)
+J (78)
+h (82)
n.t.
n.t .
n.t .
aSee Table XII for structural formulas.
bStrains TA98, TA100, TA1535, TA1537 and/or TA1538.
c"+" = positive; "-" = negative; numbers in parenthesis are references,
dStrains TA97, TA98.
eStrain TM677.
n.t. = not tested.
&Sister-chromatid exchange assay.
Mouse dominant lethal assay.
1Aspergillus nidulans.
JSperm abnormality assay in mice.
k
Escherichia coll.
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TA1537 for the detection of frameshift mutagens (63). The mutagenicity of PR
toxin was again demonstrated in this more sensitive strain (63). Other
Penicillium toxins have not been tested for their mutagenic properties in
strain TM677 or strain TA97 of _S. typhimurium.
When the genotoxicity of Penicillium toxins was studied in the rec assay
in the recombination-deficient mutant of Bacillus subtilis M45 (rec-) and in
the parent strain H17 (rec+), positive results were found with citrinin, PR
toxin, patulin, penicillic acid, luteoskyrin and rugulosin (58). Penicillin G
(76), ochratoxin A, griseofulvin and rubratoxin B (58) were not mutagenic.
The latter three compounds, as well as citrinin, patulin and penicillic acid
were also not mutagenic in Saccharomyces cerevisiae strain D3 (56). Studies
of Wei et al. (65), on the other hand, showed that PR toxin is a direct acting
mutagen toward J3. cerevisiae strains D4 and D7, causing reverse mutation, gene
conversion and mitotic crossing-over without metabolic activation. In agree-
ment with the toxicity results reported by Moule et al. (55), structure-
mutagenicity relationship analysis revealed that the aldehyde and the keto
groups but not the two epoxide moieties play the key role in the genetic
activity of PR toxin. Patulin was reported to be mutagenic in an extrachromo-
somal mutation system of a haploid strain of S. cerevisiae (67). Luteoskyrin
and rugulosin, at low concentrations, induced a high frequency of mutations in
a respiratory-deficient mutant strain of _S_. cerevisiae (84). Studies using
the Escherichia coli DNA-repair assay system showed that penicillin G is
mutagenic in the absence of microsomal activation (76).
In accord with the negative results obtained in some microbial assay
systems, which detect point mutations, cytogenetic studies showed that ochra-
toxin A (59) and griseofulvin (77) had little effects on the incidence of DNA
single-strand breaks and chromosome aberrations in mouse cells. Also, treat-
107
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raent of somatic or sperm cells of the mouse with luteoskyrin did not produce
any increase in the rate of chromosomal aberrations (59). However, in the
experiments of Kappas and Georgopoulos (80), low concentrations of griseo-
fulvin caused increased frequencies of somatic segregation due to chromosome
nondisjunction in a diploid strain of Aspergillus nidulans. Data obtained
from the sperm abnormality assay of the mouse also showed that griseofulvin is
mutagenic (78). Citrinin (62), patulin (59, 68-70), penicillic acid (59, 74)
and rubratoxin B (81) have all been demonstrated to be clastogenic in cells of
the mouse, hamster or humans. Whereas citrinin (62), patulin (68) and PR
toxin (64) are inactive in the sister-chromatid exchange (SCE) assay in
Chinese hamster V79 cells, patulin induces significantly elevated frequency of
SCE in human lymphocytes (71). Rubratoxin B (82) but not patulin (72) or
griseofulvin (79) showed any rautagenic effects in the mouse dominant lethal
assay. The result is also negative for patulin in a dominant lethal assay in
rats (73). The structural requirement for the dominant lethal effect of
rubratoxin B is the (?(, /^-unsaturated lactone ring (82).
Teratogenic Effects. Ochratoxin A, rubratoxin B, griseofulvin, PR toxin
and patulin have all been demonstrated to be embryotoxic and teratogenic in
experimental animals.
Exposure of pregnant mice during early stage of gestation (days 8 and 9)
to ochratoxin A resulted in increased prenatal mortality and a variety of
gross and skeletal abnormalities. The major abnormalities are cranio-facial
cleft associated with exencephaly and open eyelid, and skeletal defects
involving ribs and vertebrae (85). When mice were exposed to the toxin during
the 15th, 16th and 17th day of gestation, significant developmental delay was
noted in the pups as indicated by performance in several behavioral tests
(86). Teratogenic effects similar to those in mice were found in fetuses from
108
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pregnant rat given low doses (0.25, 0.50 or 0.75 mg/kg) of ochratoxin A by
gavage on day 20 of gestation (87). At doses higher than 1 mg/kg, ochratoxin
A was embryocidal in the rat (87-89). Golden hamsters are more resistant to
the fetotoxic effects of ochratoxin A. The toxin is also highly teratogenic
in this species, since high, incidence of malformations such as micrognathia,
hydrocephalus, micromelia, and heart defects occurred in offspring of pregnant
golden hamsters injected intraperitoneally with 2.5-20 mg/kg ochratoxin A on
gestation day 7, 8, 9 or 10. The highest dose (20 rag/kg) caused increased
prenatal mortality when given on day 7, 8 or 9 of gestation (90). Ochratoxin
A also induces embryotoxic and teratogenic effects in chicken. Injection of
ochratoxin A (0.5-7 yug/egg) into embryonating eggs resulted in malformations
including short and twisted limbs and neck, microphthalmia, exencephaly,
everted viscera, and decreased length of survival and body size of the chicken
(91).
