CURRENT AWARENESS DOCUMENT
ANTHRACYCLINE-TYPE, PHENOXAZONE-TYPE AND OTHER
STREPTOMYCES-GENERATED CARCINOGENS
CARCINOGEN1CITY 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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5.3.1.3 Anthracycline-type, Phenoxazone-type and Other Streptomyces-Generated
Carcinogens
5.3.1.3.1 Introduction
Since the discovery of actinomycin by Waksman (1) in 1940, numerous other
antibiotics have been isolated from the Streptomyces (formerly actinomyces) --
a group of unicellular, branching organisms morphologically resemble fungi but
are classified as bacteria. Antibiotics of this group are of particular
interest because of their use in the chemotherapy of cancer and in studies of
molecular and cellular biology. Actinomycin D, adriamycin, daunomycin, mito-
mycin C, sarkomycin, streptozotocin, azaserine and bleomycin, for instance,
all exhibit remarkable effects in repressing the growth of various neoplasms
(see rev. 2). Many of these compounds, especially actinomycin D, mitomycin C
and bleomycin, have extensively been used as tools in studies of nucleic acid
synthesis and other cellular activities, and in the elucidation of the binding
site of antibiotics on DNA (see ref. 3). Strong affinity and interaction of
these naturally occurring substances with DNA is believed to play a major role
in their antibiotic and antineoplastic activities.
The increasing number of therapeutically used agents which were found to
be carcinogenic in experimental animals (see Section 5.2.1.7.11, Vol. IIIA of
this monograph) generated considerable concern about the carcinogenic poten-
tial of these antitumor drugs in humans. Studies in rodents showed that some
naturally occurring products from Streptomyces are indeed carcinogenic (4,
5). Consistent with these findings are reports on the development of second
primary neoplasms in some cancer patients receiving chemotherapy with these
compounds (see 5-7). The structural formulas of the Streptomyces toxins,
which have been tested for carcinogenic activity, are presented in Table
XVIII.
144
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Table XVIII
Streptomyces Toxins Which Have Been Tested for Carcinogenic Activity.
Sorcosine Sorcosirw
^ \ / \
L-Prolint L-Methylvalin* L-Prolint L-Methylvolin*
Ri.
,
L-Threonine
N
A-fhreonine
NH2
0
CH,
CH,
Actinomycins
Actinomycin C( (orD)
R, =R2=D-Valine
Actinomycin C2
RI =DTAIIoisoleucine
R2:D-Valine
or R| =D-Valine
R2 = D-Alloisoleucine
Actinomycin C$
RI = R2=D-Alloisoleucine
H2N H
Daunomycin:R=-H Adriamycin: R=-OH
HO
Bleomycins
Bleomycin A2: R=-NHCH2CH2CH2-SN
®/CH3
CH,
yiNn
Bleomycin B2: R=-NHCH2CH2CH2CH2NH(f
NH2
NH
II
Bleomycin 64: R= -(NHCH2CJH2CH2CH2NHC)2-NH2
NH
II
Bleomycin Bg: R= -(NHCH2CH2CH2CH2NHC)3-NH2
-------
Table XVIII (Continued)
0
Mitomycin C
CH,OCNH,
HOOC-HC-OCH2
/ \ *
Sarkomycin
O
© © V
N = N = CH . CO. CHo . C-COOH
H
Azaserine
n-Cr,Hn-CH = CH-N
6 u
O
Elaiomycin
CH2-OCH3
CHOH
I
CH
02N
OH CH,OH 0 -'':
ii n
CH-CH-NH-C-CHCI2
Chloramphenicol
Streptozotocin
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5.3.1.3.2 Physicochemical Properties and Biological Effects
5.3.1.3.2.1 PHYSICAL AND CHEMICAL PROPERTIES
The actinomycins are chromopeptides; all contain in their molecule the
chromophore, 2-amino-4,5-dimethylphenoxazin-3-one-l,8-dicarboxylic acid,
linked to two pentapeptide lactone rings. The lactone grouping consists of
the L-methylvaline carbonyl (not shown in Table XVIII) linked to the oxygen
bridge. The various actinomycins differ chemically only in the amino acid
composition of the two cyclic polypeptide chains. For example, the difference
between actinomycin Ci (same as actinomycin D) and actinomycin C-j is that
actinomycin Co contains two molecules of D-alloisoleucine in place of two
molecules of D-valine in the pentapeptide rings. 'Actinomycin C2> on the other
hand, contains one molecule of D-alloisoleucine and one molecule of D-valine
(8). Actinomycin C is a mixture of Ci, C2 and Co, whereas actinomycin S and L
may contain C~ and Co in addition to C, (9). Although changes in the amino
acids of the polypeptide rings may alter the biological activity (3, 10), most
of the chemical reactions of the actinomycins are due to the chromophore
moiety (11). Thus, reaction with dilute alkali leads to the opening of the
ester-like oxygen bridge and the disappearance of the red-color of the
toxin. Owing to the aminoquinoneimine structure, the chromophore is believed
to form free radicals which may participate in various reactions. Substitu-
tion of the amine on the aminoquinoneimine moiety leads to the loss of the
biological activity of the actinomycins, presumably by affecting the reactions
or formation of the free radicals (see 12). Some physical and chemical pro-
perties of actinomycin D and of other carcinogenic metabolites of the
Streptomyces are given in Table XIX.
145
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Table XIX
Physical Properties of Some Carcinogenic Streptomyces Toxins3
Toxinc
Physical form ra.p. Optical rotation
Solubility
Actinomycin D
Adriamycin
(Doxorubicin)
Daunomycin
(Daunorubicin)
Bright red, 241.5
rhomboid prisms 243°C
Red, crystal- 205°C
line solid
Thin, red 188-
needles 190°C
= -315
= +248°
= +248°
Slightly soluble in
water and ether; soluble
in propylene glycol and
in water/glycol mixture;
very soluble in ethanol .
Slightly soluble in
water; insoluble in non-
polar organic solvents;
soluble in ethanol.
The hydrochloride is
soluble in water,
methanol and ethanol;
insoluble in chloroform,
ether and benzene.
Mitomycin C
Blue-violet
crystals
above
360°C
Soluble in water,
methanol, acetone, butyl
acetate and cyclohexa-
none; slightly soluble
in benzene, carbon
tetrachloride and ether.
Sarkotnycin
. Oily liquid
Streptozotocin Pointed plate-
(Streptozocin) let or prisms
Elaiomycin
Chloroamphenicol
Pale yellow oil
Greyish-white
needles
115°C
150.5-
151. 5°C
= +39
= +38.4°
\l7 = +18.6°
Soluble in water,
methanol, ethanol,
butanol, ethyl acetate.
Soluble in water, lower
alcohols and ketones;
slightly soluble in
polar organic solvents ;
insoluble in non-polar
organic solvents.
Sparingly soluble in
water; soluble in
organic solvents.
Slightly soluble in
water; very soluble in
methanol, ethanol,
butanol, ethyl acetate
and acetone.
-------
Table XIX (cont'd)
Toxinc
Physical Form
m. p.
Optical Rotation
Solubility
Azaserine
Bleomycin
Light yellow-
green crystals
Cream-colored
powder
146-
162°C
[oC]27.5 = _0.5
12.5-16C
Very soluble in water
slightly soluble in cold
methanol, ethanol and
acetone.
Very soluble in water
and methanol; slightly
soluble in ethanol;
insoluble in acetone ,
ethyl and butyl acetate
and diethyl ether.
aCompiled from IARC Monographs, Vols. 10, 17, and 26, International Agency for Research on
Cancer, Lyon, France, 1976, 1978, 1981; The Merck Index, 10th ed. , Merck and Co., Rahway,
N.J., 1983
See Table XVIII for structural formulas.
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Both adriamycin and daunomycin are anthracycline glycosides consisting of
a tetracycline ring to which an amino sugar, daunosamine, is attached through
a glycosidic linkage. The structural difference between daunomycin and adria-
mycin is the presence of an acetyl group linked to ring A in the former versus
a hydroxyacetyl group in the latter (see Table XVIII). Upon acid hydrolysis
daunomycin and adriamycin yield the respective aglycone chromophores, dauno-
mycinone and adriamycinone, in addition to the water-soluble basic amino sugar
(13).