Like ochratoxin A, rubratoxin B is also teratogenic and induces similar
abnormalities in chick embryos (92). Intraperitoneal administration of rubra-
toxin B (0.4-1.5 mg/kg) to mice resulted in a dose-related increase in early
fetal deaths as well as in the incidences of fetal defects (82, 93, 94). The
most striking developmental defects caused by rubratoxin B in the mouse are
exencephaly, malformed pinnae and jaws, umbilical hernia and "open eye"
(93). In structure-activity relationship studies it was found that saturation
of the o(.,A-unsaturated lactone ring in the molecule abolishes teratogenicity
(82).
Klein and Beall (95) administered 125-1,500 mg/kg of griseofulvin orally
to groups of pregnant rats during organogenesis. Increased frequency of
skeletal abnormalities and decreased pre- and postnatal survival rates were
observed in the offspring of dams treated with high doses of griseofulvin
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(1,250 and 1,500 mg/kg). Scott _et_ a\_. (96) reported multiple congenital
malformations in kittens of three cats given oral doses of 500 or 1,000 mg
griseofulvin at weekly intervals during pregnancy. In a chick embryotoxicity
screening test, embryonic death and abnormal development of the caudal trunk
were observed after administration of 100 ug and 10 ug of griseofulvin,
respectively (34). The corresponding doses with PR toxin to exert such
effects in this test were merely 0.1 yug and 0.01 /ug. Griseofulvin causes
embryonic death and abnormalities in newborn animals by interfering with the
formation of cell organelles, especially with the mitotic spindle (see Section
5.3.1.2.2.2).
Treatment of pregnant mice with 10-40 mg/kg citrinin (97) or 30-90 mg/kg
penicillic acid (98) caused a significant increase in prenatal mortality of
the offspring at the highest doses, but no malformations were noted in the
surviving fetus. There were no defects in the fetuses of mice (99) or rabbits
(100) given daily doses of 30-300 mg/kg (mice) or 10-100 mg/kg (rabbits)
penicillin G during pregnancy. Similarly, no evidence of teratogenicity was
found in the mouse (72) or rat (73, 101) administered patulin in the range of
1.5-15 mg/kg body weight. However, Ceigler and associates (102) observed
various skeletal abnormalities in chick embryos treated with patulin. Upon
incubation of human placenta with patulin, Fuk-Holmberg (101) noted sharp
increases in the activities of malate dehydrogenase and RNase. These effects
of patulin on placental enzymes were interpreted by the author as indicating
physiological and functional disorders in the tissue.
5.3.1.2.3 Carcinogenic ity and Structure-Activity Relationships
The carcinogenicity of Penicillium toxins were first suggested by the
observations that chronic ingest ion by mice or rats of diets containing cul-
110
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tures of molds (producing these toxins) resulted in the induction of neo-
plasms. In Swiss mice fed a rice culture of _P_. viridicatum (the fungus that
produces ochratoxin A, citrinin, penicillic acid and griseofulvin) in the diet
(7.5%), a 57% higher incidence of pulmonary tumors was observed than in the
controls (103). Similarly, administration to 30 rats of diets containing rice
cultures of P. islandicum (which produces luteoskyrin, cyclochlorotine and
islanditoxin) led to the development of hepatomas in 5 animals (104).
So far, only a small number of Penicillium toxins has been studied
adequately for carcinogenicity in long-term experiments, due probably to their
potent toxicity and to the limited production of these metabolites by fungi.
The evidence is substantial for the carcinogenicity of ochratoxin A, griseo-
fulvin, luteoskyrin and cyclochlorotine in experimental animals. Results from
preliminary studies also point to a carcinogenic potential of citrinin, PR
toxin and rugulosin. Although carcinogenicity has not been demonstrated by
other routes of administration, patulin, penicillic acid, and penicillin G are
tumorigenic in rats following subcutaneous injection. Islanditoxin, a cyclic
peptide isomeric to cyclochlorotine, was described as a carcinogenic mycotoxin
(105). The carcinogenicity studies on Penicillium toxins are summarized in
Table XVI. It is interesting to note that ochratoxin A, patulin and penicil-
lic acid all contain a lactone moiety in their molecules. Like aflatoxin and
sterigmatocin, citrinin, griseofulvin, luteoskyrin and rugulosin are biosyn-
thesized by the acetate-malonate pathway (124; rev. 9) and all contain a
phenol or quinone moiety (see Table XII).
In general, the organ or tissues which are susceptible to toxic effects
of these toxins are also the targets for tumor induction. The hepatotoxins
luteoskyrin, rugulosin, cyclochlorotine and griseofulvin all induce liver
neoplasms whereas the nephrotoxin citrinin is carcinogenic toward the kidney
111
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Table XVI
Carcinogenicity of Penicillium Toxins
Toxin3
Ochratoxin A
PR toxin
Citrinin
Patulin
Penicillic acid
Penicillin G
(sodium salt)
Griseofulvin
Rubratoxin B
Luteoskyrin
Rugulosin
Cyclochlorotine
Principal Organs Affected
Species and Strain and Route
Mouse, ddY
Rat, F344
Rat , Wistar
Rat, albino
Rat, F344
Rat , — c
Rat, Sprague-Dawley
Rat, — c
Mouse, — c
Rat, — c
Mouse, Alderley Park
Mouse, Charles River albino
Mouse , Swiss
Mouse, white, nunu
Mouse, Swiss
Rat, MRC-Wistar
Rat , Wistar
Rat, guinea pig, rabbit, — c
Hamster, Syrian
Rat , Fischer
Mouse, ddNi , ddN
Mouse, ODD
Mouse, ddYS
Rat , F344
Mouse, ddNi, ddN
Liver, kidney; oral
Liver ; oral
None ; oral , s . c .