Mitomycin C contains in its molecule three important structures: aziri-
dine, urethane and aminoquinone (see Table XVIII). Upon chemical or enzymatic
reduction of the quinone moiet-y, followed by spontaneous loss of the tertiary
methoxy group and formation of an aromatic indole ring, mitomycin C becomes a
polyfunctional alkylating agent with three possible reaction sites (14-16)
(Fig. 5).
Sarkomycin is a cyclopentanecarboxylic acid derivative having a carbonyl
conjugated with a methylene group. The vinyl carbonyl structure, which is
also present in several other mycotoxins (see Section 5.3.1.2), displays high
reactivity in free radical reactions and toward sulfhydryl groups (see 12).
Streptozotocin, the 2-deoxy-D-glucose derivative of N-methyl-N-nitroso-
urea (see Fig. 6), can undergo various reactions including acetylation,
alkylation and replacement of the methyl or nitroso group (17). Under alkali
conditions, it decomposes to diazomethane (18).
146
-------
/CH2OCNH2
HoC
Mitomycin C
OH
Fig. 5. Activation of initomycin C.
-------
r
(
•
f XH
JH
*
:=o
i
1 K 1 /~1
-
H
1
NKI — f\
— N — U
1
CH3
CH
*" H3C-N = N-OH
OH
|H3C-N=NJ
Streptozotocin
Fig. 6. Proposed mechanism for the metabolic activation of streptozo-
tocin (modified from: J.A. Miller: Naturally Occurring Substances that can
Induce Tumors. ln_ "Toxicants Occurring Naturally in Foods," National Academy
of Sciences, Washington, D.C., 1973, p. 508).
-------
Elaiomycin, 4-methoxy-3-(l-octenyl-N-0-N-azoxy)-2-butanol (see Table
XVIII), resembles chemically to cycasin, another naturally occurring carcino-
gen (see Section 5.3.2). This antibiotic is stable in neutral or slightly
acid aqueous solutions but yields a raethylating agent by metabolic activation
(see Section 5.3.1.3.4 on metabolism).
Chloramphenicol has an interesting structure, with a nitrobenzene and a
dichloroacetamide moiety in the molecule. The nitro group is readily reduced
to the amine. Both chloramphenicol and azaserine (o-diazoacetyl-L-serine) are
stable in neutral solutions (9).
The bleomycins are a group of complex glycopeptides. Each contains a
pyrimidine chromophore linked to propionamide, a R -aminoalanine side chain,
L-gulose, 3-0-carbamoyl-D-mannose and a side chain with L-histidine, L-threo-
nine, a methyl valerate residue and a bithiazole carboxylic acid connected to
a terminal amine. The chemistry of a large number of natural and synthetic
bleomycins has been discussed by Uraezawa (19) who discovered this group of
antibiotics in 1966. The clinically used drug "Blenoxane" is a mixture of
bleomycin A2 (55-70%), bleomycin B2 (25-32%), and small quantities of bleo-
mycin B/ and Bg « 1%), which differ only in their terminal amine moiety (see
Table XVIII). In the United States and England, bleomycin is available as
bleomycin sulphate, whereas in Japan it is marketed as bleomycin hydrochloride
(9).
5.3.1.3.2.2 BIOLOGICAL EFFECTS OTHER THAN CARCINOGENICITY
Toxic Effects. Owing to their ability to inhibit rapidly proliferating
cells, many of these antibiotics have become the most widely used antineo-
plastic agents for the treatment of human cancers. However, many of them are
not sufficiently selective and inhibit normal proliferating cells of vital
organ systems as well.
147
-------
Early toxic manifestations in humans common to these agents include
anoxia, nausea, vomiting, diarrhea, stomatitis and glossitis. Dermatological
reactions such as alopecia, erythema, desquamation and hyperpigmentation also
frequently occur. The most important adverse effects are, however, to the
hematopoietic system. Actinomycin D, adriamycin, daunomycin, tnitomycin,
chloramphenicol and azaserine all cause leukopenia and thrombocytopenia as a
result of bone marrow depression (see rev. 2). Adriamycin and daunomycin are
unique in their cardiac toxicity which is manifested by tachycardia,
arrhythmias, dyspnea and hypotension (20). Stimulation of cardiac microsomal
lipid peroxidation (21, 22) and effects on reactive oxygen metabolism by mito-
chondrial reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase (23)
have been suggested to play an essential role in the cardiotoxicity of these
quinone-containing compounds. Renal and pulmonary toxicity, manifesting as
glomerular sclerosis and interstitial pneumonia, have been observed in
patients treated with mitomycin C. At 10 mg/kg/day, azaserine may cause liver
damage. In contrast to other antitumor agents, bleomycin has toxic effects
primarily toward the lung rather than toward the bone marrow. A significant
incidence of pulmonary fibrosis has been noted in individuals taking high
doses of this drug (rev. 2).
Studies in animals have shown that actinomycin D as well as mitomycin C
are extremely toxic, especially when administered parenterally. Other anti-
biotics of this group are also moderately toxic, producing local and systemic
lesions, generally analogous to those observed in humans. Table XX lists the
LDijQ of the Streptomyces toxins in rats and mice by various routes of admini-
stration. Animal studies have shown that the predominant toxicity of strepto-
zotocin and azaserine is to the pancreas. Streptozotocin produces irrevers-
ible injury to the A-cells in the organ, and is diabetogenic toward dogs,
148
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Table XX
Acute Toxicity of Some Streptomyces Toxins
Toxin3
Actinomycin D
Adriamycin
Daunomycin
Mitomycin C
Sarkomycin
Streptozotocin
Elaiomycin
Chloramphenicol
Species
and Route
Rat , oral
s .c .
i.p.
i.v.
Mouse, oral
s .c .
i.p.
i .v .
Mouse , s .c .
i.p.
i .v .
Rat, i.v.
Mouse, s.c.
i.p.
i .v.
Rat , oral
i.p.
i.v .
Mouse, oral
i.p.
i .v.
Mouse, oral
s.c.
i.v.
Rat, i.v.
Mouse, oral
Mouse, s.c.
i.v.
Rat , oral
s.c.
i.p.
i.v .
Mouse, oral
s.c.
i.p.
i .v.
LD5Q
(mg/kg)
7.2
0.8
0.4
0.46
13
0.5
0.85
1
16
15
10
13
16
2.5
20
30
2.5
3
23
8
5
5,600
600
1,200
138
264
63
44
3,400
5,450
80
171
2,640
400
1,320
110
Reference
24
24
25
24
24
25
25
25
25
26
25
25
25
25
25
27
27
25
27
27
25
25
25
25
25
25
25
25
28
28
29
25
25
25
25
25
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Table XX (cont'd)
Toxin3
Azaserine
Bleomycin
Species
and Route
Rat , oral
i.p.
Mouse, oral
i.p.
1 .V .
Mo u s e , i.p.
1 . V .
LD50
(mg/kg)
170
147
150
100
62
77
53
Reference
30
30
30
30
25
25
25
aSee Table XVIII for structural formulas.
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monkeys and rodents (31; see also ref. 17). The sensitivity to streptozotocin
diabetes ranks as rat > mouse > dog > guinea pig (17). The glucose segment of
the streptozotocin molecule is believed to function as a "carrier" moiety for
transport to or across the membrane of the ^3-cells (17, 31). Azaserine
causes damage to the pancreatic acinar cells in mice, rats, cats and dogs. In
addition, it produces diverse pathological changes in the liver, kidney, and
gastrointestinal tract (30, 32). Similarly, death in rodents due to lethal
doses of elaiomycin is the result of acute lesions in the liver, lung, kidney
and stomach (see 33).
Mutagenic Effects. Since many of these antibiotics interact with DNA,
various short-term assays involving mutagenicity and related genotoxic
effects, have been used for screening for potential carcinogens. Except for
sarkomycin and elaiomycin, which do not seem to have been tested for muta-
genicity, and for chloramphenicol, which appears to produce no chromosomal
aberrations in mice in vivo (34, 35), other agents of this group have all been
shown to be mutagenic and clastogenic in more than one assay system. The
results of some current studies are summarized in Table XXI.
Actinomycin D and bleomycin are inactive in the Ames Salmonella test (36-
38) but are mutagenic in fungi (16, 46-68, 105, 109) and Drosophila (49, 107,
108) and induce sister chromatid exchange (50, 51, 70) and chromosomal aberra-
tions in various in vitro (41-45, 102, 103) and in vivo (103, 104) cytogenetic
assay systems. Actinomycin was also inactive in mutagenicity tests using
Escherichia coli (39) and Bacillus subtilus (40). On the other hand, actino-
mycin D showed positive effects in the mouse dominant lethal assay (52) and
the sperm abnormality test (38, 53). These findings led many investigators to
suggest that the chromosomal events induced by actinomycin D and bleomycin
involve intragenic and intergenic recombinations, chromosome breakage and
149
-------
Table XXI
Mutagenic and Related Genotoxic Effects of Some Streptomyces Toxins'
Toxins
Actinomycin D
Adriamycin
Salmonella
typhimurium
- (36-38)
+ (36,37,54-56)
Escherichia
^coli
- (39)
n.t.
Bacillus
subt ilis
- (40)
n.t .
Chromosomal
aberrations
+ (41-45)
+ (44,45,56-62)
Other
tests0
+ [A-G.I] (38,
H- [G-J] (50,55
46-53)
,56,
Daunomycin
Mitomycin C
Streptozotocin
Chloramphenicol
Azaserine
Bleomycin
+ (36,37,54,55,68)
+ (37,71-73)
+ (54,90-92)
? (54,95)
+ (54,96,97)
- (36,37)
+ (101)
+ (40,69)
+ (40,69,74,75)
+ (69)
+'(40)
+ (98)
n.t .
(40)
(60,70)
+ (40,76) + (44,50,70,77-80)
n.t.
- (40)
n.t.
- (34,35)
n.t.
+ (43-45,102-104)
58-60,63-67)
+ [G-I] (60,63,64,
67,70)
+ [A,D-I,K] (50,52,
53,63,70,78-89)
+ [F.H.J] (92-94)
- [D] (52)
+ [H] (99,100)
+ [A.C.E-G] (50,51,
70,105-109)
a"+" = positive; "-" = negative; "?" = chemical toxicity prevented adequate testing; n.t. = not tested; numbers in
parenthesis are references.
bSee Table XVIII for structural formulas.
CA = Saccharomyces cerevisiae; B = Neurospora crassia; C = Aspergillus nidulans; D = mouse dominant lethal assay;
E = sperm abnormality assay; F = Drosoghila melanogaster; G = sister chromatid exchange assay; H = unscheduled DNA
synthesis; I = mouse lymphoma assay; J = Chinp.se h.nmster V79 eel l/HGPRT (hypoxanth Lne-guanine phosphoribosyl transferase
deficient) assay; K = Chinese hamster ovary (CIIO) cell specific loci assay.
-------
chromosome loss, but no point mutations. However, a recent study by Podger
and Grigg (101) indicates that bleomycin A2, 82, B^ and several other struc-
turally related glycopeptide antibiotics do induce reverse-mutation in strain
trp E8/pKM101 of Salmonella typhimurium. Among these compounds, tallysomycin
A and B, which structurally resemble the bleomycins, but contain an additional
amino-sugar moiety and a methyl group, are the most potent mutagens on the
basis of dose. Phleomycin G, the chemical structure of which differs from
that of bleoraycin B^ (see Table XVIII) only in the ring structure of the
bithiazole moiety, which is partially saturated in the former, induces a
higher number of mutants per plate at the peak response than the bleomycins.
The relative mutagenic potential of the bleomycins follows the order: 62 >
A2 > B6 (101).
Both adriaraycin and daunomycin showed high potency in the frameshift
tester strains TA98 and TA1538 of Salmonella typhimurium (36, 37, 54-56).
Moreover, they have been reported to be weakly mutagenic toward the base-pair
substitution mutants of the Ames strains (37, 54, 56, 68). Metabolic ac.tiva-
tion is not required for these agents to be mutagenic. Consistent with the
results from the Ames test, daunomycin induces DNA damage and causes growth
inhibition in DNA-polymerase deficient mutant strains of _E. coli (40, 69) and
Bacillus subtilis (40). Furthermore, adriamycin brings about reversion of the
mutation at the HGPRT (hypoxanthine-guanine phosphoribosyl transferase) locus
of the Chinese hamster V79 cells (66). The DNA-damaging effects of adriamycin
and daunomycin have also been demonstrated in many in vitro and in vivo cyto-
genetic studies using tissues from humans (57-60, 70), rodents (44, 56, 61,
62) and insects (45) as well as in various mammalian rautagenicity test systems
including the sister-chromatid exchange assay (50, 56, 58-60, 63, 64, 70), the
unscheduled DNA synthesis assay (65, 70), the mouse lymphoma L5178Y cell
150
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system (55) and the 8-azaguanine resistant assay in Chinese hamster V79 cells
(67).
Consistent with these findings, a recent structure-activity study (110)
has indicated that anthracyclines with a daunosamine moiety (such as adria-
inycin, daunomycin, 4-demethoxyadriamycin, 4-deraethoxydaunomycin and carmino-
mycin) are highly mutagenic in both bacterial and mammalian cell assays.
However, N-alkylation of the primary amino group on the sugar moiety can
abolish or greatly reduce the mutagenic activity of the anthracyclines. Thus,
N,N-dimethyladriamycin, N-methyl-, N,N-dimethyl-, N,N-dibenzyl-, and morpho-
line-daunomycin are nonrautagenic or only weakly mutagenic (110, 111).
Mitoraycin C was found mutagenic by Ames and his associates (71, 72) only
in the _S_. typhimurium strain TA110, which contains the R factor plasmid pKMlOl
and is more sensitive than other standard .strains to detect fratneshift muta-
gens. However, other investigators (37, 78) observed weak mutagenic effects
of mitomycin C in several standard _£. typhimurium strains (TA1535, TA1538,
TA98, TA100 and TA92) when tested under carefully controlled conditions (e.g. ,
protected from light and freshly dissolved in ice cold water) and in the
presence of S-9 mix. Mutagenic action of this compound has also been demon-
strated in various strains of_E. coli (40, 69, 74, 75), _B. subtilus (40, 76)
and _£. cerevisiae (81) as well as in Drosophila (83), and the mouse dominant
lethal (52, 84), mouse lymphoma specific locus (85), Chinese hamster ovary
cell genetic loci (89), sperm abnormality (59) and unscheduled DNA synthesis
(70, 86-88) assays. Mitomycin C is also effective in inducing heritable
translocations (77), sister chromatid exchange (50, 63, 70, 78, 79. 82) and
chromosomal aberrations in human lymphocytes (70, 78), mouse lymphocytes (79),
bone marrow cells of rats (80) and Chinese hamster ovary cells (44, 50). In
vitro studies (44) showed that S-9 mix is not required for the clastogenic
activity of mitomycin C.
151
-------
Streptozotocin is highly toxic to S. typhimurium strains. However, at
low, non-lethal doses, extensive mutagenic effects have been observed in
strain G46 (90-92), TA100, TA1535 (54, 92) and in several other base-pair
substitution strains of J^. typhimurium (92). The carbohydrate moiety has been
shown to be important for the high mutagenic potency of Streptozotocin. For
example, when the glucose moiety was replaced by o^or ^-OCH^ glucose (in
which the -OCHo group is at C-l), the mutagenicity decreased 10 to 100 fold.
When the glucose moiety was replaced by inositol, the mutagenicity decrease
was greater than 1,000 fold. The rautagenic activity also decreased substan-
tially when the 1-methyl-l-nitrosourea moiety was attached at the C-l (instead
of the C-2) position of glucose (cited in ref. 91). Moreover, Streptozotocin
induces mutation in E^. coli (69) and in Drosophila (93), it brings about
reversion in the mutant of Chinese hamster V79 cells deficient in hypoxan-
thine-guanine phosphoribosyl transferase (94), and it induces unscheduled DNA
synthesis in primary rat hepatocyte culture (92).