Neck, uterus; oral
Kidney; oral
Local sarcoma; s.c.
None; oral
Local sarcoma; s.c.
Local sarcoma; s.c.
Local sarcoma; s.c.
Liver; oral
Liver; oral
Liver ; s.c.
Liver; — c
Liver; oral
Thyroid; oral
None ; i . p .
None; oral
None; oral
None; oral
Liver; oral
Liver; oral
Liver; oral
Liver ; oral
Liver; oral
Reference
106
107
108
109
110
111
112
111, 113,
114
114
111, 113
115
116
117, 118
119
120
120
121
115
120
29
32
122
123
107
32
See Table XII for structural formulas.
Based on the initiating and promoting activities in liver carcinogenesis.
Strain/route of administration not reported.
-------
(see Table XVI). However, studies of griseofulvin and PR toxin have also
revealed tumor induction in the thyroid, uterus and/or neck of animals, indi-
cating that several target tissues are affected by Penicillium toxins. The
histogenesis and ultrastructural changes of liver tumor cells following treat-
ment with hepatotoxic raycotoxins of this class have been fully described and
were shown to be similar to findings in human hepatomas (125).
Ochratoxin A. In 1971, a pilot study on the carcinogenicity of ochra-
toxin A was conducted in rainbow trout (Salmo garidneri) . Hepatomas were
noted in rainbow trout fed ochratoxin A at the level of 20 ppb together with
the cocarcinogen, sterculic acid. However, no tumors were found when ochra-
toxin A was fed alone at the levels of 16, 32 or 64 ppb for 8 months (126).
Ochratoxin A is a fairly strong carcinogen toward the liver and kidney of
the mouse. Feeding 40 ppm ochratoxin A in the diet for 44 weeks produced 8
hepatic cell tumors, 5 renal cell tumors, and 18 cyctic adenomas of the kidney
in 19 ddY mice. Whereas dosing with aflatoxin B, (a single dose of 20 rag/kg)
alone elicited only 2 hepatic cell tumors and no renal cell tumors in 18 mice,
administration of aflatoxin B, followed by ochratoxin feeding (40 ppm, 44
weeks) induced 15 hepatic and 3 renal cell tumors in 20 mice, indicating a
synergistic effect of aflatoxin Bi on hepatocarcinogenesis of ochratoxin
(106).
In the rat (Wistar-derived), Purchase and Van der Watt (108) failed to
induce a significant incidence of tumors by administering either 2.5 mg/kg
ochratoxin A subcutaneously twice weekly for 17.5 weeks or 0.3 rag ochratoxin A
orally 5 times/week for 50 weeks. They have noted a hamartoma of the kidney
in one of the ten rats which received ochratoxin A orally. Using F344 rats,
Imaida et al. (107) investigated initiation and promotion by ochratoxin A in
112
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liver carcinogenesis. In these bioassays, N-2-fluorenylacetamide (200 ppm in
diet) was used as an initiator (or a promoter) and ochratoxin A was given to
the rats at a dietary level of 50 ppm for 6 weeks during the initiation stage
(or the promotion stage). Ochratoxin A displayed both initiating and promot-
ing activity and was termed a hepatocarcinogen (107).
PR Toxin. The carcinogenic potential of PR toxin in the rat has been
investigated by Polonelli _e_t^ _al_. (109). A group of 10 albino weanling rats of
both sexes was given 200 ppm PR toxin in drinking water for 52 days. About 13
months after the treatment, a squamous epithelioma developed in the neck
region of one rat and after about 3 more months of observation, an uterine
sarcoma was detected in another rat. None of the 10 matched control animals
developed any tumors during the same course of the study. Although the tumor
incidences are not statistically significant and further studies are needed,
the development of these tumors, particularly the squaraous epithelioma in the
neck, was considered treatment-related on the basis of historical data showing
that spontaneous tumor of this type is rare in the rat.
Citrinin. Early investigations have demonstrated both the tumorigenesis
initiating and promoting activity of critinin in the rat. Imaida and co-
workers (107) showed that administration of citrinin to F344 rats in the
initiating stage and of N-2-fluorenylacetamide in the promoting stage signifi-
cantly increased the number and area of liver hyperplastic nodules as compared
with those in the control group (which did not receive citrinin pretreat-
ment). Whereas N-(3,5-dichlorophenyl)succinimide (NDPS) or citrinin alone did
not induce kidney tumors in Sprague-Dawley rat, feeding of NDPS for 8 weeks
followed with citrinin (0.02%) for 20 weeks resulted in renal cell tumor in 4
of 18 rats (127). Moreover, the kidney tumor incidence in rats treated with
113
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citrinin following dimethylnitrosamine (DMN) was much higher than the inci-
dence in rats treated with DMN alone (127).
Arai and Hibino (110) were the first to present direct evidence showing
that citrinin is indeed carcinogenic, producing kidney adenomas in the rat.
Among 48 male F344 rats given 0.1% citrinin in the diet for up to 80 weeks, 35
(72.9%) developed renal epithelial tumors (not found in the controls).
Patulin and Penicillic acid. Interest in the carcinogenicity studies of
patulin and penicillic acid arose in the early 1960's when F. Dickens and
H.E.H Jones of England drew attention to the possible carcinogenic activity of
chemicals having a lactone ring in the molecule (see Vol. IIIA, Section
5.2.1.1.6 on lactones). Patulin and penicillic acid, both having a five-
membered lactone ring and an ct_, B-unsaturated bond, are clearly carcinogenic
by repeated injections into rats (111, 113, 114). When 0.2-2.0 mg patulin was
injected twice weekly into subcutaneous sites in the flank of 2-month-old male
rats, local sarcomas arose in six of eight rats that survived for 1 year.