Investigation of the mutagenic potential of chloramphenicol in Salmonella
typhimurium by McCann et al. (54) was similarly hampered by the high toxicity
of the compound. Jackson et al. (95) later showed that the L(+) threo isomer
of chloramphenicol, which is less toxic than the D(-) isoraer, was mutagenic in
_£. typhimurium TA100 and TA1535 but not in TA98. Both isomers caused breakage
at different points of bacterial DNA (as analyzed by alkaline sucrose gradient
sedimentation, implying that the mutagenic effects of chloramphenicol may be
masked by its toxicity. However, except in a DNA-repair test using E. coli
(40), there is no evidence that chloramphenicol is mutagenic in other studies
with_E. coli (39, 98), Bacillus subtilus (40) or in the mouse dominant lethal
assay (52).
152
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Azaserine is a strong, direct-acting bacterial mutagen, which brings
about reversion of the mutation of S. typhimurium TAlOO (54, 96, 97, 112) and
inhibits the growth of DNA-polymerase-deficient mutant strains of JE. coli (98,
113). The compound is also effective in inducing DNA damages and repair in
rat tissues (99, 100). Interestingly, 6-diazo-5-oxo-L-norleucine:
NH9
© © | *
N = N = CH . CO. CH,. CH9 . C-COOH
2 ^ I
H
a compound similar to azaserine in both chemical reactivity and biological
activity, is virtually non-mutagenic (96). Another structurally related
chemical, alanoser [0-(N-methyl-N-nitroso- B> -alanyl)-L-serine], is also not
mutagenic and not carcinogenic (112).
Teratogenic Effects. As expected from the well-documented cytotoxic,
mutagenic and clastogenic properties of these Streptorayces toxins, a number of
them were found to be embryotoxic and teratogenic in various animal species.
In the rat, daily doses of 50-100 ug/kg/actinomycin D before the 10th day
of gestation induced 15-56% gross malformations of the central nervous system,
the viscera, and the skeleton (114, 115). Similar abnormalities were observed
in the offspring of the rabbit and the Syrian golden hamster exposed to
actinomycin D during the first trimester of pregnancy. However, the compound
is more embryotoxic and less teratogenic in these two species than in the rat
(116-119). A three-generation study showed that A/He mice treated with
actinomycin D (0.05-50 ug/kg) had fewer progeny than did the controls; how-
ever, no malformations were noted among the live progeny (120).
153
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Both adriamycin (121) and daunomycin (121, 122) are teratogenic in the
rat. Administration of adriamycin (1-2 mg/kg) or daunomycin (1-4 mg/kg) to
pregnant rats during various periods of organogenesis resulted in malforma-
tions in the progeny showing a dose-response relationship. Major malfor-
mations involve the eye, the urinary tract, the cardiovascular system and the
cephalic region. On a dose (mg/kg) basis, adriamycin is a more potent
teratogen than daunomycin (121). Neither agent appears to be teratogenic in
the rabbit (121), the mouse and chick embryos (123). As daunomycin may
produce embryopathy'in humans, the drug is not recommended for use by women
during pregnancy (123).
Mitoraycin C induced defects of the skeleton, the palate and the brain in
newborn mice when the pregnant females were given a signle dose of 5-10 mg/kg
b.w. during the gestational period from day 7 through 13 (124). No terato-
genicity was detected in the rat (125).
Daily injections of 5-11 mg/kg sarkomycin to rats during the 6th-10th day
of gestation produced abnormalities in 10% progeny (126). The principal
abnormalities observed were hydronephros and microophthalmia. Fetuses of rats
given 700-1,200 mg/kg b.w. chloramphenicol daily ffbnT'day 6 to day 10 of
gestation also showed an increased rate of the same defects (126). Similarly,
a clear-cut teratogenic effect of chloramphenicol in the rat was noted by
Fritz and Hess (127). Malformations involved a persisting umbilical hernia
associated with costal fusion. Chloraraphenicol has also embryotoxic and fetal
growth inhibitory effects in the rat, the mouse, and the rabbit (127).
The teratogenic potential of azaserine has been investigated in the rat
and the chick embryo. Skeletal and palate defects were found in the offspring
of rats treated with 2.5 rag/kg b.w. azaserine from the 8th-12th day of gesta-
154
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tion (128). Injection of 0.15-2.4 mg azaserine into the chick embryo produced
defects of the appendicular skeleton of the developed animal (129).
5.3.1.3.3 Carcinogenicity and Structure-Activity Relationships
A large number of cancer chemotherapeutic drugs are carcinogenic in
experimental animals (130; revs. 4, 5). Several of the carcinostatic anti-
biotics produced by the Streptomyces contain alkylating and/or intercalating
moieties in their molecule and are, therefore, potentially carcinogenic.
Structurally, sarkomycin, streptozotocin and eliaomycin are analogous to known
carcinogens, the c£,A-unsaturated lactones, N-methyl-N-nitrosourea, and
cycasin, respectively. The results of the carcinogenicity bioassays of
Streptomyces antibiotics are summarized in Table XXII.
As early as 1955, sarkomycin became the first antibiotic recognized to
exhibit carcinogenicity in the rat (143). Later in 1958 and 1959, actinomycin
S and actinomycin L were found to produce sarcoma in the mouse at the injec-
tion site (131, 132). Since then, an increasing number of antibiotics have
been shown to be carcinogenic in the rat and/or the mouse. In general, most
of these antibiotics are weak to moderately active carcinogens on the basis of
the tumor incidence and the latent period. For example, compared to methyl-
cholanthrene, actinomycin D and mitomycin C are less potent in inducing sar-
comas in btk strain mice (133). Actinomycin D is also less active in inducing
lung tumors in the mouse than is urethan (120).
Actinomycins. Kawamata and his associates (132) were the first to report
the emergence of sarcomas in btk and ctk strain mice following repeated sub-
cutaneous injections of actinomycin S (7.5^ug/kg body weight, twice weekly for
up to 40 weeks). These findings were confirmed in later studies by the same
group (131, 133). In addition, they found carcinogenic effects with actino-
155
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Page 1 of 2
Table XXII
Streptomyces Toxins Which Have Been Tested for Carcinogenic Activity
Toxin1
Species and Strain
Principal Organs
Affected
and Route
References
Actinomycin L
Actinomycin S
Mouse, btk
Mouse, btk, ctk,
Local sarcoma, s.c.
Local sarcoma, s.c.
131
131-133
Actinomycin C
Actinomycin D
Adriamycin
Daunomycin
Mitomycin C
Sarkomycin
Streptozotocin
ddo, C57BL, Swiss
Rat, BR-46
Mouse, Swiss, DBA/1
Mouse, btk
Mouse, A/He
Mouse, Swiss-Webster
derived
Rat, Fischer 344
Rat, Sprague-Dawley
Mouse, BALB/c
Rat, Sprague-Dawley
Mouse, XVII/RhO
Mouse, C57BL/RhO
Mouse, Swiss
Mouse, BALB/c
Rat, Sprague-Dawley
Rat, Sprague-Dawley
Rat, Sprague-Dawley
Mouse, btk, C57BL
Mouse, C3H, ddO
Mouse, Swiss-Webster
derived
Rat, BR-46
Rat, Sprague-Dawley
Rat, Wistar
Mouse, Swiss-Webster
Rat, Holtzman
Rat, Holtzman
Rat, Holtzman
None , i .v.
Skin, s.c.
Local sarcoma, s.c,
Lung, i.p. or oral
None, i.p.
Mesentary, i.p.
Peritoneum, i.p.
None, i.v.
Mammary gland, i.v,
Local sarcoma, s.c.
None, oral
None, i.p.
None, i.v.
Mammary gland, i.v.
Kidney, genital
tract, i.v.
Peritoneum, i.p.
Local sarcoma, s.c,
None, s.c.
None, i.p.
Multiple site, i.v
Peritoneum, i.p.
Local sarcoma, s.c
Kidney, lung,
uterus, i.p.
Kidney, pancreas,
i .v.
Pancreas, i.v.
Kidney, i.v.
134
135
133
120
4
136
4
137
20, 67, 138,
139
140
140
130
137
20, 67, 138,
139, 141
141
130
133
133
4
130, 134, 142
4
143, 144
4
145
146
147
-------
Table XXII (cont'd)
Page 2 of 2
Toxin
Species and Strain
Principal Organs
Affected
and Route
References
Streptozotocin
(cont'd)
Elaiomycin
Chloramphenicol
Azaserine
Bleomycin
Rat, Sprague-Dawley,
Lewis
Rat, Sprague-Dawley
Rat, Wistar
Rat, Wistar
Rat, Sprague-Dawley
Hamster, Chinese
Rat, albino
Mouse, BALB/c x AF,
Rat, Sprague-Dawley
Mouse, CD-I
Rat, Wistar
Rat, Wistar
Rat, Wistar/Lewis
Mastomys natalensis
Mystromys albicaudatus
Rat, Wistar
Rat, Sprague-Dawley
Kidney, i.v.
Kidney, pancreas,
liver, peritoneum,
i .v.
Kidney, i.v.
Kidney, pancreas,
i.v.
Kidney, i.v.
Liver, i.p.
Liver, kidney,
other sites, oral
Hematopoiet ic
t issue, i.p.
None, oral
None, i.p.
Pancreas, kidney,
i.p.
Pancreas, i.p.
Pancreas, i.p.
Pancreas, i.p.
None, i.p.
Multiple site, i.v,
Kidney, local
sarcoma, s.c.
148
4
149
150
151
152
33
154
155
156
112, 157
158
156, 159, 160
161
161
162
163
See Table XVIII for structural formulas.
-------
mycin L in the btk strain although sarcoma induction required about ten times
higher doses of actinomycin L compared with actinomycin S. Mice of the btk
and ctk strains were more susceptible to tumor induction by actinomycin S than
several other mouse strains tested (131). Similarly, btk mice showed a high
incidence (8 out of 9 mice) of sarcoma after receiving a total of 35 subcu-
taneous injections of actinomycin D (0.2/ug each, twice weekly) (133).
DiPaolo (135) also found two skin squamous cell carcinomas and one adeno-
acanthoma among 51 mice of Swiss and DBA/1 strains which survived the injec-
tions of 200 ug actinomycin D twice weekly for 16 weeks. Interestingly, when
groups of 10 female A/He mice were given 0.05, 0.5, 5 or 50yug/kg b.w. actino-
mycin D intraperitoneally or orally through five pregnancies, 85-100% of the
animals in the treated groups developed lung adenomas roughly in a dose-
response fashion (120). Such effect, however, was not found in a Swiss-
derived strain of mice administered 1/2 of the Maximum Tolerated Dose (MTD)
(180/ug/kg) or the MTD (370 ug/kg) of the drug by intraperitoneal injection 3
times/week for six months and observed for a further 12-month period (4).
The carcinogenic potential of actinomycin C (a mixture of Ci, C£ and C^)
and actinomycin D has also been studied in the rat. In a group of 48 male
BR46 rats given weekly i.v. injections of 7 mg/kg b.w. actinomycin C for one
year, and observed for life, the tumor incidence was not significantly higher
than that in the 65 control animals (134). However, in Sprague-Dawlay (CD)
rats of both sexes, receiving i.p. injections of 0.022 or 0.045 mg/kg b.w.
actinomycin D twice weekly for 26 weeks, 57 of 74 developed peritoneal sar-
comas tumor 12 months after the treatment; only one peritoneal sarcoma was
seen in 360.controls (4). Similarly, in 26 male Fischer 344 rats receiving
0.025 or 0.05 mg/kg b.w. actinomycin D intraperitoneally 2-5 times/week for up
to 18 weeks, 18 (69%) were found to bear invasive, transplantable mesenchymal
156
-------
tumors after 50 weeks. A tumor of the same histological type was also noted
after 41 weeks in 1 of 9 rats given a single injection of 2 mg/kg b.w. actino-
mycin D. No tumors occurred in 10 control rats or in groups of rats given a
single injection of actinotnycin D at doses of 0.5 or 1 mg/kg b.w. (136).
Interestingly, actinocylgramicidin S (an analog of actinomycin D that contains
the same chromophore but different peptide chains) was found inactive in
producing tumors in the rats in a long-terra study (136). However, the authors
noted high early mortality among rats treated with actinocylgramicidin S.
Adriamycin and Daunomycin. In vitro studies in mammalian cell systems
have shown that these two anthracycline antibiotics exhibit transforming
activity comparable to that of the potent carcinogen, N-methyl-N-nitro-N-
nitrosoguanidine (67). Both adriamycin and daunomycin are indeed systemic
carcinogens in the rat by parenteral administration. Daunomycin has also been
shown to induce local sarcomas in the mouse following repeated subcutaneous
injections.
Of the 20 male and 20 female XVII/Rho mice injected s.c. with 1.25 mg/kg
b.w. daunomycin weekly for 12 weeks, 5 animals of each sex were found bearing
local sarcomas when sacrificed 19 months after treatment (140). However,
chronic administration of nonlethal doses of daunomycin to mice of several
other strains orally (140) or parenterally (130, 137) did not bring about
higher tumor incidence than the spontaneous tumor incidence of the controls.
Consistent with the above findings, adriamycin induced no tumors in BALB/c
mice 12 months after a single i.v. dose of 5 mg/kg body weight (137).
In contrast to mice, the carcinogenic effects of adriaraycin and dauno-
mycin in the rat have been repeatedly confirmed. Bertazzoli and coworkers
(138) were the first to report the high incidence of mammary fibroadenomas and
157
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adenocarcinomas in groups of weanling female Sprague-Dawley rats treated with
single i.v. doses of adriamycin (4-8 mg/kg b.w.) or daunomycin (5-12.5 mg/kg
b.w.). Similar carcinogenic effects were obtained in later studies (20, 67,
139). Sternberg and associates (141), on the other hand, detected an 18%
incidence of renal carcinomas and adenomas and a 15% incidence of genital
tumors in 33 female Sprague-Dawley rats about 1 year after each receiving a
single i.v. injection of 5 or 10 mg/kg b.w. daunomycin; no such neoplasms
occurred in the controls. Weisburger (130) administered 0.11-0.45 mg/kg b.w.
daunomycin to groups of 25 male or female Sprague-Dawley rats intraperi-
toneally three times weekly for 26 weeks; 13 of 23 males and 16 of 29 females
examined 52 weeks later bore peritoneal sarcomas which are virtually nonexis-
tent in untreated animals.
Mitomycin C. Mitomycin C induces sarcomas at the injection sites of
certain mouse strains and neoplasms in various tissues in the rat following
parenteral administration.
Subcutaneous injection of 0.2 ug mitomycin C per mouse for 17.5 weeks
(twice weekly) resulted in sarcomas in 7/7 btk mice and 2/10 C57BL mice but
neither in the respective groups of 10-11 saline-treated controls nor in
groups of ten C3H and ten ddO mice (133). The compound was also noncarcino-
genic in groups of 25 male and 25 female Swiss-Webster derived strain mice
given three i.p. injections of 0.25 or 0.5 mg/kg per b.w. week for 26 weeks
followed by observation for a further 52 weeks. However, peritoneal sarcomas
occurred in almost all 29 male and 31 female Sprague-Dawley (CD) rats which
survived a similar schedule of treatments with 0.038 or 0.15 mg/kg raitomycin C
(4). Of 96 male BR-46 rats given five i.v. injections of 0.52 mg/kg b.w.
mitomycin C within two weeks, 30 developed malignant or benign tumors about 18
months following treatment. The malignant tumors were: 2 hemangioendo-
158
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theliomas, 8 subcutaneous or peritoneal fibrosarcomas, 5 mammary sarcomas, 3
lyraphosarcomas, 1 pheochromocytoma, 3 lung carcinomas, 2 bladder carcinomas, 1
gastric carcinoma, 1 esophageal carcinoma and 1 saliva-gland carcinoma. Only
1 pheochromocytoraa and 3 mammary sarcomas were seen in 89 control rats during
a 23-month observation period (134, 142).
Sarkomycin. As expected from the structural similarity between sarko-
mycin and the carcinogenic g( , B -unsaturated lactones (see Section 5.2.1.1.6,
Vol. IIIA of this monograph), sarkomycin is weakly carcinogenic in the rat.
Repeated subcutaneous injections of 2 rag sarkomycin twice weekly for 42 weeks
induced a myxosarcoma in one of six Wistar rats (144). The carcinogenicity of
this antibiotic has been confirmed in a bioassay by subcutaneous injections
into the back of rats, using a similar administration schedule (143).
Streptozotocin. There is a considerable body of evidence on the carcino-
genicity of Streptozotocin in the rat. The antibiotic induces tumors of the
kidney, pancreas and several other tissues following repeated intravenous or
intraperitoneal injections. The compound also induces neoplasms in the mouse
and the hamster.