Penicillic acid, at doses of 1 mg in arachis oil, gave rise to highly malig-
nant tumors at the injection sites in all four rats that survived 64 weeks of
treatment (111). Later experiments showed that a dose of penicillic acid as
low as 0.1 mg is sarcomatogenic in one of four animals surviving for 94 weeks
(113, 114). Subcutaneous injections of aqueous solution of penicillic acid (2
rag/0.5 ml water) also produced sarcomas in 4 of 5 surviving rats indicating
that the oil vehicle dose not play a significant role in the tumorigenicity
(113). Similarly, local sarcomas occurred in 6 of 19 mice receiving sub-
cutaneous doses of 0.2 mg penicillic acid twice weekly for 65 weeks (114).
Patulin is not carcinogenic in animals by oral administration. A total
oral dose of 358 mg patulin/kg given over a period of 64 weeks produced no
tumors in 50 female Sprague-Dawley rats (112).
114
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Penicillin G. Dickens and Jones (111, 113) have investigated the car-
cinogenic potential of penicillin G in rats by subcutaneous injection for 65
weeks. In an early study, tumors at the injection site were observed in 2 of
8 animals that survived for at least 59 weeks (111). In a subsequent study, 5
of 11 animals developed fibrosarcomas after 108 weeks; one of the tumors was
highly malignant and was transplantable (113). Interestingly, 6-aminopeni-
cillanic acid, a penicillin analog which lacks the benzyl side chain, is a
much less potent carcinogen under the same study conditions (114). The car-
cinogenic action of other members of the penicillin group (see rev. 14), which
contain various side chains, has not been tested.
Griseofulvin. The mouse is highly susceptible to the hepatocarcino-
genicity of griseofulvin. The presence of hyperplastic nodules is readily
seen in the livers of Swiss mice following griseofulvin administration (2.5%
in the diet) for 6 to 8 months (128, 129). High incidence of hepatomas have
been repeatedly reported in various strains of mice following on prolonged
administration of griseofulvin either orally (115, 116, 119, 120) or paren-
terally (117, 118). Among 13 Alderly-Park strain mice which ingested 1%
griseofulvin in the diet for 435 days, 10 were found to bear multiple hepa-
tomas; 5 of 20 mice fed a 0.5% diet also developed tumors in the liver
(115). Rustia and Shubik (120) reported that the liver tumor incidence show a
dose-response in Swiss mice given 0, 0.3, 1.5 and 3.07, griseofulvin in the
diet daily (during alternating 5-week periods for life). At the 3.0% dietary
level, the incidences of hepatoraas in male and female mice were 83.3% and
87.0%, respectively; the corresponding liver tumor incidences at the 1.5%
dietary level were 68.0% and 53.6%; no significant liver tumors were found in
the mice at the 0.3% dose level. In addition to nodular hyperplasia, neo-
plasms of the liver have also occurred in mice of "nunu" strain after 12-14
115
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months of griseofulvin treatment (119). DeMatteis and coworkers (116) noted a
marked sex difference in hepatoma incidence of Charles River albino mice which
received 1% griseofulvin orally for 12-16 months; the male animals showed a
higher incidence as well as multiplicity of these tumors than the females.
Significant incidence of hepatomas was also found in Swiss mice subcutaneously
injected a total dose of 3 mg griseofulvin at birth and infancy (117, 118).
Moreover, cocarcinogenic and promoting effects upon skin tumorigenesis in
Swiss-Webster mice were noted when low doses (10—15 mg/kg) of griseofulvin
were administered orally before, during or following topical applications of
methylcholanthrene (130). In agreement with the tumorigenesis-promoting
activity of griseofulvin in the mouse, in vitro transformation of Swiss 3T3
cells infected with tsA mutants of the virus SV40 was enhanced following
exposure to either griseofulvin or to the potent tumorigenesis promoter,
phorbol ester (131).
In MRC-Wistar rats oral administration of griseofulvin to groups of 30
males and females life resulted in significant incidence of thyroid tumors in
a dose-response manner at dietary levels of 0.2, 1.0 and 2.0% (120). However,
groups of Syrian golden hamsters given 0.3, 1.5 or 3.0% griseofulvin in the
diet for the whole lifespan did not develop tumors (120). Other studies using
rats (115, 121), guinea pigs or rabbits (115) yielded little information on
the carcinogenicity of.griseofulvin. The .failure of several experiments (115,
121) to elicit tumors in these species appears to have resulted from the too
short exposure periods and/or the small number of animals used.
Rubratoxin B. The carcinogenic potential of rubratoxin B has only been
explored by Wogan and coworkers (29) during a chronic toxicity study of rubra-
toxin B in the rat. Groups of 10-20 Fischer rats of both sexes were intubated
with rubratoxin B at a dose of 5 or 10 mg/kg 3 times weekly for 60 weeks. No
116
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evidence of preneoplastic or neoplastic lesions was observed in animals killed
after 82-87 weeks. Also, there was no enhancement of the carcinogenic activ-
ity of aflatoxin B^ by rubratoxin B when rats were exposed simultaneously to
both toxins.
Nonetheless, in view of the reported mutagenicity of the compound (see
Section 5.3.1.2.2) and the presence in the molecule of reactive carbonyl
groups and ethylenic double bonds, further exploration on the possible car-
cinogenic activity of rubratoxin B in other assay systems appears desirable.