Arison and Feudale (145) were the first to report the induction of renal
cystadenomas in 9 and of pancreatic tumors in one of 19 male Holtzman rats 8
months after a single i.v. injection of Streptozotocin at 50 rag/kg body weight
(a diabetogenic dose). Later studies showed that nicotinamide, which prevents
the diabetogenic action of Streptozotocin, enhances pancreatic tumorigenesis
but reduces the renal tumor incidence in these rats. While only a single
pancreatic islet cell tumor was noted in 26 animals treated with Streptozo-
tocin alone, a 64% (18/28) incidence of pancreatic tumors was observed in a
group of male Holtzman rats given both Streptozotocin and nicotinamide
159
-------
(146). To the contrary, adenomas of the kidney developed in 77% (21/28) of
animals treated with streptozotocin-alone, but .in only 18% (5/28) of animals
given both streptozotocin and nicotinamide (147).
The tumorigenic action of streptozotocin on the kidney and pancreas was
confirmed by several other studies in various strains of rats. Of 29 Sprague-
Dawley rats that survived for more than eight months following a single i.v.
injection of 65 mg/kg b.w. streptozotocin, 11 developed both benign and malig-
nant tumors in the rena-l -cortex.. -"The-.same-'treatment of Lewis rats gave rise
to renal tumors in 13 of 45 animals sacrificed more than eight months after
streptozotocin administration (148).
Attempts were made to explore the possible relationship between the
diabetogenic and carcinogenic effects of streptozotocin. Mauer et al. (148)
induced diabetes in 72 Sprague-Dawley rats by the administration of alloxan
and noted no neoplasms .in the surviving.animals. This led the authors to
conclude that the tumorigenic action of streptozotocin is unrelated to its
diabetogenic effect. The same conclusion was reached by Horton _et_ _al_. (149)
on the basis of a study showing that the management of the diabetic state by
insulin does not affect the incidence of renal tumors in 36 of 80 Wistar male
rats, each given a single i.v. dose (25 mg/kg) of streptozotocin.
Additional evidence for the oncogenic effects of streptozotocin toward
the kidney and other tissues of 'Sprague-Dawley (4, 151) and Wistar (150) rats
has been documented. In groups of 25 Sprague-Dawley rats of either sex
receiving repeated doses (6 or 12 mg/kg b.w.) of streptozotocin intraperi-
toneally, significant incidences of kidney tumors, pancreatic tumors, peri-
toneal sarcomas, liver tumors and muscle tumors were found (4). A study by
Kazumi et al. (150) showed that even a single i.v. dose of streptozotocin (30
160
-------
mg/kg b.w.) displays a marked tumorigenic effect toward the pancreas in male
Wistar rats.
The carcinogenicity of streptozotocin in the mouse has been documented by
the study of Weisburger and coworkers (4) who administered 6 or 12 mg/kg of
the antibiotic intraperitoneally to groups of 25 Swiss-Webster mice of either
sex, three times a week, for 6 months. About 12 months after the last injec-
tion, mice in both the male and female groups developed significant incidences
of tumors of the lung (60% in males, 85% in females) and the kidney (60% in
males, 18% in females). In addition, tumors of the uterus were noted in 6 of
39 female mice.
Streptozotocin is also carcinogenic, at least toward the liver, in the
Chinese hamster. Intraperitoneal administration of divided or single doses
(100 mg/kg b.w.) of streptozotocin to young Chinese hamsters of both sexes
gave rise to various neoplastic lesions in over 90% of the animals with latent
periods of up to 1 year. Benign or malignant neoplasms of the liver were
noted in 34% (13/35) of the hamsters; other lesions noted were undifferen-
tiated sarcomas, squamous cell carcinomas, and hyperplasia of renal, bronchial
and pancreatic epithelium. No such neoplastic lesions were found in the
matched controls (152, 153).
Elaiomycin. Elaiomycin resembles structurally to the plant carcinogen,
cycasin (discussed in Section 5.3.2). Consistent with this structural
analogy, elaiomycin induces a variety of tumors similar to those induced by
cycasin. In a study by Schoental (33), groups of weanling albino rats were
given a single oral dose of elaiomycin (10-35 mg/kg b.w.) alone or followed by
2-6 additional doses (not exceeding 30 mg/kg b.w.) of the drug over the next
10 months. Among the 34 rats surviving for 11-28 months after treatment, a
161
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few developed sarcomas of the liver, adenocarcinoraas of the upper jejunum and
oligodendrogliomas of the brain. In another experiment using newborn rats (in
the same study), one adenocarcinoma of the stomach, one oligodendroglioma of
the brain, one thymoma, two subcutaneous sarcomas and one adenocarcinoma of
the uterus were seen in 18 animals surviving for 18-22 months after receiving
the same treatment.
Chloramphenicol. The potential carcinogenicity of chloramphenicol has
been explored in the rat and the mouse, but in only one bioassay study each.
Feeding the compound at the dietary level of 0.05^ for up to 66 weeks did not
result in significant incidence of tumors in a group of 36 female Sprague-
Dawley rats (155). However, among 41 male BALB/c x AFi mice injected i.p.
with 2.5 mg chloramphenicol five times weekly for five weeks, two developed 18
weeks after treatment lymphomas which were not seen in the controls (154).
Chloramphenicol (by simultaneous oral administration) has been noted to
enhance the induction of papilloraas in mouse skin by 3-methylcholanthrene
(applied by skin painting) (164).
Azaserine. Pancreatic carcinogenesis by azaserine has been extensively
studied by the research group led by Longnecker (112, 156, 157, 159, 160).
Azaserine induces a high incidence of hyperplastic nodules, adenomas and
adenocarcinomas of the pancreas in Wistar and Wistar/Lewis rats which received
weekly or twice weekly i.p. injections of 5-25 rag/kg b.w. of the drug over a
period of 6-26 weeks; the tumors appeared in 1-2 years from the beginning of
administration. In addition, kidney adenomas and adenocarcinoraas are often
found in azaserine-treated Wistar rats (112). Mastomys natalensis (a rodent)
is also highly responsive to pancreatic carcinogenesis by azaserine (161).
Under similar study conditions, however, no significant incidence of neoplasms
162
-------
was observed in non-inbred CD-I mice (156) or in Mystromys albicaudatus (161)
treated with azaserine, or in Wistar rats treated with alanoser [0-(N-methyl-
N-nitroso-/?-alanyl)-L-serine], a compound structurally and metabolically
similar to azaserine (112).
Azaserine-induced pancreatic carcinogenesis in the rat is enhanced by
diet high in unsaturated fat (159) and by partial pancreatectomy (158).
Groups of azaserine-treated rats which were underfed or fed a diet high in
protein or supplemented with retinoids developed fewer pancreatic neoplasms
(159, 160).
Bleomycin. In addition to its mutagenic and clastogenic activity, bleo-
mycin produces a dose-dependent increase in neoplastic transformation in mouse
C3H/10T,/2 clone cell line at doses ranging between 0.1 /ag/ml and 2.5 ug/ml
(43). The drug has also been suggested to be a possible transplacentary
carcinogen on the basis of the findings that it induced both benign and malig-
nant tumors in a variety of tissues in 13% (35 of 279) of the offspring of
Wistar rats injected i.v. with bleomycin at the dose of 0.04 mg/kg body weight
at the 20th and 21st day of gestation; the neoplasms included tumors of the
mammary, liver, kidney, skin and the nervous system (162). Moreover, Habs and
Schmahl (163) reported in a preliminary communication that administration of
0.35, 0.70, 1.40 or 2.8 mg/kg b.w. bleomycin sulfate into the interscapular
region of groups of Sprague-Dawley rats resulted in significant dose-related
incidences of renal adenosarcomas and fibrosarcomas at the application site.
A structurally related chemical, peplomycin sulfate, exerted similar carcino-
genic effects under the same study conditions (163).
163
-------
5.3.1.3.4 Metabolism and Mechanism of Action
Actinomycins. Actinomycin D is absorbed slowly from the gastrointestinal
trace and does not cross the blood-brain barrier. When administered intra-
venously, it rapidly clears from the blood and accumulates in different
tissues and organs. In the rat, approximately 50% of the administered actino-
mycin D is excreted unchanged in the bile and 10% in the urine. The drug does
not seem to undergo metabolic modification (see rev. 2).
Actinomycin D selectively concentrates in the nucleus of mammalian cells
where it brings about profound nuclear and nucleolar morphological changes.
Extensive studies suggest that the biological responses to the actinomycins
are directly attributable to their interaction with DNA. The mechanisms of
the carcinogenic action of the actinomycins is still unclear. Yet, the mole-
cular nature of the actinomycin binding site in DNA has been amply explored
and described (revs. 3, 165). Studies with chemically modified actinomycins
show that the integrity of several functional moieties is important for the
formation of a stable complex with DNA. These functional moieties are: (a)
the free amino group at position 3 of the chromophore, (b) the unreduced
quinonoid oxygen and (c) the intact pentapeptide lactone rings (see rev.
165). As the condensation products of the mouse carcinogens 3-hydroxy-
anthranilic acid and 3-hydroxykynurenine (166) are also phenoxazone deriva-
tives (see also Section 5.1.2.5.4, Vol. IIB), Kawamata et_ _a_l_. (131) suggested
that the phenoxazone moiety of actinomycins may be important in tumor induc-
tion. The peptide rings of the actinomycins were postulated to function as
transport "carriers" and may account for the differences in the carcinogenic-
ity of actinomycins (see ref. 131). In accord with this hypothesis, Svoboda
and coworkers (136) noted that actinocylgramicidin S, which contains the same
phenoxazone moiety but peptide rings of different amino acid composition than
164
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the actinomycins, does not bind to DNA and is not carcinogenic in male F344
rats. The negative finding with actinocylgramicidin S could have been due,
however, to the high, early mortality of the tested animals.
Adriamycin and .Daunomycin. The metabolism and pharmacokinetics of adria-
mycin and daunomycin have been reviewed by DiMarco (13) and elsewhere (2,
9). Following intravenous injection into rodents or humans, there is rapid
uptake of adriamycin and daunomycin by the heart, kidney, lung and liver,
spleen and lymphoid tissue. Both drugs are metabolized mainly in the liver
and excreted in the bile, although urinary excretion also occurs. In general,
daunomycin is metabolized and excreted more readily than adriamycin. The
principal metabolic reaction that the two drugs undergo is the reduction of
the carbonyl group in the acetyl/hydroxyacetyl moiety. Other metabolic reac-
tions are: reductive and hydrolytic deglycosidation, 0-demethylation,
0-sulfation and 0-glucuronidation. Adriamycinol and daunorubicinol, resulting
from the metabolic reduction of the carbonyl group in the side-chain of ring
A, have been suggested to represent the reactive intermediates responsible for
the pharmacological and toxicological effects (167-169).
Complex formation of adriamycin and daunomycin with DNA has been
repeatedly demonstrated (revs. 3, 13). These anthracycline agents intercalate
between adjacent base pairs and hamper DNA replication and transcription.
Removal of the methoxy group does not affect the intercalation of dauno-
mycin. However, the amino sugar, daunosamine, is essential for the complex-
ing. Recent evidence indicates that both microsomes and nuclei of rat liver
contain metabolizing enzymes that can activate adriamycin to reactive inter-
mediate^) which then bind covalently to exogenously added nucleic acids and
microsomal proteins (170, 171). In addition to blocking the template activity
of DNA, interaction of DNA with adriamycin or daunomycin causes strand
165
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breakage, possibly through the production of reactive free radicals in the
proximity of the DNA molecule. The quinone-hydroquinone structure of the
anthracycline ring would allow the molecule to function as an electron accep-
tor and form a semiquinone. The formation of superoxide radicals ('C^)
resulting from increased rate of oxidation of NADPH has actually been demon-
strated following the addition of either adriamycin or daunomycin to liver
microsomes. The generation of free radicals is also indicated by the accumu-
lation of malonyldialdehyde (a product of oxygen free radical attack during
lipid peroxidation on unsaturated fatty acids) in tissues of mice treated with
adriamycin (see rev. 2).
Regarding the possible role that the disturbance of intercellular com-
munication may play in the mechanism of chemical carcinogenesis (see Section
5.2.2.2.4.2, Vol. IIIB), there is evidence for the disruptive effect of adria-
mycin on cell membrane function (172-174).
Mitomycin C. Mitomycin C rapidly disappears from the blood following
i.v. injection. Only traces of the injected dose (8 mg/kg) were detected in
the blood of mice 30 minutes after administration (175). The median half-life
r\
of the drug in 36 patients (receiving by i.v. infusion 10-20 mg/m body sur-
face) was 50 minutes (176). The drug is widely distributed throughout the
body and is metabolized chiefly in the liver. Only small quantities of the
administered dose are excreted in the urine or the bile (176, 177).
Following enzymatic reduction of the quinone to hydroquinone and depar-
ture of the raethoxyl group, the cleavage of the aziridine ring leads to the
formation of a bi- or poly-functional alkylating agent (see Fig. 5 in Section
5.3.1.3.2.1 above).
166
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The biological activity of mitomycin C is believed to be due to single
point alkylation of DNA and cross-linking between adjacent nucleic acid
strands. DNA model studies showed that the site of reaction is probably the
0 position of guanine (see ref. 3), Various substituted mitomycins (such as
mitomycins A and B, 7-hydroxyporfiromycin) and structurally related natural or
synthetic products may also function as bioreductive alkylating agents and
cross-link DNA (see 16). Unfortunately no carcinogenesis data of these com-
pounds are available to substantiate the view that cross linking between DNA
strands is the mechanism of carcinogenesis for this class of compound. Some
investigators still attribute the carcinogenic activity of mitomycin C and
other anti-cancer drugs to their immunedepressive effects which lead to the
loss of immune surveillance system (see 142).
Sarkomycin. The metabolism and mechanism of carcinogenic action of
sarkomycin is unknown. Based on the structural similarity between this anti-
biotic and the carcinogenic 0\,ft-unsaturated lactones (Section 5.2.1.1.6,
Vol. IIIA), it seems possible that sarkomycin may be a direct-acting agent
toward nucleophilic centers in the cell.
Streptozotocin and Elaiomycin. Streptozotocin is readily absorbed from
the gastrointestinal tract in the mouse, but not in the dog or the monkey
(178). Following intravenous injection into rats, Streptozotocin clears
rapidly from the blood, but remains localized in the liver, kidney and
pancreas for at least 6 hours (179). The half-life of Streptozotocin in
humans after i.v. infusion is about 15 minutes. Only 10-20% of administered
dose was detectable in the urine (180). No metabolic products of Streptozo-
tocin and elaiomycin have been reported. However, because of their respective
nitrosourea and azoxymethanol structure, Streptozotocin and elaiomycin are
expected to be metabolized in vivo to form alkylating species (see Sections
167
-------
5.2.1.2.3.1 and 5.2.1.2.4.1, Vol. IIIA). The proposed routes leading to these
alkylating species are shown in Fig. 6 and Fig. 7.
Bennett and Pegg (181) have studied the alkylation of DNA in rat tissue
following administration of streptozotocin. As expected, they found that
streptozotocin is a potent alkylating agent which methylates DNA in the
kidney, liver, pancreas and intestine leading to the formation of 0 - and 7-
methylguanine and 3- and 7-methyladenine. Among these adducts, 0 -methyl-
guanine is lost from DNA more slowly in the kidney and pancreas than in the
liver and intestine. The greater persistence of this alkylated base in the
kidney and pancreas probably accounts for carcinogenesis in these two organs
due to streptozotocin. It is possibly the sugar moiety of the streptozotocin
molecule which directs the alkylation preferentially toward the pancreatic
&-cells since N-methyl-N-nitrosourea, which lacks the 2-deoxy-D-glucose group
in the molecule, does not methylate DNA in the pancreas (181).