Luteoskyrin and Cyclochlorotine. Long-term feeding studies in the mouse
have shown that these two mycotoxins exhibit similar chronic effects and are
both carcinogenic toward the liver. In a series of experiments conducted by
Uraguchi and coworkers (32), significant incidences of benign and malignant
liver tumors were induced in a dose-response manner in groups of 8-30 ddNi and
ddN strain mice fed luteoskyrin (0, 50, 150 or 500 ug/day) or cyclochlorotine
(0, 40 or 60 ^ug/day) for up to 2 years. Of 26 DDD strain mice given daily
doses of 160 ug luteoskyrin in the diet for 328 days, 17 were found by Ueno et
al. (122) to bear hepatomas of various histological types.
Rugulosin. In a preliminary study in which groups of 16 DdYS male mice
were administered daily doses of 12 or 25 rag/kg rugulosin in the diet for over
800 days, 4 animals bearing hyperplastic nodules composed of hepatocytes were
found in both groups. In addition, one animal bearing a hepatocellular
adenoma was found in the high-dose group. None of the 14 control mice had
such lesions in the liver. These results led the authors (123) to suggest
that rugulosin is possibly a weak hepatocarcinogen in mice with a potency
about one tenth that of luteoskyrin. The carcinogenic potential of rugulosin
was supported by a study demonstrating that rugulosin possesses initiating as
well as promoting activity in hepatocarcinogenesis in the rat (107).
117
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5.3.1.2.4 Metabolism and Possible Mechanism of Action
Information regarding the metabolism of these mycotoxins is scanty and
their mechanisms of carcinogenic action is unknown. Previously, we have
discussed the reaction mechanisms of carcinogenic ^-lactones with nucleo-
philic centers (Section 5.2.1.1.7, Vol. IIIA). Similar reactions probably
also occur between nucleophiles and carcinogenic mycotoxins of this group
since they all (with the exception of cyclochlorotine and islanditoxin)
possess one or more lactone or ketone carbonyl groups with &(.,$ -unsaturation
which, upon metabolic oxidation, can be transformed into alkylating inter-
mediates (e.g., epoxides). Consistent with results of mutagenicity studies,
patulin, penicillic acid, rubratoxin B and luteoskyrin all form adducts with
DNA and/or chromatin. The interaction between the sulfhydryl and amino group
of proteins, on one hand, and patulin, penicillic acid, ochratoxin A, luteo-
skyrin and rubratoxin B, on the other hand, is well documented. Such reac-
tions have been postulated to account for a wide range of their biological and
biochemical activities including alteration of carbohydrate and lipid metabo-
lism, inhibition of protein and nucleic acids synthesis and impairment of cell
respiration, membrane transport, etc. It is possible that one or a constella-
tion of these activities acting in a concerted manner may bring about perma-
nent structural and functional changes in the cells, leading eventually to
neoplasia.
Ochratoxin A. The metabolism of ochratoxin A has been studied in several
animal species including the rat (132-134), the pig (135) and the cow (cited
in ref. 9). After a single intraperitoneal injection into rats, ochratoxin A
was detected in the serum, liver and kidney (132, 134). Part of ochratoxin A
was metabolized to ochratoxino(, (the isocoumarin acid derived from the loss
of the phenylalanine moiety of ochratoxin A) and 4-hydroxyochratoxin A which,
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along with the unchanged toxin, were excreted primarily in the urine. Ochra-
toxin 06 is also the major metabolite in pigs (135), cows (cited in ref. 9) or
rats (133) dosed orally with ochratoxin A. Although ochratoxin^ is much
less toxic than ochratoxin A toward chick embryos (136), ducklings (44) and
rainbow trout (137), it is more inhibitory than the parent compound to the
respiration of isolated rat liver mitochondria (138).
Ochratoxin A interacts strongly with serum albumin both in vitro (139)
and in vivo (134). There is no evidence as yet for the binding of ochratoxin
A to nucleic acids. Treatment of rats with ochratoxin A results in signifi-
cant depletion of liver glycogen and decrease of the activities of hepatic
enzymes such as cyclic AMP-protein kinase, carboxypeptidase and phenylalanine
t-RNA synthetase, etc. In certain bacteria ochratoxin A is a potent inhibitor
of protein and RNA synthesis (see 140).
Citrinin. In rats (141), rabbits or dogs (142) citrinin is rapidly
absorbed and excreted. Peak citrinin levels in the serum, liver and kidney
were attained within 30 minutes after parenteral administration. At a non-
nephrotoxic dose of 3 mg/kg, about 74% of the administered citrinin was
excreted, mostly unchanged, in the urine of rats by 24 hours after administra-
tion. However, in rats, rabbits, and dogs, which received higher doses
citrinin, a much smaller percentage of the toxin or its metabolites were
detected in the urine. The metabolites of citrinin have not been identified
as yet. Some of its metabolites are suspected to be dihydrocitrinins (142).
Disturbance of carbohydrate metabolism (143) and inhibition of
proteolysis in kidney phagolysomes (144) were noted in mice treated with
citrinin.
119
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PR Toxin. In addition to mutagenicity data indicating the genotoxicity
of PR toxin, macromolecular binding studies have shown that the compound binds
significantly to RNA, DNA and protein in cultured cells as well as in isolated
nuclei (145). Moreover, Moule et al. (145) have shown that the toxin cross-
links between DNA and protein in the chromatin. The authors implicated exclu-
sively the aldehyde group in the PR toxin molecule, which would form a
methylene bridge between an araino groups in DNA and a functional group in
chromatin protein. However, the present writers feel that cross-linking via
the reactive epoxide groupings in the PR toxin molecule cannot be dis-
counted. PR toxin has also been shown to impair liver cell metabolism by
inhibiting macromolecule synthesis (146).
Patulin and Penicillic acid. Both patulin and penicillic acid are
rapidly absorbed in the gastrointestinal tract. In metabolic studies with
[14C]-patulin (147) or [14C]-penicillic acid (148) in rats, most of the [14C]-
radioactivity was recovered from urine and feces within 24 hours after dos-
ing. However, appreciable levels of radioactivity remained in the red blood
cells, liver, kidney and lung for up to 7 days. Significant amount of radio-
activity becomes bound to DNA, RNA and protein in the liver cells following
administration of [^4C]-penicillic acid to rats (148). The metabolites of
patulin and penicillic acid have not been identified.
Patulin and penicillic acid are potent inhibitors of polymerases (149),
ATPases (150, 151) and various thiol enzymes (152, 153) in vitro. The effects
are presumed to be due to interaction of the toxins with sulfhydryl and amino
groups of these enzymes. Indeed, patulin and penicilTic acid are known to
readily combine with sulfhydryl compounds to form S-alkylated adducts by
interaction of the nucleophilic sulfhydryl group with the double bond(s) (154,
155). Penicillic acid also reacts, albeit at a slow rate, with lysine,
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arginine and histidine, at pH 7.0 (154). The inactivation of polymerases,
ATPases, and thiol enzymes probably accounts for the inhibitory effects of
patulin and penicillic acid on raacromolecular synthesis (156), active membrane
transport (157, 158) and cellular respiration (159). Although the relation-
ship between these biochemical effects and the mechanism of their carcinogenic
action is not clear, investigation of the reaction of unsaturated TT-lactones
with cysteine has shown that S-alkylated adducts are formed only with carcino-
genic lactones but not with noncarcinogenic lactones (160).
Penicillin G. In humans, about 30% of an oral dose of penicillin G is
absorbed in the small intestine, while a large quantity remains unabsorbed and
passes into the colon. The absorbed penicillin G is widely distributed in the
body fluids and tissues. Significant levels of penicillin G can be found in
the liver, bile, kidney and plasma. The compound is excreted mainly through
the kidney and bile; a small amount is excreted in milk and saliva. One of
the urinary metabolites has been identified as 6-aminopenicillanic acid (see
rev. 14), which is a less potent carcinogen than penicillin G. Since the
benzyl side chain is absent in 6-aminopenicillanic acid, Dickens and Jones
(114) speculated that the side chain might contribute to the carcinogenic
action of penicillin G. On the other hand, penicillins were suggested to act
as alkylating or acylating agents (see rev. 161) by way of the probable reac-
tion mechanisms (shown in Fig. 4), which would be influenced little if at all
by the benzyl side chain.
Griseofulvin. The metabolic fate of griseofulvin in mammalian species
have been critically reviewed by Lin and Symchowicz (162). In the mouse, rat,
121
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C02H
Penicillins
N
0' H
C02H
r-"
N
H
Alkylation
Nu:
Acylation
COoH
x VCH
pCH
HN -
^
Fig. A. Probable reaction mechanisms of alkylation
penicillins.
and acylation by
-------
rabbit, dog and human, the rate of absorption is rapid and most of the com-
pound is excreted in the urine as metabolites. Studies in the rat showed that
the highest level of griseofulvin occurs in the liver after oral administra-
tion and in the lung following subcutaneous injection. In the mouse and rat,
both A-desmethylgriseofulvin and 6-desinethylgriseofulvin are the major metabo-
lites; in rabbits, dogs and humans, on the other hand, the only major
metabolite is 6-desmethylgriseofulvin. In the rabbit, griseofulvin is also
metabolized to 3-chloro-4,5-dimethoxysalicylic acid (163). Several unidenti-
fied additional metabolites of griseofulvin have been found in human urine
(164).
In the mouse liver, griseofulvin induces the proliferation of the smooth
endoplasmic reticulum, it increases the amount of NADPH-cytochrome c reduc-
tase, and stimulates the metabolism of other exogenous chemicals (165).
Rubratoxin B. Hayes (166) studied the distribution and excretion
patterns of rubratoxin B in mice and rats. During the first 24-hour period
following administration of [C]-rubratoxin B (0.05 mg/kg, i.p.) to mice and
rats, 30-40% of the radioactivity was excreted through respiration as COj,
6-9% was recovered in the urine and a small amount was found in the feces. In
both species, the concentration of radioactive substances was higher in the
liver than in other tissues. In the liver, radioactivity was about 54-80% in
the cytosol, 14-25% in the mitochondrial fraction, 7-12% in the nuclear frac-
tion and 3-10% in the microsomal fraction. Consistent with the findings of
the subcellular distribution studies, rubratoxin B inhibits oxygen uptake,
ATPase activity and electron transport in liver mitochondria (167), binds to
DNA (cited in rev. 168) and causes disaggregation of polysomes (169).