Chloramphenicol. In humans, chloramphenicol is absorbed rapidly from the
gastrointestinal tract and distributes in body fluids. The drug is primarily
metabolized in the liver and is excreted via the bile and the urine, either
unchanged or in the form of glucuronide conjugate (182). Investigation of the
metabolic activation of chloramphenicol by rat liver microsomes in vitro (183)
showed that chloramphenicol is hydroxylated by a cytochrome P-450 monooxy-
genase to yield a hydroxydichloroacetamide intermediate which leads to an
oxamyl chloride intermediate by spontaneous loss of hydrochloric acid. The
highly reactive oxamyl chloride intermediate either hydrolyzes to chlor-
amphenicol oxamic acid or acylates microsomal proteins or other macromolecules
(Fig. 8). It is not known whether the reactive oxamyl chloride metabolite
contributes to the pharmacological and carcinogenic effects of chlorampheni-
col. An alternate metabolic route is indicated by the studies of Pohl et al.
168
-------
n-CH-CH =
CH2-OCH3
613
I
o
I
CHOH
Elaiomycin
R _
r
= CH-N = N-C-OH
* R
"
o
I!
R —C—
R,-CH = CH-N =
O
|"
RI-CH=CH-N =N-OH
t
©
r © ~\
R1-CH = CH
0
Fig. 7. Proposed mechanism of metabolic activation of elaiomycin
(modified from: j.A. Miller: Naturally Occurring Substance, that can Induce
Tumor.. _In "Toxicants Occurring Naturally in Foods," National Academy of
Sciences, Washington, D.C., 1973, p. 508).
-------
0 OH
II I
R-NH-C-C-CI
I
Cl
0 Vd**' 0
S^0 H20 II
R_NH-C-CHCI2 -^ > HO-C-CHCI2
RNH-
Chloramphenicol
R= 02N-
0 0
II II
R-NH-C-C-H
IT\
OH CH2OH
I I
-CH-CH-
HCI
o 0
II II
R-NH-C-C-CI
2HCI
0 0
H-C-C-OH
Glyoxylic
acid
Transamination
0 0
II II
R-NH-C-C-OH
Chloramphenicol
oxamic acid
0 0
II II
R-NH-C-C-Protein
NH2
H-C-C-OH
I II
H 0
Glycine
Hydroxymethylation
NH,
HO-CH2-C-C-OH
I II
H 0
Serine
Fig. 8. Proposed metabolic pathways of Chloramphenicol [modified from:
L.R. Pohl, S.D. Nelson and G. Krishna: Biochem. Pharmacol. 27, 491 (1978);
L.R. Pohl, G.B. Reddy and G. Krishna: Biochem. Pharmacol. 28, 2433 (1979)].
-------
(184). In this pathway, chloramphenicol is hydrolyzed to dichloroacetic acid
or is hydrolytically dechlorinated to an aldehyde derivative. Both of these
products yield, by further hydrolysis, glyoxylic acid which gives glycine by
transamination and serine by subsequent hydroxymethylation (Fig. 8).
Azaserine. Azaserine is a direct-acting mutagen (see Section
5.3.1.3.2.2) and probably acts as a direct-acting alkylating agent. The
compound causes DNA damage in various organs of rats, mice, hamsters and
guinea pigs (99, 156). However, alkylation of DNA following azaserine expo-
sure has not been clearly shown.
Bleomycins. The pharmacokinetics of the bleomycins have been studied in
rats (185), mice (186), rabbits (187) and humans (188). After parenteral
administration, bleomycin distributes rapidly to various organs. Most animal
tissues, except the skin and lung, have a relative abundance of a bleomycin-
inactivating enzyme, bleomycin hydrolase, which hydrolyzes the amide group of
the & -aminoalanine moiety of the bleomycins (19). Bleomycinic acid, the acid
without the terminal amine moiety, has been isolated as one of the metabolites
in the urine of humans (188).
The biological effects of bleomycins are believed to be related to inter-
action with DNA. Both in vitro and in vivo studies have shown that bleomycin
intercalates into DNA and breaks DNA strands at the glycosidic linkages, and
phosphodiester bonds (rev. 3). The bithiazole and dimethylsulfonium moieties
of bleomycin appear to be involved directly in intercalation between base
pairs of DNA (189). DNA fragmentation occurs within intact nuclei of lung
tissue of rabbits following activation of bleomycin by microsomal or nuclear
membrane mixed-function oxidases (190). The in vitro DNA cleavage requires
the presence of ferrous ions and dissolved oxygen. Sauville et al. (191, 192)
169
-------
suggested that the attack of bleomycin on DNA is mediated via the generation
of superoxide or hydroxyl free radicals.
5.3.1.3.5 Environmental Significance
The Streptomyces which produce this group of toxins occur in soil. Their
distribution and extent of occurrence in nature is unknown. Since many of
these toxins are used as antibiotics and antineoplastic drugs, they are pro-
duced in industry under conditions that maximize yields (2, 9). The micro-
organisms and the therapeutic uses of these Streptomyces toxins are listed in
Table XXIII.
In view of their carcinogenic activity in experimental animals, there has
been considerable concern that these agents may also act as human carcinogens
or cocarcinogens. Assessment of the carcinogenic potential of antineoplastic
drugs is difficult mainly because they are often used in combination with
irradiation and other chemotherapeutic agents. So far, epidemiological data
on the carcinogenic activity of these Streptomyces metabolites have been
scanty, and mostly inadequate for evaluation (see revs. 5, 9, 193, 194).
There are sporadic case reports in clinical literature describing the develop-
ment of second primary malignancies in cancer patients treated with actino-
mycin D (195) or bleomycin (cited in 193). The second primary tumors which
occurred most frequently were sarcomas, leukemias and neoplasms of the
haematopoietic system. However, most patients received also radiation therapy
and other cheraotherapeutic drugs. A follow-up study found three cases of
leukemia in 126 patients following treatment with chloraraphenicol (196).
Elaiomycin is suspected to be responsible for the high incidence of stomach
cancer among Japanese (cited in 33). Since there is sufficient evidence for
the carcinogenicity of streptozotocin in several animal species, the working
170
-------
Table XXIII
Streptomyces Toxins, the Generating Microorganisms, Therapeutic Uses3
Toxin
Producing
Microorganism
Disease
Actinomycin D
Adriamycin
Daunomycin
Mitomycin C
Sarkomycin
Streptozotocin
Elaiomycin
Chloramphenicol
Azaserine
Bleomycin
S. paruvllus
_S_. peucetius , var,
caesius
_S_. caeruleorubidus ;
S. peucetms
S. caespitosus
_S_. erythrochromogenes
S. achromogenes
_S_. hepaticus
_S. venezuelae
_S_. fragilis
S. verticillus
Choriocarcinoma; Wilms1 tumor;
testicular tumor; rhabdomyosar-
coma, Ewing's sarcoma; osteosar-
coraa and acute leukemias
Hodgkin's disease; non-Hodgkin" s
lymphoma; Wilms1 tumor; acute
leukemias; carcinomas of the
breast, lung, bladder, prostate,
ovary, testes , thyroid, head and
neck; soft tissue and other
sarcomas
Acute leukemias
Carcinomas of the breast, stomach,
colon, pancreas, cervix, bladder,
liver, lung, head and neck; malig-
nant melanoma
Malignant carcinoid and pancreatic
tumors; Hodgkin's disease and
other lymphomas
Infections caused by gram-positive
and gram-negative bacteria,
rickettsiae and some viruses
Acute leukemias
Hodgkin's disease and other
lymphomas; carcinomas of the
testis, head, neck, skin, eso-
phagus and genitourinary tract.
S
Summarized from IARC, IARC Monographs Vol. 10, International Agency for
Research on Cancer, Lyon (1976); P. Calabresi and R.E. Parks, Jr., "Anti-
proliferative Agents and Drugs Used for Immunosuppression." In "The
Pharmacological Basis of Therapeutics" (A.G. Gilraan, L.S. Goodman and A.
Oilman, eds.), MacMillan, New York, 1980, pp. 1256-1313.
Bacterial infections and cancer [see F. Dickens and H.E.H.
Cancer 19, 392 (1965)].
Jones , Brit. J.
cBacterial infections [see R.C,
Aflatoxins, and Nucleic Acids,
Garner and C.N. Martin, "Fungal Toxins,
' in "Chemical Carcinogens and DNA" (P.L.
Grover, ed.), Vol. I, Chapter 7, CRC Press, Boca Raton, Florida, 1978,
187-225.
pp.
-------
group of the International Agency for Research on Cancer (197) suggested that
streptozotocin should be regarded for practical purposes as if it were a human
carcinogen despite the lack of epidemiological data.
171
-------
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