122
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Luteoskyrin and Rugulosin. Pharmacoki.netic studies in the mouse (170,
o
171) showed slow uptake and slow excretion of H-luteoskyrin following sub-
cutaneous or oral administration. During the 18 days after dosing, only 19%
o
and 6% of the administered H-luteoskyrin were excreted in the feces and
urine, respectively. The liver accumulated 83-94% of the total organ
localized radioactivity; only a minute quantity of radioactivity was present
o
in the lung, kidney and spleen (170). The H-luteoskyrin level in the liver
of male mice is about twice as high than in the liver of females, but is only
about 15% of that in suckling mice (171). Subcellular distribution studies
showed that about 50% of the radioactivity in liver homogenate is localized in
the mitochondria; the nuclear and microsomal fractions contain only small
amounts of radioactivity. More than 80% of the radioactivity in the mito-
chondria represents unchanged H-luteoskyrin (171). Pretreatment of male mice
with 3-methylcholanthrene or promethazine inhibits considerably the accumula-
tion of luteoskyrin in the liver, suggesting that the microsomal mixed-
function oxidases play a role in the detoxification of luteoskyrin (123). The
pharmacokinetics and the distribution pattern of rugulosin in the mouse was
reported to be similar to those of luteoskyrin (cited in rev. 172).
In vitro studies with mitochondrial preparations and whole liver homo-
genates have shown that luteoskyrin inhibits oxidative phosphorylation through
a mechanism similar to that of dinitrophenol in uncoupling phosphorylation and
to oligomycin in inhibiting electron transport (173). In the presence of
divalent cations (e.g. , Mg++, Mn"1"*"), luteoskyrin forms complexes with single-
stranded as well as double-stranded nucleic acids (174-176). Flow dichroism
studies established that luteoskyrin is oriented parallel to the axis of the
double helix of native DNA (176). The binding of luteoskyrin to deoxyribo-
nucleohistone in vitro has also been reported (177). Because of its ability
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to interact with single-stranded nucleic acids, it was suggested that luteo-
skyrin binds to nicked DNA and interferes with DNA repair synthesis. Indeed,
Mouton and Fromageot (178) showed that the repair of UV-induced DNA lesions in
Tetrahymena cells is inhibited by luteoskyrin. There is also evidence that
luteoskyrin interacts with the transcription complex and inhibits the syn-
thesis of RNA in Escherichia coli (179).
Rugulosin is believed to have similar DNA-binding properties as luteo-
skyrin (180).
Cyclochlorotine. Cyclochlorotine is highly resistant to the proteolytic
effects of tissue proteases. Following subcutaneous administration to male
mice, cyclochlorotine is rapidly absorbed and transported to the liver and is
primarily excreted unchanged by the kidney. In vitro studies showed that only
specific proteolytic enzymes having an ability to hydrolyze cyclic peptides
can degrade cyclochlorotine. Removal of the two chlorine atoms of cyclo-
chlorotine by treatment with ammonia or alkali results in loss of toxicity of
the toxin (see rev. 172). Studies with liver preparations have shown that
cyclochlorotine inhibits glycogenesis, decreases the incorporation of amino
acids into proteins and enhances the incorporation of acetate into lipids
(cited in rev. 140). Cyclochlorotine inhibits Na^-dependent glycine transport
in rabbit reticulocytes (158).
5.3.1.2.5 Environmental Significance.
Penicillium toxin-producing fungi can grow at considerably low moisture
content and at wide ranges of temperature and pH, and thus occur ubiquitously
in the environment. Like the Aspergillus, the Penicillium are among the most
common storage fungi in foods throughout the world. Humans may be exposed to
Penicillium toxins by direct contact, by inhalation, by therapeutic use or by
124
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ingestion of the contaminated foodstuffs. Although mounting evidence links
liver cancer to aflatoxin contamination of food corps (see Section 5.3.1.1),
epidemiological evidence on the Penicillium toxin-induced cancer in humans is
lacking. This is not too surprising since epidemiological studies on many of
these toxins are still in their infancy. Nonetheless, fungal toxins are
increasingly suspected to be possible etiological agents of some human cancers
(see refs. 181, 182).
Table XVII summarizes the natural occurrence of several carcinogenic
Penicillium toxins, which has been the subject of many reviews (e.g., 9, 10,
183).
Ochratoxin A. Ochratoxin A has been detected in corn (0.083-0.166 ppm),
wheat (0.03-27 ppm), rye (0.24 ppm), mixed oat and barley (22 ppm), beans
(0.02-2.1 ppm) and peanuts (4.9 ppm) during surveys in Canada (184, 185) and
in the United States (186, 187). In districts of Denmark where a high inci-
dence of porcine nephropathy occurred, up to 27.5 ppm and 0.067 ppm of ochra-
toxin A were found in about 20% of the plant (cereals) and animal (pork,
poultry) products sampled, respectively. Residues of ochratoxin A have also
been detected in various food commodities of seven other European countries
(see rev. 188).
Citrinin. Citrinin was detected in 13 of 29 grain samples from Canadian
farms at concentrations of 0.07 to 80 ppm. These samples were mainly wheat,
but there were also samples of rye, oats and mixed oats, and barley containing
citrinin (185). In addition to ochratoxin A, low levels (0.16 to 2 ppm) of
citrinin were found in 3 samples of cereals from Denmark (41). There are also
reports on the presence of citrinin in moldy ground nut (189) and in rotten
apples (190). One of the citrinin-producing fungi (P_. c itrinum) was isolated
from the Japanese "yellowed rice" imported from Thailand (see rev. 17).
125
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Table XVII
Natural Occurrence of Some Penicillium Toxins'
Toxin
Producing Fungus
Occurrence
Ochratoxin A
Citrininc
PR toxin
Patulind
Penicillic acid°
P. viridicatum; P_. purpurescens ;
P. palitans; P. commune;
P. eye 1opium; P. variabile
P. citrinum; P. citreviride;
P- viridicatum; P. citreo-viride;
P. fellutanum; P. lividum; etc.
P_. jroquefort i
P. patulum; P. expansum;
£• urticae; P_. c yd opium;
P. lapidosum; P. terrestre
P. puberulum; P. viridicatum;
P. thomii; P. suavolens;
P. martensii ; _P_. palitans ;
P. expansum; P^. commune;
P. qlivino-yiride; etc.
P. griseofulvin; P. viridicatum;
_P. nigricans; _P_. urticae;
P. jpatulum
Corn, wheat, oat,
rye, barley, bean,
peanut, pork, poultry
Wheat, oat, barley,
groundnut, apple,
rice
Silage
Apple
Corn, bean, tobacco
Wheat, bean, flour
P. chrysogenum; P. notatum
—' rubrum; ,£.• purpurogenum
_P_. is land icurn
P. rugulosum; P. brunneum;
P. tardurn; P. ^yariabile
P. islandicum
P. islandicum
Wheat, flour, rice,
fermented foodstuffs
Corn, bean, peanut,
silage
Rice
Rice
Rice
Rice
Griseofulvin
Penicillin G
Rubratoxin B
Luteoskyrin
Rugulosin6
Cyclochlorotine
Islanditoxin
References cited in J.M. Hamburg, P.M. Strong and E.B. Smalley, J. Agr. Food
Chem. 17. 443 (1969); IARC Monographs, Vol. 10, 1976; P.M. Scott, Penicillium
Mycotoxins, In "Mycotoxic Fungi, Mycotoxins, Mycotoxicoses, An Encyclopedia
Handbook" (T.D. Wyllie and L.G. Morehouse, eds.), Vol. 1, Part 2, Marcel
Dekker, New York, 1977, p. 283.
Also produced by Aspergillus ochraceus, A. sulphureus, _A. alliaceus, _A.
sclerotorium, A. melleus, A. ostianus and A_. j>etrakii.
cAlso produced by Aspergillus terreus, A. niveus, A. candidus and Clavariopsis
aquatica.
Also produced by Aspergillus flavus, £. clavatus, _A. giganteus, _A. terreus
and Byssochlamys nivea.
eAlso produced by Myrothecium verrucaria.
-------
PR Toxin. PR toxin is the major fungal metabolite isolated from moldy
silage associated with cases of bovine poisoning in Wisconsin (191). Several
strains of PR toxin-producing fungus are used in the ripening of roquefort
cheese (see ref. 65).
Patulin. Patulin occurs primarily in rotten apples and related products
since patulin-producing fungi are common causes of the storage rot of apples
(see rev. 9). The toxin has been detected in 8 of 13 samples of apple juice
from the United States at levels of 49 to 309 jug/liter (192). Also, five of
11 apple juice samples from Canada contained 20 to 120 ug patulin/liter
(193). The concentration of patulin in apple cider made from rotten apples
may be as high as 45 mg/liter (194).
Penicillic acid. Penicillic acid has been identified in moldy corn (195)
and in poultry feed (196). Thorpe and Johnson (197) found the toxin in 7 of
20 samples of commercial corn (5-230/ug/kg) and in 5 of 20 samples of commer-
cial dried beans (11-179 /ug/kg) from the United States. Snow£t__aJ_. (198)
found 110 and 230 /ug/kg of penicillic acid in two samples of moldy tobacco.
Penicillin G. Penicillin was introduced for therapeutic use in the early
1940's. The drug was extracted from cultures of Penicillium notatum. Since
then, many new derivatives of the basic penicillin nucleus have been dis-
covered and produced. Presently, members of this important group of anti-
biotics remain drugs of choice against a wide variety of infectious
diseases. Penicillin G is the most effective against infectious diseases
caused by gram-positive and gram-negative cocci, gram-positive bacilli, spiro-
chetes, actinomyces and psittacosis virus. Preparations of penicillin G for
oral and parenteral administration, as well as for topical, ophthalmic and
vaginal uses are all available (see rev. 14).
126
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Penicillin G and several natural penicillins, are presently prepared from
a strain of Penicillium chrysogenum that grows on the stem of cantaloupes.
High yields of penicillin G are produced by submerged fermentation of a mutant
of the mold, induced by x-rays (see rev. 14). Penicillium chrysogenum has
been detected occasionally in wheat, rice and in some fermented foodstuffs
consumed daily by most Japanese (183, 199).
Griseofulvin. Griseofulvin is produced by many species of Penicillium
(see Table XVII). These fungi have been detected in wheat, beans and flour
(183). Griseofulvin is often used in human medicine for the treatment of
dermatophytoses. The annual sales of griseofulvin in the United States are
estimated to be in the order of 25,000 kg (see ref. 10).
Rubratoxin B. Owing to difficulties in detecting rubratoxin B in complex
substrates, there are as yet no reports about its natural occurrence in agri-
cultural products. However, fungi that produce rubratoxin B have been
repeatedly isolated from cereal and legume products, corn, peanuts and from
feeds which have caused liver disease in farm animals (see ref. 168).
Luetoskyrin, Rugulosin, Cyclochlorotine and Islanditoxin. These are
commonly referred to as "yellowed rice toxins" because they are metabolites of
predominant storage fungi associated with heavily moldy rice ("yellowed rice")
of Japan. Contamination by fungi which produce these toxins was found in rice
both originated from Japan and imported from Thailand, Burma or other Asian
countries. Since rice constitutes a major part of the diet of Asian popula-
tions, the high incidence of liver disease, including cancer, has been sus-
pected to be related to consumption of rice contaminated by these carcinogenic
toxins (see ref. 200). Yellowed rice toxin-producing fungi are also major
isolates from Danish barley as well as from various African grains (see rev.
201).
127
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